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Raymond C Harris M.D.

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Gewin L, Bulus N, Mernaugh G, Moeckel G, Harris RC, Moses HL, Pozzi A, Zent R. TGF-beta receptor deletion in the renal collecting system exacerbates fibrosis. J Am Soc Nephrol (2010) 21:1334-43
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TGF-beta plays a key role in upregulating matrix production in injury-induced renal fibrosis, but how TGF-beta signaling in distinct compartments of the kidney, such as specific segments of the nephron, affects the response to injury is unknown. In this study, we determined the role of TGF-beta signaling both in development of the renal collecting system and in response to injury by selectively deleting the TGF-beta type II receptor in mice at the initiation of ureteric bud development. These mice developed normally but demonstrated a paradoxic increase in fibrosis associated with enhanced levels of active TGF-beta after unilateral ureteral obstruction. Consistent with this observation, TGF-beta type II receptor deletion in cultured collecting duct cells resulted in excessive integrin alphavbeta6-dependent TGF-beta activation that increased collagen synthesis in co-cultured renal interstitial fibroblasts. These results suggest that inhibiting TGF-beta receptor-mediated function in collecting ducts may exacerbate renal fibrosis by enhancing paracrine TGF-beta signaling between epithelial and interstitial cells.

Cheng H, Harris RC. The glomerulus--a view from the outside--the podocyte. Int J Biochem Cell Biol (2010) 42:1380-7
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In the past decade, podocyte research has been greatly aided by the development of powerful new molecular, cellular and animal tools, leading to elucidation of an increasing number of proteins involved in podocyte function and identification of mutated genes in hereditary glomerulopathies. Accumulating evidence indicates that podocyte disorders may not only underlie these hereditary glomerulopathies but also play crucial role in a broad spectrum of acquired glomerular diseases. Genetic susceptibility, environmental influence and systemic responses are all involved in the mediation of the pathogenesis of podocytopathies. Injured podocytes may predisopose to further injury of other podocytes and other adjacent/distant renal cells in a vicious cycle, leading to inexorable progression of glomerular injury. The classic view is that podocytes have a limited ability to proliferate in the normal mature kidney. However, recent research in rodents has provided suggestive evidence for podocyte regeneration resulting from differentiation of progenitor cells within Bowman's capsule. CI Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Chen X, Whiting C, Borza C, Hu W, Mont S, Bulus N, Zhang MZ, Harris RC, Zent R, Pozzi A. Integrin alpha1beta1 regulates epidermal growth factor receptor activation by controlling peroxisome proliferator-activated receptor gamma-dependent caveolin-1 expression. Mol Cell Biol (2010) 30:3048-58
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Integrin alpha1beta1 negatively regulates the generation of profibrotic reactive oxygen species (ROS) by inhibiting epidermal growth factor receptor (EGFR) activation; however, the mechanism by which it does this is unknown. In this study, we show that caveolin-1 (Cav-1), a scaffolding protein that binds integrins and controls growth factor receptor signaling, participates in integrin alpha1beta1-mediated EGFR activation. Integrin alpha1-null mesangial cells (MCs) have reduced Cav-1 levels, and reexpression of the integrin alpha1 subunit increases Cav-1 levels, decreases EGFR activation, and reduces ROS production. Downregulation of Cav-1 in wild-type MCs increases EGFR phosphorylation and ROS synthesis, while overexpression of Cav-1 in the integrin alpha1-null MCs decreases EGFR-mediated ROS production. We further show that integrin alpha1-null MCs have increased levels of activated extracellular signal-regulated kinase (ERK), which leads to reduced activation of peroxisome proliferator-activated receptor gamma (PPARgamma), a transcription factor that positively regulates Cav-1 expression. Moreover, activation of PPARgamma or inhibition of ERK increases Cav-1 levels in the integrin alpha1-null MCs. Finally, we show that glomeruli of integrin alpha1-null mice have reduced levels of Cav-1 and activated PPARgamma but increased levels of phosphorylated EGFR both at baseline and following injury. Thus, integrin alpha1beta1 negatively regulates EGFR activation by positively controlling Cav-1 levels, and the ERK/PPARgamma axis plays a key role in regulating integrin alpha1beta1-dependent Cav-1 expression and consequent EGFR-mediated ROS production.

Peti-Peterdi J, Harris RC. Macula densa sensing and signaling mechanisms of renin release. J Am Soc Nephrol (2010) 21:1093-6
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Macula densa cells in the distal nephron, according to the classic paradigm, are salt sensors that generate paracrine chemical signals in the juxtaglomerular apparatus to control vital kidney functions, including renal blood flow, glomerular filtration, and renin release. Renin is the rate-limiting step in the activation of the renin-angiotensin system, a key modulator of body fluid homeostasis. Here, we discuss recent advances in understanding macula densa sensing and suggest these cells, in addition to salt, also sense various chemical and metabolic signals in the tubular environment that directly trigger renin release.

He W, Wang Y, Zhang MZ, You L, Davis LS, Fan H, Yang HC, Fogo AB, Zent R, Harris RC, Breyer MD, Hao CM. Sirt1 activation protects the mouse renal medulla from oxidative injury. J Clin Invest (2010) 120:1056-68
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Sirtuin 1 (Sirt1) is a NAD+-dependent deacetylase that exerts many of the pleiotropic effects of oxidative metabolism. Due to local hypoxia and hypertonicity, the renal medulla is subject to extreme oxidative stress. Here, we set out to investigate the role of Sirt1 in the kidney. Our initial analysis indicated that it was abundantly expressed in mouse renal medullary interstitial cells in vivo. Knocking down Sirt1 expression in primary mouse renal medullary interstitial cells substantially reduced cellular resistance to oxidative stress, while pharmacologic Sirt1 activation using either resveratrol or SRT2183 improved cell survival in response to oxidative stress. The unilateral ureteral obstruction (UUO) model of kidney injury induced markedly more renal apoptosis and fibrosis in Sirt1+/mice than in wild-type controls, while pharmacologic Sirt1 activation substantially attenuated apoptosis and fibrosis in wild-type mice. Moreover, Sirt1 deficiency attenuated oxidative stress-induced COX2 expression in cultured mouse renal medullary interstitial cells, and Sirt1+/mice displayed reduced UUO-induced COX2 expression in vivo. Conversely, Sirt1 activation increased renal medullary interstitial cell COX2 expression both in vitro and in vivo. Furthermore, exogenous PGE2 markedly reduced apoptosis in Sirt1-deficient renal medullary interstitial cells following oxidative stress. Taken together, these results identify Sirt1 as an important protective factor for mouse renal medullary interstitial cells following oxidative stress and suggest that the protective function of Sirt1 is partly attributable to its regulation of COX2 induction. We therefore suggest that Sirt1 provides a potential therapeutic target to minimize renal medullary cell damage following oxidative stress.

Zhang MZ, Su Y, Yao B, Zheng W, Decaestecker M, Harris RC. Assessing the application of tissue microarray technology to kidney research. J Histochem Cytochem (2010) 58:413-20
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Tissue microarray (TMA) is a new high-throughput method that enables simultaneous analysis of the profiles of protein expression in multiple tissue samples. TMA technology has not previously been adapted for physiological and pathophysiological studies of rodent kidneys. We have evaluated the validity and reliability of using TMA to assess protein expression in mouse and rat kidneys. A representative TMA block that we have produced included: (1) mouse and rat kidney cortex, outer medulla, and inner medulla fixed with different fixatives; (2) rat kidneys at different stages of development fixed with different fixatives; (3) mouse and rat kidneys with different physiological or pathophysiological treatments; and (4) built-in controls. As examples of the utility, immunostaining for cyclooxygenase-2, renin, Tamm Horsfall protein, aquaporin-2, connective tissue growth factor, and synaptopodin was carried out with kidney TMA slides. Quantitative analysis of cyclooxygense-2 expression in kidneys confirms that individual cores provide meaningful representations comparable to whole-kidney sections. These studies show that kidney TMA technique is a promising and useful tool for investigating the expression profiles of proteins of interest in rodent kidneys under different physiological and pathophysiological conditions.

Yao B, Harris RC, Zhang MZ. Intrarenal dopamine attenuates deoxycorticosterone acetate/high salt-induced blood pressure elevation in part through activation of a medullary cyclooxygenase 2 pathway. Hypertension (2009) 54:1077-83
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Locally produced dopamine in the renal proximal tubule inhibits salt and fluid reabsorption, and a dysfunctional intrarenal dopaminergic system has been reported in essential hypertension and experimental hypertension models. Using catechol-O-methyl-transferase knockout (COMT(-/-)) mice, which have increased renal dopamine because of deletion of the major renal dopamine-metabolizing enzyme, we investigated the effect of intrarenal dopamine on the development of hypertension in the deoxycorticosterone acetate/high-salt (DOCA/HS) model. DOCA/HS led to significant increases in systolic blood pressure in wild-type mice (from 115+/-2 to 153+/-4 mm Hg), which was significantly attenuated in COMT(-/-) mice (from 114+/-2 to 135+/-3 mm Hg). In DOCA/HS COMT(-/-) mice, the D1-like receptor antagonist SCH-23390 increased systolic blood pressure (156+/-2 mm Hg). DOCA/HS COMT(-/-) mice also exhibited more urinary sodium excretion (COMT(-/-) versus wild-type: 3038+/-430 versus 659+/-102 micromol/L per 24 hours; P<0.01). Furthermore, DOCA/HS-induced renal oxidative stress was significantly attenuated in COMT(-/-) mice. COX-2-derived prostaglandins in the renal medulla promote sodium excretion, and dopamine stimulates medullary prostaglandin production. Renal medullary COX-2 expression and urinary prostaglandin E2 excretion were significantly higher in COMT(-/-) than in wild-type mice after DOCA/HS treatment. In DOCA/HS-treated COMT(-/-) mice, the COX-2 inhibitor SC-58236 reduced urinary sodium and prostaglandin E(2) excretion and increased systolic blood pressure (153+/-2 mm Hg). These studies indicate that an activated renal dopaminergic system attenuates the development of hypertension, at least in large part through activating medullary COX-2 expression/activity, and also decreases oxidative stress resulting from DOCA/HS.

Brosius FC 3rd, Alpers CE, Bottinger EP, Breyer MD, Coffman TM, Gurley SB, Harris RC, Kakoki M, Kretzler M, Leiter EH, Levi M, McIndoe RA, Sharma K, Smithies O, Susztak K, Takahashi N, Takahashi T. Mouse models of diabetic nephropathy. J Am Soc Nephrol (2009) 20:2503-12
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Diabetic nephropathy is a major cause of ESRD worldwide. Despite its prevalence, a lack of reliable animal models that mimic human disease has delayed the identification of specific factors that cause or predict diabetic nephropathy. The Animal Models of Diabetic Complications Consortium (AMDCC) was created in 2001 by the National Institutes of Health to develop and characterize models of diabetic nephropathy and other complications. This interim report and our online supplement detail the progress made toward that goal, specifically in the development and testing of murine models. Updates are provided on validation criteria for early and advanced diabetic nephropathy, phenotyping methods, the effect of background strain on nephropathy, current best models of diabetic nephropathy, negative models, and views of future directions. AMDCC investigators and other investigators in the field have yet to validate a complete murine model of human diabetic kidney disease. Nonetheless, the critical analysis of existing murine models substantially enhances our understanding of this disease process.

Zhang X, Mernaugh G, Yang DH, Gewin L, Srichai MB, Harris RC, Iturregui JM, Nelson RD, Kohan DE, Abrahamson D, Fassler R, Yurchenco P, Pozzi A, Zent R. beta1 integrin is necessary for ureteric bud branching morphogenesis and maintenance of collecting duct structural integrity. Development (2009) 136:3357-66
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The kidney collecting system develops from branching morphogenesis of the ureteric bud (UB). This process requires signaling by growth factors such as glial cell line derived neurotrophic factor (GDNF) and fibroblast growth factors (FGFs) as well as cell extracellular matrix interactions mediated by integrins. The importance of integrin signaling in UB development was investigated by deleting integrin beta1 at initiation (E10.5) and late (E18.5) stages of development. Deletion at E10.5 resulted in a severe branching morphogenesis phenotype. Deletion at E18.5 did not alter renal development but predisposed the collecting system to severe injury following ureteric obstruction. beta1 integrin was required for renal tubular epithelial cells to mediate GDNFand FGF-dependent signaling despite normal receptor localization and activation in vitro. Aberrations in the same signaling molecules were present in the beta1-null UBs in vivo. Thus beta1 integrins can regulate organ branching morphogenesis during development by mediating growth-factor-dependent signaling in addition to their well-defined role as adhesion receptors.

Ray KC, Blaine SA, Washington MK, Braun AH, Singh AB, Harris RC, Harding PA, Coffey RJ, Means AL. Transmembrane and soluble isoforms of heparin-binding epidermal growth factor-like growth factor regulate distinct processes in the pancreas. Gastroenterology (2009) 137:1785-94
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BACKGROUND & AIMS: Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is produced as a type-I, single-pass transmembrane protein that can be cleaved to release a diffusible peptide. HB-EGF, often overexpressed in damaged or diseased epithelium, is normally expressed in pancreatic islets, but its function is not understood. METHODS: To understand the function of each isoform of HB-EGF, we made transgenes expressing either a constitutively transmembrane or a constitutively secreted protein. RESULTS: The transmembrane isoform was not an inert precursor protein, but a functional molecule, downregulating the glucose-sensing apparatus of pancreatic islets. Conversely, the secreted form of HB-EGF improved islet function, but had severe fibrotic and neoplastic effects on surrounding tissues. Each isoform had a more severe phenotype than that of full-length HB-EGF, even though the full-length protein was efficiently cleaved, thus producing both isoforms, suggesting that a level of regulation was lost by separating the isoforms. CONCLUSIONS: This work demonstrates that islet function depends on the ratio of cleaved to uncleaved HB-EGF and that the transmembrane intermediate, while deleterious to islet function, is necessary to restrict action of soluble HB-EGF away from surrounding tissue.

Cheng H, Fan X, Guan Y, Moeckel GW, Zent R, Harris RC. Distinct roles for basal and induced COX-2 in podocyte injury. J Am Soc Nephrol (2009) 20:1953-62
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Transgenic mice that overexpress cyclooxygenase-2 (COX-2) selectively in podocytes are more susceptible to glomerular injury by adriamycin and puromycin (PAN). To investigate the potential roles of COX-2 metabolites, we studied mice with selective deletion of prostanoid receptors and generated conditionally immortalized podocyte lines from mice with either COX-2 deletion or overexpression. Podocytes that overexpressed COX-2 were virtually indistinguishable from wild-type podocytes but were significantly more sensitive to PAN-induced injury, produced more prostaglandin E(2) and thromboxane B(2), and had greater expression of prostaglandin E(2) receptor subtype 4 (EP(4)) and thromboxane receptor (TP). Treatment of COX-2-overexpressing podocytes with a TP antagonist reduced apoptosis, but treatment with an EP(4) antagonist did not. In contrast, podocytes from COX-2-knockout mice exhibited increased apoptosis, markedly decreased cell adhesion, and prominent stress fibers. In vivo, selective deletion of podocyte EP(4) did not alter the increased sensitivity to adriamycin-induced injury observed in mice overexpressing podocyte COX-2. In contrast, genetic deletion of TP in these mice prevented adriamycin-induced injury, with attenuated albuminuria and foot process effacement. These results suggest that basal COX-2 may be important for podocyte survival, but overexpression of podocyte COX-2 increases susceptibility to podocyte injury, which is mediated, in part, by activation of the thromboxane receptor.

Chen JK, Chen J, Thomas G, Kozma SC, Harris RC. null Am J Physiol Renal Physiol (2009) null:null
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Removal of one kidney stimulates synthesis of RNA and protein, with minimal DNA replication, in all nephron segments of the remaining kidney, resulting in cell growth (increase in cell size) with minimal cell proliferation (increase in cell number). In addition to the compensatory renal hypertrophy caused by nephron loss, pathophysiologic renal hypertrophy can occur as a consequence of early uncontrolled diabetes. However, the molecular mechanism underlying renal hypertrophy in these conditions remains unclear. In the present study, we report that deletion of S6 kinase 1 (S6K1) inhibited renal hypertrophy seen following either contralateral nephrectomy or induction of diabetes. In wild type mice, hypertrophic stimuli increased phosphorylation of 40S ribosomal protein S6 (rpS6), a known target of S6K1. Immunoblotting analysis revealed that S6K1(-/-) mice exhibited moderately elevated basal levels of rpS6, which did not increase further in response to the hypertrophic stimuli. Northern blotting indicated a moderate up-regulation of S6K2 expression in the kidneys of S6K1(-/-) mice. Phosphorylation of the eukaryotic translation initiation factor (eIF) 4E-binding protein 1 (4E-BP1), another downstream target of the mammalian target of rapamycin (mTOR), was stimulated to equivalent levels in S6K1(-/-) and S6K1(+/+) littermates during renal hypertrophy, indicating that mTOR was still activated in the S6K1(-/-) mice. The highly selective mTOR inhibitor, rapamycin, inhibited increased phosphorylation of rpS6 and blocked 60-70% of the hypertrophy seen in wild type mice but failed to prevent the ~10% hypertrophy seen in S6K1(-/-) mice in response to uninephrectomy (UNX), although it did inhibit the basal rpS6 phosphorylation. Thus, the present study provides the first genetic evidence that S6K1 plays a major role in the development of compensatory renal hypertrophy as well as diabetic renal hypertrophy and indicates that UNXand diabetes-mediated mTOR activation can selectively activate S6K1 without activating S6K2. Key words: Compensatory renal hypertrophy, streptozotocin, mTORC1 signaling, S6 kinases.

Fujita H, Fujishima H, Chida S, Takahashi K, Qi Z, Kanetsuna Y, Breyer MD, Harris RC, Yamada Y, Takahashi T. Reduction of renal superoxide dismutase in progressive diabetic nephropathy. J Am Soc Nephrol (2009) 20:1303-13
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Superoxide excess plays a central role in tissue damage that results from diabetes, but the mechanisms of superoxide overproduction in diabetic nephropathy (DN) are incompletely understood. In the present study, we investigated the enzyme superoxide dismutase (SOD), a major defender against superoxide, in the kidneys during the development of murine DN. We assessed SOD activity and the expression of SOD isoforms in the kidneys of two diabetic mouse models (C57BL/6-Akita and KK/Ta-Akita) that exhibit comparable levels of hyperglycemia but different susceptibility to DN. We observed down-regulation of cytosolic CuZn-SOD (SOD1) and extracellular CuZn-SOD (SOD3), but not mitochondrial Mn-SOD (SOD2), in the kidney of KK/Ta-Akita mice which exhibit progressive DN. In contrast, we did not detect a change in renal SOD expression in DN-resistant C57BL/6-Akita mice. Consistent with these findings, there was a significant reduction in total SOD activity in the kidney of KK/Ta-Akita mice compared with C57BL/6-Akita mice. Finally, treatment of KK/Ta-Akita mice with a SOD mimetic, tempol, ameliorated the nephropathic changes in KK/Ta-Akita mice without altering the level of hyperglycemia. Collectively, these results indicate that down-regulation of renal SOD1 and SOD3 may play a key role in the pathogenesis of DN.

Zhang MZ, Xu J, Yao B, Yin H, Cai Q, Shrubsole MJ, Chen X, Kon V, Zheng W, Pozzi A, Harris RC. Inhibition of 11beta-hydroxysteroid dehydrogenase type II selectively blocks the tumor COX-2 pathway and suppresses colon carcinogenesis in mice and humans. J Clin Invest (2009) 119:876-85
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Colorectal cancer (CRC) is a leading cause of cancer death, yet primary prevention remains the best approach to reducing overall morbidity and mortality. Studies have shown that COX-2-derived PGE2 promotes CRC progression, and both nonselective COX inhibitors (NSAIDs) and selective COX-2 inhibitors (such as glucocorticoids) reduce the number and size of colonic adenomas. However, increased gastrointestinal side effects of NSAIDs and increased cardiovascular risks of selective COX-2 inhibitors limit their use in chemoprevention of CRC. We found that expression of 11beta-hydroxysteroid dehydrogenase type II (11betaHSD2), which converts active glucocorticoids to inactive keto-forms, increased in human colonic and Apc+/min mouse intestinal adenomas and correlated with increased COX-2 expression and activity. Furthermore, pharmacologic inhibition or gene silencing of 11betaHSD2 inhibited COX-2-mediated PGE2 production in tumors and prevented adenoma formation, tumor growth, and metastasis in mice. Inhibition of 11betaHSD2 did not reduce systemic prostacyclin production or accelerate atherosclerosis in mice, thereby avoiding the major cardiovascular side effects seen with systemic COX-2 inhibitors. Therefore, 11betaHSD2 inhibition represents what we believe to be a novel approach for CRC chemoprevention and therapy by increasing tumor glucocorticoid activity, which in turn selectively blocks local COX-2 activity.

Smith JP, Pozzi A, Dhawan P, Singh AB, Harris RC. Soluble HB-EGF induces epithelial-to-mesenchymal transition in inner medullary collecting duct cells by upregulating Snail-2. Am J Physiol Renal Physiol (2009) 296:F957-65
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Animal models of acute renal injury suggest that the epidermal growth factor receptor (EGFR) axis may have a beneficial role in the recovery from acute renal injury, but recent reports describe detrimental effects of EGFR activation in chronic renal injury. Expression of the EGFR ligand heparin-binding EGF-like growth factor (HB-EGF) increases following renal injury, but the effects of this sustained upregulation have not been well studied. Here, stable overexpression of soluble HB-EGF (sHB-EGF) in mouse inner medullary collecting duct (IMCD) cells led to marked phenotypic changes: sHB-EGF-expressing cells demonstrated a fibroblast-like morphology, did not form epithelial sheets, exhibited cytoplasmic projections, decreased expression of epithelial markers, and increased expression of fibroblast-specific protein-1. They also demonstrated anchorage-independent growth and formed tumors when injected subcutaneously into nude mice. Quantitative RT-PCR and a luciferase reporter assay suggested that sHB-EGF repressed transcription of E-cadherin, and a concomitant TGF-beta-independent upregulation of the E-cadherin repressor Snail-2 was observed. Stable downregulation of Snail-2 in sHB-EGF-overexpressing cells restored epithelial characteristics (E-cadherin and cytokeratin expression) but did not alter their anchorage-independent growth. In summary, sustained exposure to sHB-EGF induces epithelial-to-mesenchymal transition of IMCD cells, in part by upregulating the E-cadherin transcriptional repressor Snail-2.

Zeng F, Xu J, Harris RC. Nedd4 mediates ErbB4 JM-a/CYT-1 ICD ubiquitination and degradation in MDCK II cells. FASEB J (2009) 23:1935-45
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ErbB4, a type I transmembrane receptor tyrosine kinase, is a member of the epidermal growth factor receptor family. Its cleavage releases an intracellular C-terminal domain (ICD), which can be either degraded following ubiqitination or translocated to the nucleus and regulate gene expression. There are 2 ErbB4 ICD isoforms: CYT-1 and CYT-2. We and others have previously reported that following cleavage, CYT-2 selectively translocates to the nucleus. In the current study we found that following cleavage, the intracellular levels of CYT-1 ICD decreased rapidly, while levels of CYT-2 ICD remained relatively stable. CYT-1 ICD degradation could be prevented by administration of either the proteasome inhibitor lactacystin or the lysosome inhibitor chloroquine, indicating both proteasomal and lysosomal degradation. Further studies implicated Nedd4, an E3 ubiquitin ligase, as a mediator of CYT-1 ubiquitination and degradation. The interaction of Nedd4 with CYT-1 was shown by coimmnunoprecipitation, an in vitro direct binding assay, and an in vitro ubiquitination assay. Three PPxY or PY motifs present in the CYT-1 C terminus are necessary for binding by Nedd4 WW domains, because impaired interactions are seen in mutation of any of the PY motifs. Nedd4-CYT-1 binding was associated with increased CYT-1 ubiquitination following proteasome inhibitor treatment. Impaired Nedd4 binding to CYT-1 by PY motif mutations led to increased CYT-1 ICD stability, whereas only one of the PY motif mutations (Y1056A), which disrupts the binding sites for both a WW domain and an SH2 domain of PI3 kinase, demonstrated enhanced nuclear translocation following HB-EGF treatment. These studies indicate that Nedd4 mediates ErbB4 CYT-1 ICD ubiquitination and degradation, and the prevention of both WW binding and PI3 kinase activity are required for ErbB4 nuclear translocation.

Zhang MZ, Yao B, Fang X, Wang S, Smith JP, Harris RC. Intrarenal dopaminergic system regulates renin expression. Hypertension (2009) 53:564-70
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Dopamine is a major regulator of proximal tubule salt reabsorption and is a modulator of renin release. Dopamine has been reported to stimulate renin release in vitro through activation of D1-like receptors. However, previous studies investigating dopamine regulation of renin release in vivo have provided contradictory results, indicating stimulation, inhibition, or no effect. We have reported previously that macula densa cyclooxygenase-2 (COX-2) is suppressed by dopamine. Because macula densa COX-2 stimulates renal renin expression, our current studies investigated dopamine regulation of renal renin release and synthesis in vivo. Acute treatment with a D1-like receptor agonist, fenoldopam, significantly inhibited renin release, as did acute inhibition of proximal tubule salt reabsorption with acetazolamide. In catechol-O-methyl transferase knockout (COMT(-/-)) mice, which have increased kidney dopamine levels because of deletion of the major intrarenal dopamine metabolizing enzyme, there was attenuation in response to a low-salt diet of the increases of renal cortical COX-2 and renin expression and renin release. A high-salt diet led to significant decreases in renal renin expression but much less significant decreases in COMT(-/-) mice than wild type mice, resulting in higher renal renin expression in COMT(-/-) mice. In high salt-treated wild-type mice or COX-2 knockout mice on a normal salt diet, fenoldopam stimulated renal renin expression. These results suggest that dopamine predominantly inhibits renal renin expression and release by inhibiting macula densa COX-2, but suppression of renal cortical COX-2 activity reveals a contrasting effect of dopamine to stimulate renal renin expression through activation of D1-like receptors.

Zeng F, Singh AB, Harris RC. The role of the EGF family of ligands and receptors in renal development, physiology and pathophysiology. Exp Cell Res (2009) 315:602-10
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Mammalian kidney expresses all of the members of the ErbB family of receptors and their respective ligands. Studies support a role for ErbB family receptor activation in kidney development and differentiation. Under physiologic conditions, EGFR activation appears to play an important role in the regulation of renal hemodynamics and electrolyte handling by the kidney, while in different pathophysiologic states, EGFR activation may mediate either beneficial or detrimental effects to the kidney. This article provides an overview of the expression profile of the ErbB family of ligands and receptors in the mammalian kidney and summarizes known physiological and pathophysiological roles of EGFR activation in the organ.

Tchekneva EE, Khuchua Z, Davis LS, Kadkina V, Dunn SR, Bachman S, Ishibashi K, Rinchik EM, Harris RC, Dikov MM, Breyer MD. Single amino acid substitution in aquaporin 11 causes renal failure. J Am Soc Nephrol (2008) 19:1955-64
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A screen of recessive mutations generated by the chemical mutagen n-ethyl-n-nitrosourea (ENU) mapped a new mutant locus (5772SB) termed sudden juvenile death syndrome (sjds) to chromosome 7 in mice. These mutant mice, which exhibit severe proximal tubule injury and formation of giant vacuoles in the renal cortex, die from renal failure, a phenotype that resembles aquaporin 11 (Aqp11) knockout mice. In this report, the ENU-induced single-nucleotide variant (sjds mutation) is identified. To determine whether this variant, which causes an amino acid substitution (Cys227Ser) in the predicted E-loop region of aquaporin 11, is responsible for the sjds lethal renal phenotype, Aqp11-/sjds compound heterozygous mice were generated from Aqp11 +/sjds and Aqp11 +/intercrosses. The compound heterozygous Aqp11 -/sjds offspring exhibited a lethal renal phenotype (renal failure by 2 wk), similar to the Aqp11 sjds/sjds and Aqp11-/phenotypes. These results demonstrate that the identified mutation causes renal failure in Aqp11 sjds/sjds mutant mice, providing a model for better understanding of the structure and function of aquaporin 11 in renal physiology.

Han BG, Hao CM, Tchekneva EE, Wang YY, Lee CA, Ebrahim B, Harris RC, Kern TS, Wasserman DH, Breyer MD, Qi Z. Markers of glycemic control in the mouse: comparisons of 6-hand overnight-fasted blood glucoses to Hb A1c. Am J Physiol Endocrinol Metab (2008) 295:E981-6
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The present studies examined the relationship between fasting blood glucose and Hb A(1c) in C57BL/6J, DBA/2J, and KK/HlJ mice with and without diabetes mellitus. Daily averaged blood glucose levels based on continuous glucose monitoring and effects of 6-h vs. overnight fasting on blood glucose were determined. Daily averaged blood glucose levels were highly correlated with Hb A(1c), as determined with a hand-held automated device using an immunodetection method. R(2) values were 0.90, 0.95, and 0.99 in KK/HIJ, C57BL/6J, and DBA/2J, respectively. Six-hour fasting blood glucose correlated more closely with the level of daily averaged blood glucose and with Hb A(1c) than did blood glucose following an overnight fast. To validate the immunoassay-determined Hb A(1c), we also measured total glycosylated hemoglobin using boronate HPLC. Hb A(1c) values correlated well with total glycosylated hemoglobin in all three strains but were relatively lower than total glycosylated hemoglobin in diabetic DBA/2J mice. These results show that 6-h fasting glucose provides a superior index of glycemic control and correlates more closely with Hb A(1c) than overnight-fasted blood glucose in these strains of mice.

Chen JK, Chen J, Imig JD, Wei S, Hachey DL, Guthi JS, Falck JR, Capdevila JH, Harris RC. Identification of novel endogenous cytochrome p450 arachidonate metabolites with high affinity for cannabinoid receptors. J Biol Chem (2008) 283:24514-24
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Arachidonic acid is an essential constituent of cell membranes that is esterified to the sn-2-position of glycerophospholipids and is released from selected lipid pools by phospholipase cleavage. The released arachidonic acid can be metabolized by three enzymatic pathways: the cyclooxygenase pathway forming prostaglandins and thromboxanes, the lipoxygenase pathway generating leukotrienes and lipoxins, and the cytochrome P450 (cP450) pathway producing epoxyeicosatrienoic acids and hydroxyeicosatetraenoic acids. The present study describes a novel group of cP450 epoxygenase-dependent metabolites of arachidonic acid, termed 2-epoxyeicosatrienoylglycerols (2-EG), including two regioisomers, 2-(11,12-epoxyeicosatrienoyl)glycerol (2-11,12-EG) and 2-(14,15-epoxyeicosatrienoyl)glycerol (2-14,15-EG), which are both produced in the kidney and spleen, whereas 2-11,12-EG is also detected in the brain. Both 2-11,12-EG and 2-14,15-EG activated the two cannabinoid (CB) receptor subtypes, CB1 and CB2, with high affinity and elicited biological responses in cultured cells expressing CB receptors and in intact animals. In contrast, the parental arachidonic acid and epoxyeicosatrienoic acids failed to activate CB1 or CB2 receptors. Thus, these cP450 epoxygenase-dependent metabolites are a novel class of endogenously produced, biologically active lipid mediators with the characteristics of endocannabinoids. This is the first evidence of a cytochrome P450-dependent arachidonate metabolite that can activate G-protein-coupled cell membrane receptors and suggests a functional link between the cytochrome P450 enzyme system and the endocannabinoid system.

Srichai MB, Hao C, Davis L, Golovin A, Zhao M, Moeckel G, Dunn S, Bulus N, Harris RC, Zent R, Breyer MD. Apoptosis of the thick ascending limb results in acute kidney injury. J Am Soc Nephrol (2008) 19:1538-46
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Ischemiaor toxin-induced acute kidney injury is generally thought to affect the cells of the proximal tubule, but it has been difficult to define the involvement of other tubular segments because of the widespread damage caused by ischemia/reperfusion or toxin-induced injury in experimental models. For evaluation of whether thick ascending limb (TAL)-specific epithelial injury results in acute kidney injury, a novel transgenic mouse model that expresses the herpes simplex virus 1 thymidine kinase gene under the direction of the TAL-specific Tamm-Horsfall protein promoter was generated. After administration of gancyclovir, these mice demonstrated apoptosis only in TAL cells, with little evidence of neutrophil infiltration. Compared with control mice, blood urea nitrogen and creatinine levels were at least five-fold higher in the transgenic mice, which also developed oliguria and impaired urinary concentrating ability. These findings suggest that acute injury targeted only to the TAL is sufficient to cause severe acute kidney injury in mice with features similar to those observed in humans.

Pozzi A, Jarad G, Moeckel GW, Coffa S, Zhang X, Gewin L, Eremina V, Hudson BG, Borza DB, Harris RC, Holzman LB, Phillips CL, Fassler R, Quaggin SE, Miner JH, Zent R. Beta1 integrin expression by podocytes is required to maintain glomerular structural integrity. Dev Biol (2008) 316:288-301
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Integrins are transmembrane heteromeric receptors that mediate interactions between cells and extracellular matrix (ECM). beta1, the most abundantly expressed integrin subunit, binds at least 12 alpha subunits. beta1 containing integrins are highly expressed in the glomerulus of the kidney; however their role in glomerular morphogenesis and maintenance of glomerular filtration barrier integrity is poorly understood. To study these questions we selectively deleted beta1 integrin in the podocyte by crossing beta1(flox/flox) mice with podocyte specific podocin-cre mice (pod-Cre), which express cre at the time of glomerular capillary formation. We demonstrate that podocyte abnormalities are visualized during glomerulogenesis of the pod-Cre;beta1(flox/flox) mice and proteinuria is present at birth, despite a grossly normal glomerular basement membrane. Following the advent of glomerular filtration there is progressive podocyte loss and the mice develop capillary loop and mesangium degeneration with little evidence of glomerulosclerosis. By 3 weeks of age the mice develop severe end stage renal failure characterized by both tubulointerstitial and glomerular pathology. Thus, expression of beta1 containing integrins by the podocyte is critical for maintaining the structural integrity of the glomerulus.

Breyer MD, Tchekneva E, Qi Z, Takahashi T, Fogo AB, Harris RC. Examining diabetic nephropathy through the lens of mouse genetics. Curr Diab Rep (2007) 7:459-66
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Although diabetic nephropathy occurs in only a minority of patients with diabetes, it is the major cause of end-stage renal disease in the United States. Hyperglycemia and hypertension are important factors predisposing patients to diabetic nephropathy, but accumulating evidence points to critical genetic factors predisposing only a subset of patients with diabetes to nephropathy. It has been challenging to define the genes conferring risk for nephropathy in human populations. Comparative genomics using the robust genetic reagents available in laboratory mice should provide a complementary approach to defining genes that may predispose to diabetic nephropathy in mice and humans. This article reviews new studies to identify genetic risk factors for diabetic nephropathy and the unique approaches that may be used to elucidate the genetic pathogenesis of this disorder in mice.

Breyer MD, Qi Z, Tchekneva EE, Harris RC. Insight into the genetics of diabetic nephropathy through the study of mice. Curr Opin Nephrol Hypertens (2008) 17:82-6
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PURPOSE OF REVIEW: To discuss mouse models of diabetic nephropathy and their use in discovering genetic risk factors predisposing to diabetic nephropathy. RECENT FINDINGS: Despite occurring in only 10-40% of diabetic patients, diabetic nephropathy is the largest single cause of end stage renal disease in the USA. Accumulated evidence points to critical genetic factors that predispose a subset of diabetic patients to nephropathy.Defining the genes that confer risk for nephropathy in human populations has proven challenging. The use of robust genetic reagents available in the laboratory mouse provides a complementary approach to defining genes that predispose to diabetic nephropathy in mice and humans. These findings support the existence of dominant mutations predisposing to diabetic nephropathy in mice as well as substantiating an important role for eNOS in forestalling the development of diabetic nephropathy. SUMMARY: When studied for a sufficient duration of diabetic hyperglycemia, some strains of mice exhibit changes similar to those of human diabetic nephropathy. The unique genetic reagents in mice should help accelerate the identification of genes predisposing to diabetic nephropathy.

Harris RC. An update on cyclooxygenase-2 expression and metabolites in the kidney. Curr Opin Nephrol Hypertens (2008) 17:64-9
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PURPOSE OF REVIEW: This review highlights recent studies examining the expression and function of cyclooxygenase-2 and its metabolites in the kidney. RECENT FINDINGS: Expression of cyclooxygenase-2 is regulated by both physiologic and pathophysiologic perturbations, with volume depletion upregulating macula-densa expression and volume expansion upregulating medullary expression. Macula densa cyclooxygenase-2 is a modulator of juxtaglomerular renin expression, and there is increasing evidence that cyclooxygenase-2 expression is modulated by multiple components of the renin-angiotensin system, including angiotensin II, through both AT1 and AT2 receptors. There are also indications that macula densa cyclooxygenase-2 expression may be regulated by the prorenin/renin receptor. Medullary cyclooxygenase-2 metabolites modulate salt and water excretion, and cyclooxygenase-2 inhibitors lead to sodium and volume retention and may raise blood pressure. There is also increasing evidence that cyclooxygenase-2 expression increases in progressive renal injury. Given their cardiovascular and renal side effects, cyclooxygenase-2 inhibitors are not a feasible intervention for long-term therapy against progressive renal damage, but further delineation of the downstream receptors and synthases involved may provide therapeutic targets. SUMMARY: Recent studies have highlighted the important role that cyclooxygenase-2 metabolites play both in regulation of normal renal function and as potential mediators of acute and chronic renal injury.

Bing Y, Xu J, Harris RC, Zhang MZ. null Am J Physiol Renal Physiol (2007) null:null
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Tissue prostaglandin levels are determined by both biosynthesis and catabolism. The current studies report the expression and localization of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a key enzyme in prostaglandin catabolism in the kidneys. We also investigated potential interactions between 15-PGDH and cyclooxygenase (COX), a key enzyme in prostaglandin biosynthesis. Both 15-PGDH mRNA and protein levels were significantly higher in kidney cortex than papilla, which is opposite to the expression pattern of COX-2. In situ hybridization indicated that 15-PGDH mRNA was mainly localized to the tubular epithelial cells in kidney cortex and outer medulla, but not in the glomerulus or papilla. Dual immunofluorescent staining indicated that 15-PGDH was expressed in the proximal tubule, medullary and cortical thick ascending limbs and cortical and outer medullary collecting duct but not in the macula densa or papilla. 15-PGDH levels were significantly lower in a macula densa cell line (MMDD1) than a proximal tubule cell line. Although a high salt-diet decreased COX-2 expression in macula densa, it increased macula densa 15-PGDH expression in both mouse and rat kidneys. In MMDD1 cells, a COX-2 inhibitor increased 15-PGDH while a COX-1 inhibitor had no effect. Furthermore, intense 15-PGDH immunofluorescent staining was found in both macula densa and glomerulus in COX-2 knockout mice. The intrarenal distribution of 15-PGDH and its interactions with COX-2 suggest that differential regulation of COX-2 and 15-PGDH may play an important role in determining levels of prostaglandins involved in regulation of salt, volume, and blood pressure homeostasis. Key words: prostaglandin, cyclooxygenase, macula densa.

Jo YI, Cheng H, Wang S, Moeckel GW, Harris RC. Puromycin induces reversible proteinuric injury in transgenic mice expressing cyclooxygenase-2 in podocytes. Nephron Exp Nephrol (2007) 107:e87-94
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Previous studies from our own group and others have demonstrated that cyclooxygenase-2 (COX-2) inhibitors could reduce proteinuria in some experimental models of progressive renal disease. To investigate a possible role of COX-2 in podocytes during the course of self-limited glomerular injury, we administered puromycin nucleoside (PAN) on day 1 (15 mg/100 g BW) and day 3 (30 mg/100 g BW) to wild-type and transgenic mice with podocyte-specific COX-2 expression driven by a nephrin promoter. An additional group received both PAN and the COX-2-specific inhibitor, SC58236 (6 mg/l in drinking water). There was no significant difference in the albumin (microg)/creatinine (mg) ratio between wild-type (26.3 +/- 4.2, n = 8) and transgenic (28.9 +/2.3, n = 8) mice under baseline conditions. PAN induced significant albuminuria only in the transgenic mice with a peak at day 3: 72.1 +/8.9 microg/mg creatinine (n = 12, p < 0.05, compared with basal level), which remitted by day 10 (37.4 +/4.4 microg/mg, n = 7, p < 0.05, compared with day 3). Electron microscopy demonstrated that PAN caused 56.7 +/4.2% foot process effacement in transgenic mice compared with 38.8 +/4.1% in wild type at day 3. PAN increased immunoreactive COX-2 in glomeruli from transgenic mice (day 3: 1.47 +/0.08 fold; day 10: 1.25 +/0.16 fold, n = 5-9, p < 0.05 compared with basal level), which was restricted to podocytes. Real time PCR indicated that endogenous COX-2 mRNA increased (2.6 +/0.1 fold of wild-type control at day 3 and 2.2 +/0.2 at day 10, n = 4, p < 0.05), while the nephrin-driven COX-2 mRNA was unchanged. Nephrin mRNA and protein expression were decreased by PAN in the transgenic mice. The COX-2-specific inhibitor, SC58236, reduced foot process effacement in transgenic mice administered PAN to 21.7 +/5.2% and significantly reduced the albuminuria at day 3 (42.2 +/3.8, n = 13, p < 0.05 compared with untreated) without significantly altering COX-2 expression. In summary, in transgenic mice with podocyte COX-2 overexpression, PAN increased albuminuria and induced foot process fusion. Thus, increased COX-2 expression increased podocyte susceptibility to further injury. CI Copyright 2007 S. Karger AG, Basel.

Singh AB, Sugimoto K, Dhawan P, Harris RC. Juxtacrine activation of EGFR regulates claudin expression and increases transepithelial resistance. Am J Physiol Cell Physiol (2007) 293:C1660-8
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Heparin-binding (HB)-EGF, a ligand for EGF receptors, is synthesized as a membrane-anchored precursor that is potentially capable of juxtacrine activation of EGF receptors. However, the physiological importance of such juxtacrine signaling remains poorly described, due to frequent inability to distinguish effects mediated by membrane-anchored HB-EGF vs. mature "secreted HB-EGF." In our studies, using stable expression of a noncleavable, membrane-anchored rat HB-EGF isoform (MDCK(rat5aa) cells) in Madin-Darby canine kidney (MDCK) II cells, we observed a significant increase in transepithelial resistance (TER). Similar significant increases in TER were observed on stable expression of an analogous, noncleavable, membrane-anchored human HB-EGF construct (MDCK(human5aa) cells). The presence of noncleavable, membrane-anchored HB-EGF led to alterations in the expression of selected claudin family members, including a marked decrease in claudin-2 in MDCK(rat5aa) cells compared with the control MDCK cells. Reexpression of claudin-2 in MDCK(rat5aa) cells largely prevented the increases in TER. Ion substitution studies indicated decreased paracellular ionic permeability of Na(+) in MDCK(rat5aa) cells, further indicating that the altered claudin-2 expression mediated the increased TER seen in these cells. In a Ca(2+)-switch model, increased phosphorylation of EGF receptor and Akt was observed in MDCK(rat5aa) cells compared with the control MDCK cells, and inhibition of these pathways inhibited TER changes specifically in MDCK(rat5aa) cells. Therefore, we hypothesize that juxtacrine activation of EGFR by membrane-anchored HB-EGF may play an important role in the regulation of tight junction proteins and TER.

Singh AB, Sugimoto K, Harris RC. Juxtacrine activation of epidermal growth factor (EGF) receptor by membrane-anchored heparin-binding EGF-like growth factor protects epithelial cells from anoikis while maintaining an epithelial phenotype. J Biol Chem (2007) 282:32890-901
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Loss of cell-matrix adhesion is often associated with acute epithelial injury, suggesting that "anoikis" may be an important contributor to cell death. Resistance against anoikis is a key characteristic of transformed cells. When nontransformed epithelia are injured, activation of the epidermal growth factor (EGF) receptor (EGFR) by paracrine/autocrine release of soluble ligands can induce a prosurvival program, but there is generally evidence for concomitant dedifferentiation. The EGFR ligand, heparin-binding EGF-like growth factor (HB-EGF), is synthesized as a membrane-anchored precursor that can activate the EGFR via juxtacrine signaling or can be released and act as a soluble growth factor. In Madin-Darby canine kidney cells, expression of membrane-anchored HB-EGF increases cell-cell and cell-matrix adhesion. Therefore, these studies were designed to test the effects of juxtacrine HB-EGF signaling upon cell survival and epithelial integrity when cells are denied proper cell-matrix interactions. Cells expressing a noncleavable mutated form of membrane-anchored HB-EGF demonstrated increased survival from anoikis, formed larger cell aggregates, and maintained epithelial characteristics even following prolonged detachment from the substratum. Physical association between membrane-anchored HB-EGF and EGFR was observed. Signaling studies indicated synergistic effects of EGFR activation and phosphatidylinositol 3-kinase signaling to regulate apoptotic and survival pathways. In contrast, although administration of exogenous EGF partially suppressed anoikis in wild type cells, it also led to an increased expression of mesenchymal markers, suggesting dedifferentiation. Taken together, we propose a novel role for membrane-anchored HB-EGF in the cytoprotection of epithelial cells.

Zeng F, Zhang MZ, Singh AB, Zent R, Harris RC. ErbB4 isoforms selectively regulate growth factor induced Madin-Darby canine kidney cell tubulogenesis. Mol Biol Cell (2007) 18:4446-56
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ErbB4, a member of the epidermal growth factor (EGF) receptor family that can be activated by heregulin beta1 and heparin binding (HB)-EGF, is expressed as alternatively spliced isoforms characterized by variant extracellular juxtamembrane (JM) and intracellular cytoplasmic (CYT) domains. ErbB4 plays a critical role in cardiac and neural development. We demonstrated that ErbB4 is expressed in the ureteric buds and developing tubules of embryonic rat kidney and in collecting ducts in adult. The predominant isoforms expressed in kidney are JM-a and CYT-2. In ErbB4-transfected MDCK II cells, basal cell proliferation and hepatocyte growth factor (HGF)-induced tubule formation were decreased by all four isoforms. Only JM-a/CYT-2 cells formed tubules upon HB-EGF stimulation. ErbB4 was activated by both HRG-beta1 and HB-EGF stimulation; however, compared with HRG-beta1, HB-EGF induced phosphorylation of the 80-kDa cytoplasmic cleavage fragment of the JM-a/CYT-2 isoform. HB-EGF also induced early activation of ERK1/2 in JM-a/CYT-2 cells and promoted nuclear translocation of the JM-a/CYT-2 cytoplasmic tail. In summary, our data indicate that JM-a/CYT-2, the ErbB4 isoform that is proteinase cleavable but does not contain a PI3K-binding domain in its cytoplasmic tail, mediates important functions in renal epithelial cells in response to HB-EGF.

Xu J, Yao B, Fan X, Langworthy MM, Zhang MZ, Harris RC. Characterization of a putative intrarenal serotonergic system. Am J Physiol Renal Physiol (2007) 293:F1468-75
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Serotonin [5-hydroxytryptamine (5HT)] acts through multiple G protein-coupled 5-HT receptors, and its activity is also regulated by the 5-HT transporter. The current studies report the expression and localization of the 5-HT receptors and transporter in the kidney. In addition, the enzymatic pathway mediating 5-HT synthesis is present in renal cortex, especially in the proximal tubules and glomerular epithelial cells and mesangial cells. Expression of the 5-HT receptors and 5-HT transporter was detected by RT-PCR in cell lines of these cell types. In cultured proximal tubule cells and podocytes, 5-HT activated ERK1/2 and increased the expression of connective tissue growth factor and transforming growth factor-beta, two key mediators of extracellular matrix accumulation. Immunohistochemistry and real-time RT-PCR studies also indicated that 5-HT stimulated expression of vascular endothelial growth factor in podocytes in vitro and in vivo. Therefore, these results indicate the presence of an integrated intrarenal serotonergic system and suggest a possible role for 5-HT as a mediator of renal fibrosis in the kidney.

Harris RC, Breyer MD. Update on cyclooxygenase-2 inhibitors. Clin J Am Soc Nephrol (2006) 1:236-45
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Nonsteroidal anti-inflammatory drugs represent the most commonly used medications for the treatment of pain and inflammation, but numerous well-described side effects can limit their use. Cyclooxygenase-2 (COX-2) inhibitors were initially touted as a therapeutic strategy to avoid not only the gastrointestinal but also the renal and cardiovascular side effects of nonspecific nonsteroidal anti-inflammatory drugs. However, in the kidney, COX-2 is constitutively expressed and is highly regulated in response to alterations in intravascular volume. COX-2 metabolites have been implicated in mediation of renin release, regulation of sodium excretion, and maintenance of renal blood flow. This review summarizes the current state of knowledge about both renal and cardiovascular side effects that are attributed to COX-2 selective inhibitors.

Kanetsuna Y, Takahashi K, Nagata M, Gannon MA, Breyer MD, Harris RC, Takahashi T. Deficiency of endothelial nitric-oxide synthase confers susceptibility to diabetic nephropathy in nephropathy-resistant inbred mice. Am J Pathol (2007) 170:1473-84
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Recent studies have implicated dysfunctional endothelial nitric-oxide synthase (eNOS) as a common pathogenic pathway in diabetic vascular complications. However, functional consequences are still incompletely understood. To determine the role of eNOS-derived nitric oxide (NO) in diabetic nephropathy, we induced diabetes in eNOS knockout (KO) and wild-type (WT) mice on the C57BL6 background, using low-dose streptozotocin injection, and we investigated their glomerular phenotype at up to 20 weeks of diabetes. Although the severity of hyperglycemia in diabetic eNOS KO mice was similar to diabetic WT mice, diabetic eNOS KO mice developed overt albuminuria, hypertension, and glomerular mesangiolysis, whereas diabetic WT and nondiabetic control mice did not. Glomerular hyperfiltration was also significantly reduced in diabetic eNOS KO mice. Electron micrographs from diabetic eNOS KO mice revealed an injured endothelial morphology, thickened glomerular basement membrane, and focal foot process effacement. Furthermore, the anionic sites at glomerular endothelial barrier estimated by cationic ferritin binding were reduced in diabetic eNOS KO mice. In aggregate, these results demonstrate that deficiency of eNOS-derived NO causes glomerular endothelial injury in the setting of diabetes and results in overt albuminuria and glomerular mesangiolysis in nephropathy-resistant inbred mice. The findings indicate a vital role for eNOS-derived NO in the pathogenesis of diabetic nephropathy.

Breyer MD, Tchekneva E, Qi Z, Takahashi T, Fogo AB, Zhao HJ, Harris RC. Genetics of diabetic nephropathy: lessons from mice. Semin Nephrol (2007) 27:237-47
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Although diabetic nephropathy occurs only in a minority of diabetic patients (approximately 30%), it is the major single cause of end-stage renal disease in the United States. Hyperglycemia and hypertension are important factors predisposing patients to nephropathy, however, accumulating evidence points to critical genetic factors that predispose only a subset of diabetic patients to nephropathy. Defining the genes responsible for nephropathy risk in human populations has proven challenging. Comparative genomics using the robust genetic reagents available in the laboratory mouse should provide a complementary approach to defining genes that may predispose to diabetic nephropathy in mice and human beings. In this article we review studies that have started to identify genetic risk factors for diabetic nephropathy in mice and the multiple approaches that may be used to elucidate the genetic pathogenesis of this disorder.

Chen X, Abair TD, Ibanez MR, Su Y, Frey MR, Dise RS, Polk DB, Singh AB, Harris RC, Zent R, Pozzi A. Integrin alpha1beta1 controls reactive oxygen species synthesis by negatively regulating epidermal growth factor receptor-mediated Rac activation. Mol Cell Biol (2007) 27:3313-26
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Integrins control many cell functions, including generation of reactive oxygen species (ROS) and regulation of collagen synthesis. Mesangial cells, found in the glomerulus of the kidney, are able to produce large amounts of ROS via the NADPH oxidase. We previously demonstrated that integrin alpha1-null mice develop worse fibrosis than wild-type mice following glomerular injury and this is due, in part, to excessive ROS production by alpha1-null mesangial cells. In the present studies, we describe the mechanism whereby integrin alpha1-null mesangial cells produce excessive ROS. Integrin alpha1-null mesangial cells have constitutively increased basal levels of activated Rac1, which result in its increased translocation to the cell membrane, excessive ROS production, and consequent collagen IV deposition. Basal Rac1 activation is a direct consequence of ligand-independent increased epidermal growth factor receptor (EGFR) phosphorylation in alpha1-null mesangial cells. Thus, our study demonstrates that integrin alpha1beta1-EGFR cross talk is a key step in negatively regulating Rac1 activation, ROS production, and excessive collagen synthesis, which is a hallmark of diseases characterized by irreversible fibrosis.

Chen J, Chen JK, Falck JR, Guthi JS, Anjaiah S, Capdevila JH, Harris RC. Mitogenic activity and signaling mechanism of 2-(14,15- epoxyeicosatrienoyl)glycerol, a novel cytochrome p450 arachidonate metabolite. Mol Cell Biol (2007) 27:3023-34
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Arachidonic acid is an essential constituent of cell membranes that is esterified to the sn-2 position of glycerophospholipids and is released from selected phospholipid pools by tightly regulated phospholipase cleavage. Metabolism of the released arachidonic acid by the cytochrome P450 enzyme system (cP450) generates biologically active compounds, including epoxyeicosatrienoic acids (EETs) and hydroxyeicosatetraenoic acids. Here we report that 2-(14,15-epoxyeicosatrienoyl)glycerol (2-14,15-EG), a novel cP450 arachidonate metabolite produced in the kidney, is a potent mitogen for renal proximal tubule cells. This effect is mediated by activation of tumor necrosis factor alpha-converting enzyme (ADAM17), which cleaves membrane-bound transforming growth factor alpha (proTGF-alpha) and releases soluble TGF-alpha as a ligand that binds and activates epidermal growth factor receptor (EGFR). The present studies additionally demonstrate that the structurally related 14,15-EET stimulates release of soluble heparin-binding EGF-like growth factor as an EGFR ligand by activation of ADAM9, another member of the ADAM family. Thus, in addition to the characterization of 2-14,15-EG's mitogenic activity and signaling mechanism, our study provides the first example that two structurally related biologically active lipid mediators can activate different metalloproteinases and release different EGFR ligands in the same cell type to activate EGFR and stimulate cell proliferation.

Cheng H, Wang S, Jo YI, Hao CM, Zhang M, Fan X, Kennedy C, Breyer MD, Moeckel GW, Harris RC. null J Am Soc Nephrol (2007) null:null
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Increased podocyte cyclooxygenase-2 (COX-2) expression is seen in rats after renal ablation and Thy-1 nephritis and in cultured murine podocytes in response to mechanical stress. For investigation of whether COX-2 overexpression plays a role in podocyte injury, transgenic B6/D2 mice in which COX-2 expression was driven by a nephrin promoter were established. Selective upregulation of COX-2 expression in podocytes of transgenic mouse kidneys was confirmed by immunoblotting and immunohistochemistry. Whether upregulation of podocyte-specific COX-2 expression enhanced sensitivity to the development of Adriamycin nephropathy was examined. Adriamycin administration induced dramatically more albuminuria and foot process effacement and reduced glomerular nephrin mRNA and immunoreactivity in transgenic mice compared with wild-type littermates. Adriamycin also markedly increased immunoreactive COX-2 expression in podocytes from transgenic mice compared with the wild-type mice. Reverse transcriptase-PCR indicated that this increase represented a stimulation of endogenous COX-2 mRNA expression rather than COX-2 mRNA driven by the nephrin promoter. Balb/C mice, which are susceptible to renal injury by Adriamycin, also increased podocyte COX-2 expression and reduced nephrin expression in response to administration of the drug. Long-term treatment with the COX-2-specific inhibitor SC58236 ameliorated the albuminuria that was induced by Adriamycin in the transgenic mice. SC58236 also reduced Adriamycin-induced foot process effacement in both the COX-2 transgenic mice and Balb/C mice. Therefore, overexpression of COX-2 may predispose podocytes to further injury.

Zhang MZ, Yao B, Cheng HF, Wang SW, Inagami T, Harris RC. Renal cortical cyclooxygenase 2 expression is differentially regulated by angiotensin II AT(1) and AT(2) receptors. Proc Natl Acad Sci U S A (2006) 103:16045-50
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Macula densa cyclooxygenase 2 (COX-2)-derived prostaglandins serve as important modulators of the renin-angiotensin system, and cross-talk exists between these two systems. Cortical COX-2 induction by angiotensin-converting enzyme (ACE) inhibitors or AT(1) receptor blockers (ARBs) suggests that angiotensin II may inhibit cortical COX-2 by stimulating the AT(1) receptor pathway. In the present studies we determined that chronic infusion of either hypertensive or nonhypertensive concentrations of angiotensin II attenuated cortical COX-2. Angiotensin II infusion reversed cortical COX-2 elevation induced by ACE inhibitors. However, we found that angiotensin II infusion further stimulated cortical COX-2 elevation induced by ARBs, suggesting a potential role for an AT(2) receptor-mediated pathway when the AT(1) receptor was inhibited. Both WT and AT(2) receptor knockout mice were treated for 7 days with either ACE inhibitors or ARBs. Cortical COX-2 increased to similar levels in response to ACE inhibition in both knockout and WT mice. In WT mice ARBs increased cortical COX-2 more than ACE inhibitors, and this stimulation was attenuated by the AT(2) receptor antagonist PD123319. In the knockout mice ARBs led to significantly less cortical COX-2 elevation, which was not attenuated by PD123319. PCR confirmed AT(1a) and AT(2) receptor expression in the cultured macula densa cell line MMDD1. Angiotensin II inhibited MMDD1 COX-2, and CGP42112A, an AT(2) receptor agonist, stimulated MMDD1 COX-2. In summary, these results demonstrate that macula densa COX-2 expression is oppositely regulated by AT(1) and AT(2) receptors and suggest that AT(2) receptor-mediated cortical COX-2 elevation may mediate physiologic effects that modulate AT(1)-mediated responses.

Zhao HJ, Wang S, Cheng H, Zhang MZ, Takahashi T, Fogo AB, Breyer MD, Harris RC. Endothelial nitric oxide synthase deficiency produces accelerated nephropathy in diabetic mice. J Am Soc Nephrol (2006) 17:2664-9
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Functionally significant polymorphisms in endothelial nitric oxide synthase (eNOS) and reduced vascular eNOS activity have been associated with increased human diabetic nephropathy (DN), but the pathogenic role of eNOS deficiency in the development of DN has not yet been confirmed. This study characterizes the severity of DN in eNOS(-/-) mice that were backcrossed to C57BLKS/J db/db mice. Although the severity of hyperglycemia was similar to C57BLKS/J db/db mice, by 26 wk, eNOS(-/-) C57BLKS/J db/db mice exhibited dramatic albuminuria, arteriolar hyalinosis, increased glomerular basement membrane thickness, mesangial expansion, mesangiolysis, and focal segmental and early nodular glomerulosclerosis. Even more remarkable, eNOS(-/-) C57BLKS db/db exhibited decreases in GFR to levels <50% of that in eNOS(+/+) C57BLKS db/db, as confirmed by increased serum creatinine. In summary, eNOS(-/-) db/db mice provide the most robust model of type II DN that has been described to date and support a role for deficient eNOS-derived NO production in the pathogenesis of DN.

Cheng H, Harris RC. Potential side effects of renin inhibitors--mechanisms based on comparison with other renin-angiotensin blockers. Expert Opin Drug Saf (2006) 5:631-41
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Angiotensin (Ang) II plays important roles in the development of hypertension and cardiovascular and renal injury. Pharmaceutical approaches to block its activity led to the development of angiotensin-converting enzyme inhibitors and angiotensin receptor blockers. Numerous trials have documented their efficacy in controlling blood pressure, minimising left ventricular remodelling, preventing progression to heart failure, ameliorating proteinuria and retarding renal disease progression. Although they are considered safe in general, there remain concerns about the potential for adverse events in certain target populations. Recently, several novel, low molecular weight renin inhibitors without the extended peptide-like backbone of previous renin inhibitors were developed with favourable pharmacokinetic properties. They have been shown to successfully reduce Ang II levels in normal volunteers and to lower blood pressure in patients with mild-to-moderate hypertension. In this review, the authors summarise current knowledge about these renin inhibitors.

Harris RC. COX-2 and the kidney. J Cardiovasc Pharmacol (2006) 47 Suppl 1:S37-42
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Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used for the treatment of pain and inflammation. Nonselective NSAIDs inhibit both cyclooxygenase (COX)-1 and COX-2. Nephrotoxicity of nonselective NSAIDs has been well documented. The effects of selective COX-2 inhibitors on renal function and blood pressure are attracting increasing attention. In the kidney, COX-2 is constitutively expressed and is highly regulated in response to alterations in intravascular volume. COX-2 metabolites have been implicated in the mediation of renin release, regulation of sodium excretion, and maintenance of renal blood flow. Similar to nonselective NSAIDs, inhibition of COX-2 may cause edema and modest elevations in blood pressure in a minority of subjects. COX-2 inhibitors may also exacerbate preexisting hypertension or interfere with other antihypertensive drugs. Occasional acute renal failure has also been reported. Caution should be taken when COX-2 inhibitors are prescribed, especially in high-risk patients (including elderly patients and patients with volume depletion).

Kanetsuna Y, Hirano K, Nagata M, Gannon MA, Takahashi K, Harris RC, Breyer MD, Takahashi T. Characterization of diabetic nephropathy in a transgenic model of hypoinsulinemic diabetes. Am J Physiol Renal Physiol (2006) 291:F1315-22
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Genetic mouse models provide a unique opportunity to investigate gene function in the natural course of the disease. Although diabetic nephropathy (DN) in models of type II diabetes has been well characterized, diabetic renal disease in hypoinsulinemic diabetic mice is still incompletely understood. Here, we characterized renal changes in the pdx1(PB)-HNF6 transgenic mouse that exhibits beta-cell dysfunction and nonobese hypoinsulinemic diabetes. Male transgenic mice developed hyperglycemia by the age of 7 wk and survived for over 1 yr without insulin treatment. Diabetes ensued earlier and progressed more severely in the HNF6 males than the females. The HNF6 males exhibited albuminuria as early as 10 wk of age, and the urinary albumin excretion increased with age, exceeding 150 microg/24 h at 11 mo of age. Diabetic males developed renal hypertrophy after 7 wk of age, whereas glomerular hyperfiltration was not observed in the mice. Hypertension and hyperlipidemia were not observed in the diabetic mice. Histological analysis of the HNF6 kidneys displayed diabetic glomerular changes, including glomerular enlargement, diffuse mesangial proliferation and matrix expansion, thickened glomerular basement membrane, and arteriolar hyalinosis. Mesangial matrix accumulation increased with age, resulting in nodular lesions by 44 wk of age. Immunohistochemistry showed accumulation of type IV collagen and TGF-beta1 in the mesangial area. No significant immune complex deposition was observed in the HNF6 glomeruli. Thus the HNF6 mouse exhibits diabetic renal changes that parallel the early phase of human DN. The model should facilitate studies of genetic and environmental factors that may affect DN in hypoinsulinemic diabetes.

Chen J, Chen JK, Neilson EG, Harris RC. Role of EGF receptor activation in angiotensin II-induced renal epithelial cell hypertrophy. J Am Soc Nephrol (2006) 17:1615-23
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For determination of the molecular mechanisms underlying the induction of epithelial cell hypertrophy by angiotensin II (Ang II), a well-characterized porcine renal proximal tubular cell line LLCPKcl4, which does not express endogenous Ang II receptor subtypes, was transfected with cDNA encoding Ang II subtype 1 receptor (AT1R/Cl4). Ang II transactivated the EGF receptor (EGFR) in these AT1R/Cl4 cells, which was blocked by the selective AT1R antagonist losartan but not by the selective AT2R antagonist PD123319. Ang II did not transactivate EGFR in empty vector-transfected LLCPKcl4 cells (Vector/Cl4). Ang II elicited release of soluble heparin-binding EGF-like growth factor (HB-EGF) from AT1R/Cl4 cells, and Ang II-induced EGFR activation was prevented by pretreatment with the specific HB-EGF inhibitor CRM197 or the metalloproteinase inhibitors batimastat or phenanthroline, none of which had any effect on EGFR activation by exogenously administered EGF. Ang II stimulated protein synthesis and cell hypertrophy in AT1R/Cl4 cells without increasing cell number, and signaling studies revealed that Ang II stimulated phosphorylation of the 40S ribosomal protein S6 and the eukaryotic translation initiation factor 4E-binding protein 1, the two downstream target proteins of the mammalian target of rapamycin, which is a central regulator of protein synthesis and cell size. Ang II-induced mammalian target of rapamycin activation, [3H]leucine incorporation, and cellular hypertrophy were inhibited by pretreatment with either batimastat or CRM197 or by pretreatment with rapamycin or the EGFR tyrosine kinase inhibitor AG1478. Ang II also stimulated Smad 2/3 phosphorylation, which was blocked by a selective TGF-beta receptor I kinase inhibitor but not by CRM197. With blockade of TGF-beta receptor, Ang II-mediate

Yao B, Xu J, Qi Z, Harris RC, Zhang MZ. Role of renal cortical cyclooxygenase-2 expression in hyperfiltration in rats with high-protein intake. Am J Physiol Renal Physiol (2006) 291:F368-74
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Renal cortical cyclooxygenase-2 (COX-2) is restricted to the macula densa and adjacent cortical thick ascending limbs (MD/cTALH). Renal cortical COX-2 increases in response to diabetes and renal ablation, both of which are characterized by hyperfiltration and reduced NaCl delivery to the MD due to increased proximal NaCl reabsorption. High-protein intake also induces hyperfiltration and decreases NaCl delivery to the MD due to increased NaCl reabsorption proximally. We investigated whether high protein induces cortical COX-2 and whether cortical COX-2 contributes to high protein-induced hyperfiltration and increased intrarenal renin biosynthesis. Cortical COX-2 increased after protein loading but decreased after protein restriction. COX-2 inhibition attenuated high protein-induced hyperfiltration but had no effect on high protein-induced intrarenal renin elevation. Therefore, induction of cortical COX-2 contributed to high protein-induced hyperfiltration but not intrarenal renin elevation. In the kidney cortex, neuronal nitric oxide synthase (nNOS) is also localized to the MD, and interactions between intrarenal nNOS and COX-2 systems have been proposed. Cortical COX-2 elevation seen in salt restriction was blocked by nNOS inhibiton. Cortical nNOS expression also increased after protein loading, and inhibition of nNOS activity completely reversed high protein-induced cortical COX-2 elevation and hyperfiltration. These results indicate that NO is a mediator of high protein-induced cortical COX-2 elevation and suggest that both intrarenal nNOS and COX-2 systems appear to regulate afferent arteriolar tone and subsequent hyperfiltration seen in high-protein intake.

Harris RC, Neilson EG. Toward a unified theory of renal progression. Annu Rev Med (2006) 57:365-80
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Various disciplines within nephrology investigate the mechanisms by which kidneys fail. Progress in the areas of glomerular hemodynamics, proteinuria, tubular biology, interstitial nephritis, fibroblast formation, and fibrosis have added kernels of information that together support a unified theory of renal progression. Prevention of progression to end-stage disease has largely focused on control of systemic and glomerular hypertension. Current success in delaying a decline in glomerular filtration rate underlines the promise of a more comprehensive approach. New knowledge about the cell biology of progression also suggests that other adjunctive therapies may be possible. We describe the progress and highlight those spheres where new-targeted interventions may arise.

Cheng HF, Zhang MZ, Harris RC. Nitric oxide stimulates cyclooxygenase-2 in cultured cTAL cells through a p38-dependent pathway. Am J Physiol Renal Physiol (2006) 290:F1391-7
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To examine the interaction of nitric oxide (NO) and cyclooxygenase (COX-2) and the signaling pathway involved, primary cultured rabbit cortical thick ascending limb (cTAL) were used. In these cells, immunoreactive COX-2 and vasodilatory prostaglandins were increased by a NO donor, S-nitros-N-acetylpenicillamine (SNAP; 2.5 +/0.3-fold control, n = 6, P < 0.01). SNAP increased expression of phosphorylated p38 (pp38; 2.4 +/- 0.3-fold control; n = 5; P < 0.01), which was inhibited by the p38 inhibitor SB-203580 (1.3 +/0.1-fold control, n = 5, P < 0.01). SB-203580 inhibited SNAP-induced COX-2 expression [1.4 +/0.2-fold control, n = 6, not significant (NS) vs. control] and levels of PGE2 significantly. In cTAL cells transfected with a luciferase reporter driven by the wild-type mouse COX-2 promoter, SNAP stimulated luciferase activity, which was reversed by SB-203580 (control vs. SNAP vs. SNAP + SB-203580: 1.4 +/- 0.2-, 8.3 +/1.4-, and 0.4 +/0.1-fold control, respectively, n = 4, P < 0.01). Electrophoretic mobility shift assay indicated that SNAP stimulated nuclear factor (NF)-kappaB binding activity in cTAL that was also inhibited by the p38 inhibitor. SNAP was not able to stimulate a mutant COX-2 promoter construct that is not activated by NF-kappaB (0.9 +/0.1, 1.2 +/0.1, and 1.0 +/0.2 respectively, n = 4, NS). Low chloride increased COX-2 expression (2.7 +/0.4-fold control, n = 6, P < 0.01) and pp38 expression (2.8 +/0.3-fold; n = 5, P < 0.01), which were reversed by the specific NO synthase (NOS) inhibitor 7-nitroindazole. Administration of a low-salt diet increased immunoreactive COX-2 and neuronal NOS (nNOS) in the macula densa and surrounding cTAL of kidneys of wild-type mice but did not significantly elevate COX-2 expression in nNOS-/mice. In summary, these studies indicate that, in cTAL, NO c

Cheng H, Zhang M, Moeckel GW, Zhao Y, Wang S, Qi Z, Breyer MD, Harris RC. Expression of mediators of renal injury in the remnant kidney of ROP mice is attenuated by cyclooxygenase-2 inhibition. Nephron Exp Nephrol (2005) 101:e75-85
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To investigate the effects of cyclooxygenase-2 (COX-2) inhibition on renal injury of mice, ROP mice were subjected to subtotal ablation ('remnant'). A subset of the remnant group was treated with a selective COX-2 inhibitor, SC58236, in the drinking water. At 12 weeks the remnant group developed significant albuminuria (181.3 +/15.8 microg/24 h), which was blunted by SC58236 treatment (138.9 +/17.1; p < 0.05 compared to remnant). SC58236 did not alter systemic blood pressure or GFR significantly. Immunoreactive COX-2 was upregulated in remnant (1.88 +/- 0.35 fold sham, n = 8, p < 0.05), which was blunted by SC58236 (to 1.26 +/0.31 fold sham). Collagen IV mRNA increased significantly in remnant kidneys (2.69 +/0.34 fold sham, n = 8, p < 0.05), and this increase was inhibited by SC58236 treatment (to 1.84 +/0.32 fold control). Immunoreactive TGF-beta1, connective tissue growth factor, HGF receptor, c-Met, and fibronectin all increased in remnant (2.85 +/0.51, 3.83 +/- 0.55, 2.56 +/0.31, and 2.80 +/0.39 fold sham respectively, n = 4-8, p < 0.05), and SC58236 blunted the increases (to 1.45 +/0.34, 1.85 +/- 0.13, 1.75 +/0.30, and 1.60 +/0.32 fold sham). Immunohistochemistry indicated that the major localization for these progression factors was in the tubulointerstitium, especially in the scar area, which is in agreement with the expression of a macrophage marker, F4/80. Therefore, these results indicate that in a mouse model of subtotal renal ablation, COX-2 inhibition blocks expression of mediators of renal tubulointerstitial injury. CI Copyright 2005 S. Karger AG, Basel.

Singh AB, Harris RC. Autocrine, paracrine and juxtacrine signaling by EGFR ligands. Cell Signal (2005) 17:1183-93
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Receptor and cytoplasmic protein tyrosine kinases play prominent roles in the control of a range of cellular processes during embryonic development and in the regulation of many metabolic and physiological processes in a variety of tissues and organs. The epidermal growth factor receptor (EGFR) is a well-known and versatile signal transducer that has been highly conserved during evolution. It functions in a wide range of cellular processes, including cell fate determination, proliferation, cell migration and apoptosis. The number of ligands that can activate the EGF receptor has increased during evolution. These ligands are synthesized as membrane-anchored precursor forms that are later shed by metalloproteinase-dependent cleavage to generate soluble ligands. In certain circumstances the membrane anchored isoforms as well as soluble growth factors may also act as biologically active ligands; therefore depending on the circumstances these ligands may induce juxtacrine, autocrine, paracrine and/or endocrine signaling. In this review, we discuss the different ways that EGFR ligands can activate the receptor and the possible biological implications.

Cheng HF, Harris RC. Renal effects of non-steroidal anti-inflammatory drugs and selective cyclooxygenase-2 inhibitors. Curr Pharm Des (2005) 11:1795-804
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Nonsteroidal antiinflammatory drugs (NSAID) are one of the most commonly used medications worldwide to inhibiting COX activity for the treatment of pain and inflammation. Their nephrotoxicity has been well documented. With the development and clinical implementation of new COX-2 inhibitors, the safety, including the effects on renal function and blood pressure, is attracting increasing attention. In the kidney, COX-2 is constitutively expressed and is highly regulated in response to alterations in intravascular volume. COX-2 metabolites have been implicated in mediation of renin release, regulation of sodium excretion and maintenance of renal blood flow. Similar to conventional NSAIDs, inhibition of COX-2 may cause edema and modest elevations in blood pressure in a minority of subjects. COX-2 inhibitors may also exacerbate preexisting hypertension or interfere with other antihypertensive drugs. Occasional acute renal failure has also been reported. Caution should be taken when COX-2 inhibitors are prescribed, especially in high-risk patients (including elderly and patients with volume depletion). Recently, agents with combined lipooxygenase/COX inhibition and agents that combine NSAIDs with a nitric oxide (NO) donor have been reported to reduce adverse renal effects.

Breyer MD, Bottinger E, Brosius FC, Coffman TM, Fogo A, Harris RC, Heilig CW, Sharma K. Diabetic nephropathy: of mice and men. Adv Chronic Kidney Dis (2005) 12:128-45
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Accumulating evidence supports intrinsic genetic susceptibility as an important variable in the progression of diabetic nephropathy in people. Mice provide an experimental platform of unparalleled power for dissecting the genetics of mammalian diseases; however, phenotypic analysis of diabetic mice lags behind that already established for humans. Standardized benchmarks of hyperglycemia, albuminuria, and measurements of renal failure remain to be developed for different inbred strains of mice. The most glaring deficiency has been the lack of a diabetic mouse model that develops progressively worsening renal insufficiency, the sine qua non of diabetic nephropathy in humans. Differences in susceptibility of these inbred strains to complications of diabetes mellitus provide a possible avenue to dissect the genetic basis of diabetic nephropathy; however, the identification of those strains and/or mutants most susceptible to renal injury from diabetes mellitus is lacking. Identification of a mouse model that faithfully mirrors the pathogenesis of DN in humans will undoubtedly facilitate the development of new diagnostic and therapeutic interventions.

Chen JK, Chen J, Neilson EG, Harris RC. Role of mammalian target of rapamycin signaling in compensatory renal hypertrophy. J Am Soc Nephrol (2005) 16:1384-91
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Loss of functioning nephrons stimulates the growth of residual kidney tissue to augment work capacity and maintain normal renal function. This growth largely occurs by hypertrophy rather than from hyperplasia of the remaining nephrons. The signaling mechanisms that increase RNA and protein synthesis during compensatory renal hypertrophy are unknown. This study found that the remaining kidney hypertrophied 42% by 16 d after unilateral nephrectomy (UNX) in DBA/2 mice. Immunoblotting analysis revealed increased phosphorylation of the 40S ribosomal protein S6 (rpS6) and the eukaryotic translation initiation factor (eIF) 4E-binding protein 1 (4E-BP1), the two downstream effectors of the mammalian target of rapamycin (mTOR). The highly specific mTOR inhibitor rapamycin blocked UNX-increased phosphorylation of both rpS6 and 4E-BP1. UNX increased the content of not only 40S and 60S ribosomal subunits but also 80S monosomes and polysomes in the remaining kidney. Administration of rapamycin decreased UNX-induced polysome formation and shifted the polysome profile in the direction of monosomes and ribosomal subunits. Pretreatment of the mice with rapamycin inhibited UNX-induced hypertrophy. These studies demonstrate that activation of the mTOR signaling pathway in the remaining kidney after UNX plays an essential role in modulating RNA and protein synthesis during development of compensatory renal hypertrophy.

Yao B, Harris RC, Zhang MZ. Interactions between 11beta-hydroxysteroid dehydrogenase and COX-2 in kidney. Am J Physiol Regul Integr Comp Physiol (2005) 288:R1767-73
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The syndrome of apparent mineralocorticoid excess (SAME) is an autosomal recessive form of salt-sensitive hypertension caused by deficiency of the kidney type 2 11beta-hydroxysteroid dehydrogenase (11betaHSD2). In this disorder, cortisol is not inactivated by 11betaHSD2, occupies mineralocorticoid receptors (MRs), and causes excessive sodium retention and hypertension. In renal medulla, prostaglandins derived from cyclooxygenase-2 (COX-2) stimulate sodium and water excretion, and renal medullary COX-2 expression increases after mineralocorticoid administration. We investigated whether medullary COX-2 also increases in rats with 11betaHSD2 inhibition and examined its possible role in the development of hypertension. 11betaHSD2 inhibition increased medullary and decreased cortical COX-2 expression in adult rats and induced high blood pressure in high-salt-treated rats. COX-2 inhibition had no effect on blood pressure in control animals but further increased blood pressure in high-salt-treated rats with 11betaHSD2 inhibition. COX-1 inhibition had no effect on blood pressure in either control or experimental animals. 11betaHSD2 inhibition also led to medullary COX-2 increase and cortical COX-2 decrease in weaning rats, primarily through activation of MRs. In the suckling rats, medullary COX-2 expression was very low, consistent with a urinary concentrating defect. 11betaHSD2 inhibition had no effect on either cortical or medullary COX-2 expression in the suckling rats, consistent with low levels of circulating corticosterone in these animals. These data indicate that COX-2 plays a modulating role in the development of hypertension due to 11betaHSD2 deficiency and that 11betaHSD2 regulates renal COX-2 expression by preventing glucocorticoid access to MRs during postnatal development.

Chang IJ, Harris RC. Are all COX-2 inhibitors created equal? Hypertension (2005) 45:178-80
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Zhang MZ, Yao B, McKanna JA, Harris RC. Cross talk between the intrarenal dopaminergic and cyclooxygenase-2 systems. Am J Physiol Renal Physiol (2005) 288:F840-5
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In mammalian kidney, dopamine produced in the proximal tubule (PT) acts as an autocrine/paracrine natriuretic hormone that inhibits salt and fluid reabsorption in the PT. In high-salt-treated animals, PT dopamine activity increases and inhibits reabsorption, leading to increased salt and fluid delivery to the macula densa (MD) and subsequent natriuresis and diuresis. Regulated cyclooxygenase-2 (COX-2) in the MD represents another intrinsic system mediating renal salt and water homeostasis. Renal cortical COX-2 is inversely related to salt intake, and decreased extracellular NaCl stimulates COX-2 expression in cultured MD/cortical thick ascending limb cells. The current study investigated interactions between renal dopamine and cortical COX-2 systems. In rats fed a control diet, the dopamine precursor l-dihydroxyphenylalanine (l-DOPA) or the DA1 receptor agonist SKF-81297 suppressed cortical COX-2 expression. High salt suppressed cortical COX-2 expression, which was attenuated by inhibition of dopamine production with benserazide or the DA1 receptor antagonist, SCH-23390. In contrast, l-DOPA or the dopamine-metabolizing enzyme inhibitor entacapone suppressed low-salt-induced cortical COX-2 expression. Inhibition of PT reabsorption with the carbonic anhydrase inhibitor acetazolamide suppressed cortical COX-2 expression. In contrast, treatment with distally acting diuretics led to elevation of cortical COX-2. These results indicate that dopamine modulates renal cortical COX-2 expression by modifying PT reabsorption.

Rao R, Zhang MZ, Zhao M, Cai H, Harris RC, Breyer MD, Hao CM. Lithium treatment inhibits renal GSK-3 activity and promotes cyclooxygenase 2-dependent polyuria. Am J Physiol Renal Physiol (2005) 288:F642-9
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The use of LiCl in clinical psychiatry is routinely complicated by overt nephrogenic diabetes insipidus (NDI), the mechanism of which is incompletely understood. In vitro studies indicate that lithium can induce renal medullary interstitial cell cyclooxygenase 2 (COX2) protein expression via inhibition of glycogen synthase kinase-3beta (GSK-3beta). Both COX1 and COX2 are expressed in the kidney. Renal prostaglandins have been suggested to play an important role in lithium-induced polyuria. The present studies examined whether induction of the COX2 isoform contributes to LiCl-induced polyuria. Four days after initiation of lithium treatment in C57 BL/6J mice, urine volume increased in LiCl-treated mice by fourfold compared with controls (P < 0.0001) and was accompanied by decreased urine osmolality. This was temporally associated with increased renal COX2 protein expression and increased urinary PGE(2) excretion, whereas COX1 levels remained unchanged. COX2 inhibition significantly blunted lithium-induced polyuria (P < 0.0001) and reduced urinary PGE(2) levels. Lithium-associated polyuria was also seen in COX1-/mice and was associated with increased urinary PGE(2). COX2 inhibition completely prevented polyuria and PGE(2) excretion in COX1-/mice, suggesting that COX2, but not COX1, plays a critical role in lithium-induced polyuria. Lithium also induced renal medullary COX2 protein expression in congenitally polyuric antidiuretic hormone (AHD)-deficient rats, demonstrating that lithium-induced COX2 protein expression is not secondary to altered ADH levels or polyuria. Lithium also decreased renal medullary GSK-3beta activity, and this was temporally related to increased COX2 expression in the kidney from lithium-treated mice, consistent with a tonic in vivo suppression of COX2 expression by GSK-3

Breyer MD, Bottinger E, Brosius FC 3rd, Coffman TM, Harris RC, Heilig CW, Sharma K. Mouse models of diabetic nephropathy. J Am Soc Nephrol (2005) 16:27-45
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Mice provide an experimental model of unparalleled flexibility for studying mammalian diseases. Inbred strains of mice exhibit substantial differences in their susceptibility to the renal complications of diabetes. Much remains to be established regarding the course of diabetic nephropathy (DN) in mice as well as defining those strains and/or mutants that are most susceptible to renal injury from diabetes. Through the use of the unique genetic reagents available in mice (including knockouts and transgenics), the validation of a mouse model reproducing human DN should significantly facilitate the understanding of the underlying genetic mechanisms that contribute to the development of DN. Establishment of an authentic mouse model of DN will undoubtedly facilitate testing of translational diagnostic and therapeutic interventions in mice before testing in humans.

Harris RC, Almada AL, Harris DB, Dunnett M, Hespel P. The creatine content of Creatine Serum and the change in the plasma concentration with ingestion of a single dose. J Sports Sci (2004) 22:851-7
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Three samples of Creatine Serum ATP Advantage from Muscle Marketing USA, Inc. were assayed for creatine by two different techniques by four independent laboratories, and for creatinine by two different techniques by two laboratories. A further sample was assayed for phosphorylcreatine. Dry weight and total nitrogen were also analysed. Six male volunteers ingested in random order, over 3 weeks: (A) water; (B) 2.5 g creatine monohydrate (Cr.H2O) in solution; and (C) 5 ml Creatine Serum (reportedly containing an equivalent amount of Cr.H2O). Blood samples were collected before and up to 8 h after each treatment and plasma was analysed for creatine and creatinine. Eight-hour urine samples were analysed for creatine. Ingestion of 2.5 g creatine monohydrate in solution resulted in a significant increase in plasma creatine (from 59.1+/-11.8 micromol.l(-1) to 245.3+/-74.6 microM micromol.l(-1); mean+/-s) and urinary creatine excretion. No increase in plasma or urinary creatine or creatinine was found on ingestion of Creatine Serum or water. Analysis showed 5 ml of Creatine Serum to contain <10 mg Cr.H2O and approximately 90 mg creatinine. Phosphorylcreatine was not detectable and only a trace amount of phosphorous was present. Total nitrogen analysis ruled out significant amounts of other forms of creatine. We conclude that the trace amounts of creatine in the product would be too little to affect the muscle content even with multiple dosing.

Shivakumar BR, Wang Z, Hammond TG, Harris RC. EP24.15 interacts with the angiotensin II type I receptor and bradykinin B2 receptor. Cell Biochem Funct (2005) 23:195-204
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The carboxyl-terminal cytoplasmic domain of the angiotensin II type 1 receptor (AT1) is known to interact with several classes of intracellular proteins that may modulate receptor function. Employing yeast two-hybrid screening of a human embryonic kidney cDNA library with the carboxyl-terminal cytoplasmic domain of the AT1 receptor as a bait, we have isolated EP24.15 (EC 3.4.24.15, thimet oligopeptidase) as a potentially interacting protein. EP24.15 is widely distributed and is known to degrade bioactive peptides such as angiotensin I and II and bradykinin. In addition, EP24.15 was previously identified as a putative soluble angiotensin II binding protein. Two-hybrid screening also determined that EP24.15 can interact with the B2 bradykinin receptor. Transient expression of EP24.15 in a porcine kidney epithelial cell line stably expressing full length AT1 and full length B2 followed by affinity chromatography and co-immunoprecipitation confirmed EP24.15 association with both AT1 and B2 receptors. EP24.15 was also co-immunoprecipitated with AT1 and B2 in rat kidney brush border membranes (BBM) and basolateral membranes (BLM). Both AT1 and B2 undergo ligand-induced endocytosis. Analysis of endosomal fractions following immunoprecipitation with AT1 or B2 antibodies detected strong association of EP24.15 with the receptors in both light and heavy endosomal populations. Therefore, the present study indicates that EP24.15 associates with AT1 and B2 receptors both at the plasma membrane and after receptor internalization and suggests a possible mechanism for endosomal disposition of ligand that may facilitate receptor recycling.

Harris RC, Zhang MZ, Cheng HF. Cyclooxygenase-2 and the renal renin-angiotensin system. Acta Physiol Scand (2004) 181:543-7
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In the kidney, cyclooxygenase-2 (COX-2) is expressed in the macula densa/cTALH and medullary interstitial cells. The macula densa is involved in regulating afferent arteriolar tone and renin release by sensing alterations in luminal chloride via changes in the rate of Na(+)/K(+)/2Cl(-) cotransport, and administration of non-specific cyclooxygenase inhibitors will blunt increases in renin release mediated by macula densa sensing of decreases in luminal NaCl. High renin states [salt deficiency, angiotensin converting enzyme (ACE) inhibitors or angiotensin receptor blockers, diuretic administration or experimental renovascular hypertension] are associated with increased macula densa/cTALH COX-2 expression. Furthermore, there is evidence that angiotensin II and/or aldosterone may inhibit COX-2 expression. In AT1 receptor knockout mice, COX-2 expression is increased similar to increases with ACE inhibitors or AT1 receptor blockers. Direct administration of angiotensin II inhibits macula densa COX-2 expression. Previous studies demonstrated that alterations in intraluminal chloride concentration are the signal for macula densa regulation of tubuloglomerular feedback and renin secretion, with high chloride stimulating tubuloglomerular feedback and low chloride stimulating renin release. When cultured cTALH or macula densa cells were incubated in media with selective substitution of chloride ions, COX-2 expression and prostaglandin production were significantly increased. A variety of studies have indicated a role for COX-2 in the macula densa mediation of renin release. In isolated perfused glomerular preparations, renin release induced by macula densa perfusion with a low chloride solution was inhibited by a COX-2 inhibitor but not a COX-1 inhibitor. In vivo studies in rats indicated that increased r

Chen X, Moeckel G, Morrow JD, Cosgrove D, Harris RC, Fogo AB, Zent R, Pozzi A. Lack of integrin alpha1beta1 leads to severe glomerulosclerosis after glomerular injury. Am J Pathol (2004) 165:617-30
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Severity of fibrosis after injury is determined by the nature of the injury and host genetic susceptibility. Metabolism of collagen, the major component of fibrotic lesions, is, in part, regulated by integrins. Using a model of glomerular injury by adriamycin, which induces reactive oxygen species (ROS) production, we demonstrated that integrin alpha1-null mice develop more severe glomerulosclerosis than wild-type mice. Moreover, primary alpha1-null mesangial cells produce more ROS both at baseline and after adriamycin treatment. Increased ROS synthesis leads to decreased cell proliferation and increased glomerular collagen IV accumulation that is reversed by antioxidants both in vivo and in vitro. Thus, we have identified integrin alpha1beta1 as a modulator of glomerulosclerosis. In addition, we showed a novel pathway where integrin alpha1beta1 modulates ROS production, which in turn controls collagen turnover and ultimately fibrosis. Because integrin alpha1beta1 is expressed in many cell types this may represent a generalized mechanism of controlling matrix accumulation, which has implications for numerous diseases characterized by fibrosis.

Chen D, Roberts R, Pohl M, Nigam S, Kreidberg J, Wang Z, Heino J, Ivaska J, Coffa S, Harris RC, Pozzi A, Zent R. Differential expression of collagenand laminin-binding integrins mediates ureteric bud and inner medullary collecting duct cell tubulogenesis. Am J Physiol Renal Physiol (2004) 287:F602-11
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Inner medullary collecting ducts (IMCD) are terminally differentiated structures derived from the ureteric bud (UB). UB development is mediated by changes in the temporal and spatial expression of integrins and their respective ligands. We demonstrate both in vivo and in vitro that the UB expresses predominantly laminin receptors (alpha3beta1-, alpha6beta1-, and alpha6beta(4-integrins), whereas the IMCD expresses both collagen (alpha1beta1and alpha2beta1-integrins) and laminin receptors. Cells derived from the IMCD, but not the UB, undergo tubulogenesis in collagen-I (CI) gels in an alpha1beta1and alpha2beta1-dependent manner. UB cells transfected with the alpha2-integrin subunit undergo tubulogenesis in CI, suggesting that collagen receptors are required for branching morphogenesis in CI. In contrast, both UB and IMCD cells undergo tubulogenesis in CI/Matrigel gels. UB cells primarily utilize alpha3beta1- and alpha6-integrins, whereas IMCD cells mainly employ alpha1beta1 for this process. These results demonstrate a switch in integrin expression from primarily laminin receptors in the early UB to both collagen and laminin receptors in the mature IMCD, which has functional consequences for branching morphogenesis in three-dimensional cell culture models. This suggests that temporal and spatial changes in integrin expression could help organize the pattern of branching morphogenesis of the developing collecting system in vivo.

Quan A, Chakravarty S, Chen JK, Chen JC, Loleh S, Saini N, Harris RC, Capdevila J, Quigley R. Androgens augment proximal tubule transport. Am J Physiol Renal Physiol (2004) 287:F452-9
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The proximal tubule contains an autonomous renin-angiotensin system that regulates transport independently of circulating angiotensin II. Androgens are known to increase expression of angiotensinogen, but the effect of androgens on proximal tubule transport is unknown. In this in vivo microperfusion study, we examined the effect of androgens on proximal tubule transport. The volume reabsorptive rate in Sprague-Dawley rats given dihydrotestosterone (DHT) injections was significantly higher than in control rats given vehicle injections (4.57 +/0.31 vs. 3.31 +/0.23 nl x min(-1) x mm(-1), P < 0.01). Luminally perfusing with either enalaprilat (10(-4) M) to inhibit production of angiotensin II or losartan (10(-8) M) to block the angiotensin receptor decreased the proximal tubule volume reabsorptive rate in DHT-treated rats to a significantly greater degree than in control vehicle-injected rats. The renal expression of angiotensinogen was shown to be higher in the DHT-treated animals, using Northern blot analysis. The expression of angiotensin receptors, determined by specific binding of angiotensin II, was not different in the two groups of animals. Brush-border membrane protein abundance of the Na/H exchanger, a membrane transport protein under angiotensin II regulation, was also higher in DHT-treated rats vs. control rats. Rats that received DHT had higher blood pressures than the control rats but had no change in their glomerular filtration rate. In addition, serum angiotensin II levels were lower in DHT-treated vs. control rats. These results suggest that androgens may directly upregulate the proximal tubule renin-angiotensin system, increase the volume reabsorptive rate, and thereby increase extracellular volume and blood pressure and secondarily decrease serum angiotensin II levels.

Singh AB, Tsukada T, Zent R, Harris RC. Membrane-associated HB-EGF modulates HGF-induced cellular responses in MDCK cells. J Cell Sci (2004) 117:1365-79
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In MDCK cells, hepatocyte growth factor/scatter factor (HGF/SF) induces epithelial cell dissociation, scattering, migration, growth and formation of branched tubular structures. By contrast, these cells neither scatter nor form tubular structures in response to the epidermal growth factor (EGF) family of growth factors. Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors and is synthesized as a membrane-associated precursor molecule (proHB-EGF). ProHB-EGF is proteolytically cleaved to release a soluble ligand (sHB-EGF) that activates the EGF receptor. Although recent studies suggest possible physiological functions, the role of proHB-EGF remains largely undefined. Using MDCK cells stably expressing proHB-EGF, a noncleavable deletion mutant of proHB-EGF or soluble HB-EGF, we show that epithelial cell functions differ depending on the form of HB-EGF being expressed. Expression of noncleavable membrane-anchored HB-EGF promoted cell-matrix and cell-cell interactions and decreased cell migration, HGF/SF-induced cell scattering and formation of tubular structures. By contrast, expression of soluble HB-EGF induced increased cell migration, decreased cell-matrix and cell-cell interactions and promoted the development of long unbranched tubular structures in response to HGF/SF. These findings suggest that HB-EGF can not only modulate HGF/SF-induced cellular responses in MDCK cells but also that membrane-bound HB-EGF and soluble HB-EGF give rise to distinctly different effects on cell-cell and cell-extracellular matrix interactions.

Zhang MZ, Mai W, Li C, Cho SY, Hao C, Moeckel G, Zhao R, Kim I, Wang J, Xiong H, Wang H, Sato Y, Wu Y, Nakanuma Y, Lilova M, Pei Y, Harris RC, Li S, Coffey RJ, Sun L, Wu D, Chen XZ, Breyer MD, Zhao ZJ, McKanna JA, Wu G. PKHD1 protein encoded by the gene for autosomal recessive polycystic kidney disease associates with basal bodies and primary cilia in renal epithelial cells. Proc Natl Acad Sci U S A (2004) 101:2311-6
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Mutations of the polycystic kidney and hepatic disease 1 (PKHD1) gene have been shown to cause autosomal recessive polycystic kidney disease (ARPKD), but the cellular functions of the gene product (PKHD1) remain uncharacterized. To illuminate its properties, the spatial and temporal expression patterns of PKHD1 were determined in mouse, rat, and human tissues by using polyclonal Abs and mAbs recognizing various specific regions of the gene product. During embryogenesis, PKHD1 is widely expressed in epithelial derivatives, including neural tubules, gut, pulmonary bronchi, and hepatic cells. In the kidneys of the pck rats, the rat model of which is genetically homologous to human ARPKD, the level of PKHD1 was significantly reduced but not completely absent. In cultured renal cells, the PKHD1 gene product colocalized with polycystin-2, the gene product of autosomal dominant polycystic disease type 2, at the basal bodies of primary cilia. Immunoreactive PKHD1 localized predominantly at the apical domain of polarized epithelial cells, suggesting it may be involved in the tubulogenesis and/or maintenance of duct-lumen architecture. Reduced PKHD1 levels in pck rat kidneys and its colocalization with polycystins may underlie the pathogenic basis for cystogenesis in polycystic kidney diseases.

Becker BN, Cheng HF, Hammond TG, Harris RC. The type 1 angiotensin II receptor tail affects receptor targeting, internalization, and membrane fusion properties. Mol Pharmacol (2004) 65:362-9
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Endocytosis modulates cell responses by removing and recycling receptors from the cell surface. Type I angiotensin II receptors (AT1R) are somewhat unique in that they are expressed at apical (AP) and basolateral (BL) membranes in proximal tubule cells and both receptor sites undergo endocytosis. We analyzed AT1R cytoplasmic (-COOH) tail deletion mutants to determine whether classic AT1R endocytosis motifs functioned similarly in polarized cells and simultaneously altered receptor properties. Serially truncating the AT1R tail had little effect on AP/BL AT1R distribution as determined by 125I-angiotensin II binding in LLCPK(Cl4) cells transfected with an AT1R transcript. AP AT1R expression required the proximal 12 amino acids in the AT1R-COOH tail. Deleting all but the proximal 12 aa of the AT1R-COOH tail (T316L mutant) decreased AP AT1R internalization at 20 min (17 +/6%; p < 0.05 versus full-length; n = 5) and inhibited AP AT1R-stimulated arachidonic acid release (counts released per milligram of protein at 20 min: full-length, 18,762 +/4018; T316L, 2430 +/1711; n = 4; p < 0.02). Endosomal fusion assays were performed using peptide sequences of regions in the AT1R tail involved in endocytosis (YFLQLLKYIPP [LL] and LSTKMSTLSY [STL]). Peptide STL significantly inhibited endosomal fusion (22 +/10% of control; n = 5; p < 0.05 versus positive control). Peptide LL had no significant inhibitory effect. AT(1)R in polarized cells contain dominant endocytosis signals but these motifs do not correlate with AP or BL AT1R expression. Moreover, peptide sequences within the AT1R-COOH tail necessary for endocytosis also modulate endosomal fusion properties.

Zhang MZ, Sanchez Lopez P, McKanna JA, Harris RC. Regulation of cyclooxygenase expression by vasopressin in rat renal medulla. Endocrinology (2004) 145:1402-9
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The antagonism between prostaglandin and vasopressin represents a classic negative feedback loop. It is not clear whether cyclooxygenase (COX)-2 and/or COX-1 expression is involved in elevated prostaglandin production stimulated by vasopressin in vivo. In the present study, we explored vasopressin regulation of medullary COX-2 and COX-1 expression acutely and chronically in rats. Medullary COX-1 expression was moderately lower and COX-2 expression was significantly lower in adult male Brattleboro rats than age-matched Long-Evans controls. Chronic treatment of Brattleboro rats with vasopressin for 1 wk led to a decrease in urine volume and a moderate increase in medullary COX-1; in contrast, medullary COX-2 expression was almost undetectable in untreated rats but was dramatically up-regulated with vasopressin treatment and was accompanied by increased urinary prostaglandin E(2) excretion. Further investigation revealed that both V1 and V2 receptors were involved in chronic medullary COX-1 and COX-2 up-regulation. Acute treatment with specific V1 or V2 receptor agonists resulted in specific increases in medullary COX-2, which was prevented by furosemide. Vasopressin did not affect COX-2 expression in cultured renomedullary interstitial cells. These data demonstrate that vasopressin stimulates medullary COX-2 expression through activation of both V1 and V2 receptors, and this stimulation is indirect and probably involves increased medullary electrolyte tonicity.

Wang Z, Chen JK, Wang SW, Moeckel G, Harris RC. Importance of functional EGF receptors in recovery from acute nephrotoxic injury. J Am Soc Nephrol (2003) 14:3147-54
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Previous studies have demonstrated increased renal expression of EGF receptor (EGFR) and EGFR ligands in response to acute toxic or ischemic renal tubular injury and have indicated that exogenous administration of EGF accelerates recovery from such injury. However, no studies to date have proved definitively an essential role for EGFR-mediated responses in regeneration after tubule injury. To this end, waved-2 (wa-2) mice, which contain a point mutation in EGFR that reduces receptor tyrosine kinase activity by >90%, were studied. These mice have a mild phenotype (wavy coat, curly whiskers, and runted stature) and normally developed kidneys. Acute nephrotoxic injury was induced in wa-2 and wild-type mice with HgCl(2). One day after HgCl(2) injection, functional renal compromise was comparable in wild-type and wa-2 mice. However, the rates of recovery of serum blood urea nitrogen and creatinine levels were markedly slower in wa-2 mice. Histologic evidence of tubular injury also was more severe and persisted longer in wa-2 mice. Furthermore, their kidneys demonstrated reduced levels of DNA synthesis and increased TdT-mediated dUTP nick-end labeling staining. These studies indicate that functional EGFR activity is an essential component of the kidney's ability to recover from acute injury and that EGFR may regulate genes involved in growth, repair, and cell survival in the kidney.

Qi Z, Whitt I, Mehta A, Jin J, Zhao M, Harris RC, Fogo AB, Breyer MD. Serial determination of glomerular filtration rate in conscious mice using FITC-inulin clearance. Am J Physiol Renal Physiol (2004) 286:F590-6
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Two nonradioactive methods for determining glomerular filtration rate (GFR) in conscious mice using FITC-labeled inulin (FITC-inulin) were evaluated. The first method measured GFR using clearance kinetics of plasma FITC-inulin after a single bolus injection. Based on a two-compartment model, estimated GFR was 236.69 +/16.55 and 140.20 +/- 22.27 microl/min in male and female C57BL/6J mice, respectively. Total or (5/6) nephrectomy reduced inulin clearance to 0 or 32.80 +/9.32 microl/min, respectively. Conversely, diabetes mellitus induced by streptozotocin was associated with increased GFR. The other approach measured urinary inulin clearance using intraperitoneal microosmotic pumps to deliver FITC-inulin and metabolic cages to collect timed urine samples. This approach yielded similar GFR values of 211.11 +/26.56 and 157.36 +/20.02 microl/min in male and female mice, respectively. These studies demonstrate the feasibility of repeated nonisotopic measurement of inulin clearance in conscious mice.

Singh AB, Harris RC. Epidermal growth factor receptor activation differentially regulates claudin expression and enhances transepithelial resistance in Madin-Darby canine kidney cells. J Biol Chem (2004) 279:3543-52
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Tight junctions (TJs) are the most apical cell-cell junctions, and claudins, the recently identified TJ proteins, are critical for maintaining cell-cell adhesion in epithelial cell sheets. Based on their in vivo distribution and the results of overexpression studies, certain claudins, including claudin-1 and -4, are postulated to increase, whereas other claudins, especially claudin-2, are postulated to decrease the overall transcellular resistance. The overall ratio among claudins expressed in a cell/tissue has been hypothesized to define the complexity of TJs. Disruption of the TJs contributes to various human diseases, and a correlation between reduction of TJ function and tumor dedifferentiation has been postulated. The epidermal growth factor (EGF) receptor (EGFR) is overexpressed in a wide spectrum of epithelial cancers, and its expression correlates with a more metastatic cancer phenotype. However, normal functioning of EGFR is essential for normal epithelial cell proliferation and differentiation. The role of EGFR-dependent signaling in the development and maintenance of epithelial TJ integrity has not been studied in detail. This study demonstrates that, in polarized Madin-Darby canine kidney II cells, EGF-induced EGFR activation significantly inhibited claudin-2 expression while simultaneously inducing cellular redistribution and increased expression of claudin-1, -3, and -4. Accompanying these EGF-induced changes in claudin expression was a 3-fold increase in transepithelial resistance, a functional measure of TJs. In contrast, there were no alterations in protein expression and/or intracellular localization of other TJ-related proteins (ZO-1 and occludin) or adherens junction-associated proteins (E-cadherin and beta-catenin), suggesting that EGF regulates TJ function through selectiv

Zhang MZ, Wang SW, Cheng H, Zhang Y, McKanna JA, Harris RC. Regulation of renal cortical cyclooxygenase-2 in young rats. Am J Physiol Renal Physiol (2003) 285:F881-8
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Cyclooxygenase-2 (COX-2) is involved in kidney morphogenesis and is transiently elevated in the immature kidney. In adult rats, renal cortical COX-2 expression is tonically suppressed by mineralocorticoids (MC) and glucocorticoids (GC) and induced by chronic salt restriction. Young rats have low levels of GC and are in a state of relative volume depletion. The present study was designed to investigate the mechanisms underlying elevated cortical COX-2 expression in the immature kidney. Supplementation of GC or MC suppressed cortical COX-2 expression in suckling rats. GC suppression was significantly, but not completely, prevented by either an MC receptor antagonist or a GC receptor antagonist. MC suppression was completely prevented by a mineralocorticoid receptor antagonist. Salt supplementation suppressed cortical COX-2 expression in a doseand time-dependent pattern in the suckling rats. Cortical COX-2 expression in the weanling rats was upregulated by a low-salt diet and downregulated by a high-salt diet. These results suggest that relative volume depletion and reduced GC levels are involved in elevated cortical COX-2 expression in the immature rodent kidney.

Harris RC. Cyclooxygenase-2 and the kidney: functional and pathophysiological implications. J Hypertens Suppl (2002) 20:S3-9
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In adult mammalian kidney, cyclooxygenase-2 (COX-2) expression is found in restricted subpopulations of cells. High levels of expression can be detected in the macula densa (MD) and associated cortical thick ascending limb of Henle (cTALH) cells and medullary interstitial cells (MICs). In human biopsy specimens, COX-2 expression is also detected in glomerular podocytes and increased podocyte expression is seen in experimental models of progressive glomerular injury. Physiological regulation of COX-2 in these cellular compartments suggests functional roles for eicosanoid products of the enzyme. COX-2 expression increases in high-renin states (salt restriction, angiotensin-converting enzyme inhibition, renovascular hypertension) and selective COX-2 inhibitors significantly decrease plasma renin levels, renal renin activity and mRNA expression. There is evidence for negative regulation of MD/cTALH COX-2 by angiotensin II and by glucocorticoids and mineralocorticoids. Conversely, nitric oxide (NO) generated by neuronal nitric oxide synthase (nNOS) is a positive modulator of COX-2 expression. Decreased extracellular chloride increases COX-2 expression in cultured cTALH, an effect mediated by increased p38 MAP kinase activity and, in vivo, a sodium-deficient diet increases expression of activated p38 in MD/cTALH. In contrast to COX-2 in MD/cTALH, COX-2 expression in MICs increases in response to a high-salt diet, as well as water deprivation. Studies in cultured MICs confirm that expression is increased in response to hypertonicity, and expression is mediated at least in part by nuclear factor-kappaB (NFkappaB) activation. COX-2 inhibition leads to apoptosis of MICs in response to hypertonicity in vitro and following water deprivation in vivo. In addition, COX-2 metabolites appear to be important med

Means AL, Ray KC, Singh AB, Washington MK, Whitehead RH, Harris RC Jr, Wright CV, Coffey RJ Jr, Leach SD. Overexpression of heparin-binding EGF-like growth factor in mouse pancreas results in fibrosis and epithelial metaplasia. Gastroenterology (2003) 124:1020-36
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BACKGROUND & AIMS: Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is expressed in both normal pancreatic islets and in pancreatic cancers, but its role in pancreatic physiology and disease is not known. This report examines the effects of HB-EGF overexpression in mouse pancreas. METHODS: Transgenic mice were established using a tissue-specific promoter to express an HB-EGF complementary DNA in pancreatic beta cells, effectively elevating HB-EGF protein 3-fold over endogenous levels. RESULTS: Mice overexpressing HB-EGF in pancreatic islets showed both endocrine and exocrine pancreatic defects. Initially, islets from transgenic mice failed to segregate alpha, beta, delta, and PP cells appropriately within islets, and had impaired separation from ducts and acini. Increased stroma was detected within transgenic islets, expanding with age to cause fibrosis of both endocrine and exocrine compartments. In addition to these structural abnormalities, subsets of transgenic mice developed profound hyperglycemia and/or proliferation of metaplastic ductal epithelium. Both conditions were associated with severe stromal expansion, suggesting a role for islet/stromal interaction in the onset of the pancreatic disease initiated by HB-EGF. Supporting this conclusion, primary mouse fibroblasts adhered to transgenic islets when the 2 tissues were cocultured in vitro, but did not interact with nontransgenic islets. CONCLUSIONS: An elevation in HB-EGF protein in pancreatic islets led to altered interactions among islet cells and among islets, stromal tissues, and ductal epithelium. Many of the observed phenotypes appeared to involve altered cell adhesion. These data support a role for islet factors in the development of both endocrine and exocrine disease.

Harris RC, Chung E, Coffey RJ. EGF receptor ligands. Exp Cell Res (2003) 284:2-13
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null

Harris RC. Interactions between COX-2 and the renin-angiotensin system in the kidney. Acta Physiol Scand (2003) 177:423-7
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AIM: In adult mammalian kidney, COX-2 expression is found in a restricted subpopulation of cells. The two sites of renal COX-2 localization detected in all species to date are the macula densa (MD) and associated cortical thick ascending limb cells (cTALH) and medullary interstitial cells. Physiological regulation of COX-2 in these cellular compartments suggests functional roles for eicosanoid products of the enzyme. In the MD region, COX-2 expression increases in high renin states [salt restriction, angiotensin converting enzyme (ACE) inhibition, renovascular hypertension], and selective COX-2 inhibitors significantly decrease plasma renin levels and renal renin activity and mRNA expression. An important role for COX-2-derived prostanoids in regulation of renin expression and secretion has also been determined by using mice with selective genetic deletion of either the COX-1 or COX-2 gene. There is evidence for negative regulation of MD/cTALH COX-2 by angiotensin II and by glucocorticoids and mineralocorticoids, suggesting that in the kidney, cortical COX-2 expression is regulated in part by alterations in activity of the renin-angiotensin system.

Cheng HF, Harris RC. Does cyclooxygenase-2 affect blood pressure? Curr Hypertens Rep (2003) 5:87-92
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With the development and clinical implementation of the new cyclooxygenase (COX)-2 inhibitors, their safety, including the effects on renal function and blood pressure, is attracting increasing attention. In the kidney, COX-2 is constitutively expressed and is highly regulated in response to alterations in intravascular volume. COX-2 metabolites have been implicated in mediation of renin release, regulation of sodium excretion, and maintenance of renal blood flow. Similar to conventional nonsteroidal anti-inflammatory drugs, inhibition of COX-2 may cause modest elevations in blood pressure in a minority of subjects. COX-2 inhibitors may also exacerbate pre-existing hypertension or interfere with other antihypertensive drugs. Special caution should be taken in patients with volume depletion or decreased organ perfusion.

Cheng HF, Harris RC. Cyclooxygenase-2 expression in cultured cortical thick ascending limb of Henle increases in response to decreased extracellular ionic content by both transcriptional and post-transcriptional mechanisms. Role of p38-mediated pathways. J Biol Chem (2002) 277:45638-43
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We showed previously that decreased extracellular salt or chloride up-regulates the cortical thick ascending limb of Henle (cTALH) COX-2 expression via a p38-dependent pathway. The present studies determined that low salt medium increased COX-2 mRNA expression 3.9-fold control by 6 h in cultured cTALH, which was blocked by actinomycin D pretreatment, suggesting transcriptional regulation. Luciferase activity (normalized to beta-galactosidase activity) of the full-length (-3400) COX-2 promoter in cTALH increased from 1.8 +/0.3 in control media to 5.8 +/0.7 in low salt (n = 9; p < 0.01). Low chloride medium had similar effects as low salt has on COX-2 promoter activity. Deletion constructs -815, -512, and -410 were similarly stimulated, but -385 could not be stimulated significantly by low salt (1.8 +/0.3 versus 2.4 +/0.5, n = 10). This suggested involvement of an NF-kappaB cis-element located in this region, which was confirmed by utilizing a construct with a point mutation of this NF-kappaB-binding site that was not stimulated by low salt medium. Co-incubation of the specific p38 inhibitor, SB203580 or PD169316, inhibited a low salt-induced increase in luciferase activity of the intact COX-2 promoter (5.8 +/0.7 versus 1.1 +/0.2, n = 8 and 1.4 +/0.4, n = 4 respectively, p < 0.01). Mobility shift assays indicated that the low salt medium stimulated NF-kappaB binding activity, and this stimulation was inhibited by p38 inhibitors. To test whether p38 also increased COX-2 expression by increasing mRNA stability, cTALH were incubated in low salt for 2 h, and actinomycin was then added with or without SB203580. p38 inhibition led to a decreased half-life of COX-2 mRNA (from 68 to 18 min, n = 4-7, p < 0.05). Therefore, these studies indicate that p38 stimulates COX-2 expression in cTALH and macula dens

Cheng HF, Wang SW, Zhang MZ, McKanna JA, Breyer R, Harris RC. Prostaglandins that increase renin production in response to ACE inhibition are not derived from cyclooxygenase-1. Am J Physiol Regul Integr Comp Physiol (2002) 283:R638-46
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It is well known that nonselective, nonsteroidal anti-inflammatory drugs inhibit renal renin production. Our previous studies indicated that angiotensin-converting enzyme inhibitor (ACEI)-mediated renin increases were absent in rats treated with a cyclooxygenase (COX)-2-selective inhibitor and in COX-2 -/mice. The current study examined further whether COX-1 is also involved in mediating ACEI-induced renin production. Because renin increases are mediated by cAMP, we also examined whether increased renin is mediated by the prostaglandin E(2) receptor EP(2) subtype, which is coupled to G(s) and increases cAMP. Therefore, we investigated if genetic deletion of COX-1 or EP(2) prevents increased ACEI-induced renin expression. Ageand gender-matched wild-type (+/+) and homozygous null mice (-/-) were administered captopril for 7 days, and plasma and renal renin levels and renal renin mRNA expression were measured. There were no significant differences in the basal level of renal renin activity from plasma or renal tissue in COX-1 +/+ and -/- mice. Captopril administration increased renin equally [plasma renin activity (PRA): +/+ 9.3 +/2.2 vs. 50.1 +/10.9; -/13.7 +/1.5 vs. 43.9 +/6.6 ng ANG I x ml(-1) x h(-1); renal renin concentration: +/+ 11.8 +/1.7 vs. 35.3 +/3.9; -/13.0 +/3.0 vs. 27.8 +/2.7 ng ANG I x mg protein(-1) x h(-1); n = 6; P < 0.05 with or without captopril]. ACEI also increased renin mRNA expression (+/+ 2.4 +/0.2; -/2.1 +/0.2 fold control; n = 6-10; P < 0.05). Captopril led to similar increases in EP(2) -/compared with +/+. The COX-2 inhibitor SC-58236 blocked ACEI-induced elevation in renal renin concentration in EP(2) null mice (+/+ 24.7 +/1.7 vs. 9.8 +/0.4; -/21.1 +/3.2 vs. 9.3 +/0.4 ng ANG I x mg protein(-1) x h(-1); n = 5) as well as in COX-1 -/mice (SC-58236-treated PRA: +/+

Zhang MZ, Hao CM, Breyer MD, Harris RC, McKanna JA. Mineralocorticoid regulation of cyclooxygenase-2 expression in rat renal medulla. Am J Physiol Renal Physiol (2002) 283:F509-16
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The renal inner medulla and its distal one-third, the papilla, are major sites of prostanoid synthesis involved in water and electrolyte homeostasis. These sites contain variable levels of cyclooxygenase (COX)-2, a key prostaglandin synthase enzyme that is sensitive to adrenal steroids. Immunoreactive renal medullary COX-2, restricted to interstitial cells in control adult rats, shows a gradient of intense staining at the tip of the papilla that gradually diminishes to undetectable levels in the proximal inner medulla. We used adrenalectomy (ADX) and steroid replacement to investigate the effects of steroids on papillary COX-2. Immunoblots demonstrate that papillary COX-2 was reduced by one-half after 2 wk ADX; glucocorticoid replacement ameliorated the decline but not to control levels. Mineralocorticoid (deoxycorticosterone acetate; DOCA) replacement stimulated papillary COX-2 more than fivefold over control; both the intensity of immunostaining and the numbers of COX-2-positive cells in the inner medulla increased. Similar stimulation of papillary COX-2 resulted from DOCA treatment of normal control rats, but the response was blunted in rats fed a low-salt diet and absent in Brattleboro rats. DOCA treatment of mouse renal medullary interstitial cells in culture had no effect, but increased tonicity of the culture medium with NaCl caused strong upregulation of COX-2. Urea, a permeant molecule, had no effect. Together, these results suggest that mineralocorticoids lead to upregulation of COX-2 in rat renal medulla by indirect pathways, probably involving induced electrolyte hypertonicity in the interstitial fluid.

Cheng HF, Wang CJ, Moeckel GW, Zhang MZ, McKanna JA, Harris RC. Cyclooxygenase-2 inhibitor blocks expression of mediators of renal injury in a model of diabetes and hypertension. Kidney Int (2002) 62:929-39
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BACKGROUND: We previously reported that renal cortical cyclooxygenase (COX-2) expression increased following subtotal nephrectomy, and chronic treatment with a selective COX-2 inhibitor, SC58236, reduced proteinuria and retarded the development of glomerulosclerosis. The present studies were designed to examine the effects of COX-2 inhibition in a model of diabetic nephropathy. METHODS: Rats were divided into three groups: control, diabetic (streptozotocin-induced diabetic animals with superimposed DOCA/salt hypertension; right nephrectomy and DOCA treatment), and treated (administration of the selective COX-2 inhibitor, SC58236, to a subset of diabetic/DOCA/salt rats). Insulin was administered to maintain blood glucose in the 200 to 300 mg/dL range. RESULTS: Systolic blood pressure in the two diabetic groups was elevated within one week and remained elevated until sacrifice at six weeks (control, 108 +/2 mm Hg; diabetic, 158 +/4 mm Hg; treated, 156 +/7 mm Hg). When measured at six weeks, immunoreactive COX-2 expression in the renal cortex of the diabetic rats was 2.5 +/0.3-fold of control animals (N = 7). Immunohistochemical localization indicated increased expression in macula densa and surrounding cortical thick ascending limb of Henle (cTALH). The COX-2 inhibitor decreased COX-2 expression in diabetic rats to 1.3 +/0.1-fold control. In addition, SC58236 decreased expression of PAI-1 (diabetic vs. treated, 3.2 +/0.5 vs. 1.7 +/0.2-fold control, N = 7, P < 0.05), vascular endothelial growth factor (VEGF; 2.0 +/0.2 vs. 1.2 +/0.2; N = 7, P < 0.05), fibronectin (2.4 +/0.3 to 1.3 +/0.1; N = 7, P < 0.05) and transforming growth factor-beta (TGF-beta; 2.1 +/0.2 vs. 1.3 +/- 0.2; N = 7, P < 0.05). Proteinuria at six weeks was decreased in the SC58236-treated rats (149 +/8 vs. 92 +/8 mg/24 h; N = 7, P

Capdevila JH, Harris RC, Falck JR. Microsomal cytochrome P450 and eicosanoid metabolism. Cell Mol Life Sci (2002) 59:780-9
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The demonstration of a role for microsomal P450 in the metabolism of endogenous pools of arachidonic acid established this enzyme system as a member of the arachidonic acid cascade and characterized a new an important metabolic function for this enzyme system. Studies from several laboratories documenting the powerful biological activities of the P450-derived eicosanoids have suggested important roles for the P450 arachidonic acid monooxygenase in renal and vascular physiology, and in the pathophysiology of experimental hypertension. These studies provide significant evidence to indicate that in addition to its recognized traditional toxicological and pharmacological roles, microsomal P450s also play important physiological roles in the control of tissue and body homeostasis.

Wolf G, Wenzel U, Burns KD, Harris RC, Stahl RA, Thaiss F. Angiotensin II activates nuclear transcription factor-kappaB through AT1 and AT2 receptors. Kidney Int (2002) 61:1986-95
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BACKGROUND: Recent evidence suggests that angiotensin II (Ang II) induces a variety of proinflammatory mediators including chemokines. Nuclear factor-kappaB (NF-kappaB) activation plays an important role in Ang II-mediated inflammation. The present study investigated which Ang II receptor subtype is involved in NF-kappaB activation. We focused particularly on the Ang II subtype 2 (AT2) receptor because we previously observed that Ang II-induction of the chemokine RANTES in vitro and in vivo is mediated through AT2 receptors.METHODS: AT1 or AT2 receptors were selectively overexpressed in COS7 cells that normally do not express Ang II receptors. In addition, rat glomerular endothelial cells (GER) that express AT1 and AT2 receptors and PC12 cells that exclusively exhibit AT2 receptors were studied also. Ang II-receptor expression was confirmed by Western blots of membrane lysates. NF-kappaB DNA binding in vitro was detected by electrophoretic shift assays. In addition, in vivo transactivation of a reporter gene construct with kappa enhancer coupled to luciferase also was investigated. Expression of the inhibitor of kappaB alpha (IkappaB-alpha) was detected by Western blots.RESULTS: In AT1 or AT2 receptor transfected cells, but not untransfected COS7 cells, 10-7 mol/L Ang II induced NF-kappaB DNA binding in vitro, as detected by electrophoretic shift assays and in vivo transactivation of a reporter gene construct. The AT2 receptor antagonist PD 123319 but not losartan attenuated Ang II-mediated NF-kappaB activation in COS7 cells transfected with AT2 receptors. While Ang II also induced NF-kappaB activation in PC12 cells, this activation was blocked by PD 123319. Finally, stimulation of GERs with Ang II led to the activation of NF-kappaB through both subtypes of Ang II receptors. Nuclear extracts fr

Takemura T, Hino S, Okada M, Murata Y, Yanagida H, Ikeda M, Yoshioka K, Harris RC. Role of membrane-bound heparin-binding epidermal growth factor-like growth factor (HB-EGF) in renal epithelial cell branching. Kidney Int (2002) 61:1968-79
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Role of membrane-bound heparin-binding epidermal growth factor-like growth factor (HB-EGF) in renal epithelial cell branching.BACKGROUND: The developing metanephros is characterized by growth and differentiation of the ureteric bud and the surrounding mesenchymal tissue. These processes can be influenced by several growth factors, including epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha). We examined whether another member of the EGF family of growth factors, heparin-binding epidermal growth factor (HB-EGF), might act as a morphogen in renal epithelial tubulogenesis.METHODS: Expression of HB-EGF mRNA and immunoreactive protein were examined in fetal, neonatal and adult rat kidneys. For in vitro studies of tubulogenesis, a rat renal epithelial cell line (NRK52E) stably transfected with proHB-EGF (NRKproHB-EGF) was treated with TPA for 30 minutes, washed with 2 mol/L NaCl to remove soluble HB-EGF trapped by cell surface heparan sulfate proteoglycan and replated onto plastic dishes in the absence of fetal calf serum. In further experiments, NRKproHB-EGF were suspended in a type I collagen gel in serum-free media.RESULTS: Northern blot analysis indicated that HB-EGF was strongly expressed in embryonic rat kidney (embryonic days 18-20) and was still increased in the neonatal kidney (day 10), compared to the low basal levels in adult kidney. Immunohistochemical analysis confirmed that immunoreactive HB-EGF expression in the fetal rat kidney was localized predominantly to the ureteric bud. When NRKproHB-EGF were plated onto plastic substrata, they became progressively flattened and enlarged and exhibited filopoidia. By 10 hours after plating, NRKproHB-EGF began to migrate and subsequently developed cell-cell contact and fully established tubular-like structures. Immunoel

Chen JK, Capdevila J, Harris RC. Heparin-binding EGF-like growth factor mediates the biological effects of P450 arachidonate epoxygenase metabolites in epithelial cells. Proc Natl Acad Sci U S A (2002) 99:6029-34
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In addition to its important functions in detoxification of foreign chemicals and biosynthesis of steroid hormones, the cytochrome P450 enzyme system metabolizes arachidonate to 14,15-epoxyeicosatrienoic acid (14,15-EET). This study demonstrates that a P450 arachidonate epoxygenase metabolite can activate cleavage of heparin-binding epidermal growth factor-like growth factor (HB-EGF) and delineates an essential role for HB-EGF in the mitogenic effects of this lipid mediator. Blockade of HB-EGF processing or EGF receptor (EGFR) inhibited 14,15-EET-stimulated early mitogenic signals and DNA synthesis. 14,15-EET failed to induce mitogenesis in cell lines expressing minimal HB-EGF, whereas 14,15-EET induced soluble HB-EGF release into the conditioned media of cell lines that both express high levels of HB-EGF and display mitogenic response to this lipid mediator. Moreover, transfection of a bacterial 14,15-epoxygenase established intracellular endogenous 14,15-EET biosynthesis in cultured cell systems, which allowed direct confirmation of involvement of EGFR transactivation in the endogenous 14,15-EET-mediated mitogenic signaling pathway. This mechanism involves EET-dependent activation of metalloproteinases and release of the potent mitogenic EGFR ligand, HB-EGF.

Burleigh ME, Babaev VR, Oates JA, Harris RC, Gautam S, Riendeau D, Marnett LJ, Morrow JD, Fazio S, Linton MF. Cyclooxygenase-2 promotes early atherosclerotic lesion formation in LDL receptor-deficient mice. Circulation (2002) 105:1816-23
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BACKGROUND: Atherosclerosis has features of an inflammatory disease. Because cyclooxygenase (COX)-2 is expressed in atherosclerotic lesions and promotes inflammation, we tested the hypotheses that selective COX-2 inhibition would reduce early lesion formation in LDL receptor-deficient (LDLR-/-) mice and that macrophage COX-2 expression contributes to atherogenesis in LDLR-/mice. METHODS AND RESULTS: Treatment of male LDLR-/mice fed the Western diet with rofecoxib or indomethacin for 6 weeks resulted in significant reductions in atherosclerosis in the proximal aorta (25% and 37%) and in the aorta en face (58% and 57%), respectively. Rofecoxib treatment did not inhibit platelet thromboxane production, a COX-1-mediated process, but it significantly reduced the urinary prostacyclin metabolite 2,3-dinor-6-keto-PGF1alpha. Fetal liver cell transplantation was used to generate LDLR-/mice null for expression of the COX-2 gene by macrophages. After 8 weeks on the Western diet, COX-2-/--> LDLR-/mice developed significantly less (33% to 39%) atherosclerosis than control COX-2+/+ --> LDLR-/mice. In both the inhibitor studies and the transplant studies, serum lipids did not differ significantly between groups. CONCLUSIONS: The present studies provide strong pharmacological and genetic evidence that COX-2 promotes early atherosclerotic lesion formation in LDLR-/mice in vivo. These results support the potential of anti-inflammatory approaches to the prevention of atherosclerosis.

Harris RC Jr. Cyclooxygenase-2 inhibition and renal physiology. Am J Cardiol (2002) 89:10D-17D
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In the adult mammalian kidney, high levels of cyclooxygenase (COX)-2 expression can be detected in the macula densa and associated cortical thick ascending limb cells and medullary interstitial cells. In the renal cortex, COX-2 expression increases in high renin states, and selective COX-2 inhibitors significantly decrease plasma renin levels. In the medullary region of the kidney, the expression of COX-2 increases in response to a high-salt diet and water deprivation. The most important prostanoids in the kidney are prostaglandin (PG)I(2), or prostacyclin, and PGE(2). PGE(2) diminishes sodium reabsorption; thereby, its inhibition results in sodium retention that can manifest clinically in a variety of ways, such as peripheral edema, increased blood pressure (mainly in treated hypertensive patients), weight gain, and occasionally deterioration of heart failure. PGI(2) increases potassium secretion. As such, its inhibition can result in hyperkalemia, particularly in patients with underlying renal insufficiency. PGI(2) is also a potent vasodilator and helps maintain renal perfusion in conditions of decreased actual or effective circulating volume; its inhibition in such patients can result in acute renal failure. A variety of studies has been conducted to examine the effects of celecoxib and rofecoxib on renal function. These incorporate various study designs directly, making it virtually impossible to compare data across studies. It is apparent from such studies, coupled with published case reports, that the impact of both celecoxib and rofecoxib on renal function (including development of edema and hypertension) is similar to that of nonselective nonsteroidal anti-inflammatory drugs (NSAIDs). Studies comparing the 2 COX-2 inhibitors conflict in their interpretation. Overall, the data suggest si

Harris RC, Nevill M, Harris DB, Fallowfield JL, Bogdanis GC, Wise JA. Absorption of creatine supplied as a drink, in meat or in solid form. J Sports Sci (2002) 20:147-51
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We examined the plasma concentration curve obtained over 6 h after the ingestion of 2 g of creatine (Cr) (equivalent to 2.3 g Cr x H2O) contained in meat or in solution in five non-users of creatine supplements. Peak plasma creatine concentration was lower after the ingestion of meat but was maintained close to this for a longer period. Measurements of the area under the plasma concentration curve indicated approximate bioequivalence of creatine contained in meat with the same dose supplied in a solution. In a separate study, we examined the plasma concentration time curve after ingestion of solid Cr x H2O. Creatine ingested as a lozenge (crushed in the mouth and swallowed) or as a crystalline suspension in ice cold water resulted in a 20% lower peak concentration and 30-35% smaller area under the plasma creatine concentration curve than the same dose administered in solution. Despite a possibly lower bioavailability, 2.3 g Cr x H2O supplied in either solid form was nonetheless sufficient to raise the plasma concentration fiveto six-fold in individuals with a mean body mass of 75.6 kg. We conclude that creatine administered as meat or in solid form is readily absorbed but may result in slightly lower peak concentrations than when the same dose is ingested as a solution.

Chen JK, Capdevila J, Harris RC. Cytochrome p450 epoxygenase metabolism of arachidonic acid inhibits apoptosis. Mol Cell Biol (2001) 21:6322-31
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The ubiquitous cytochrome P450 hemoproteins play important functional roles in the metabolism and detoxification of foreign chemicals. However, other than established roles in cholesterol catabolism and steroid hormone biosynthesis, their cellular and/or organ physiological functions remain to be fully characterized. Here we show that the cytochrome P450 epoxygenase arachidonic acid metabolite 14,15-epoxyeicosatrienoic acid (14,15-EET) inhibits apoptosis induced by serum withdrawal, H(2)O(2), etoposide, or excess free arachidonic acid (AA), as determined by DNA laddering, Hoechst staining, and fluorescein isothiocyanate-labeled annexin V binding. In the stable transfectants (BM3 cells) expressing a mutant bacterial P450 AA epoxygenase, F87V BM3, which was genetically engineered to metabolize arachidonic acid only to 14,15-EET, AA did not induce apoptosis and protected against agonist-induced apoptosis. Ceramide assays demonstrated increased AA-induced ceramide production within 1 h and elevated ceramide levels for up to 48 h, the longest time tested, in empty-vector-transfected cells (Vector cells) but not in BM3 cells. Inhibition of cytochrome P450 activity by 17-octadecynoic acid restored AA-induced ceramide production in BM3 cells. Exogenous C2-ceramide markedly increased apoptosis in quiescent Vector cells as well as BM3 cells, and apoptosis was prevented by pretreatment of Vector cells with exogenous 14,15-EET and by pretreatment of BM3 cells with AA. The ceramide synthase inhibitor fumonisin B1 did not affect AA-induced ceramide production and apoptosis; in contrast, these effects of AA were blocked by the neutral sphingomyelinase inhibitor scyphostatin. The pan-caspase inhibitor Z-VAD-fmk had no effect on AA-induced ceramide generation but abolished AA-induced apoptosis. The antiapoptoti

Harris RC, Breyer MD. Physiological regulation of cyclooxygenase-2 in the kidney. Am J Physiol Renal Physiol (2001) 281:F1-11
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In adult mammalian kidney, cyclooxygenase-2 (COX-2) expression is found in a restricted subpopulation of cells. The two sites of renal COX-2 localization detected in all species to date are the macula densa (MD) and associated cortical thick ascending limb (cTALH) and medullary interstitial cells (MICs). Physiological regulation of COX-2 in these cellular compartments suggests functional roles for eicosanoid products of the enzyme. COX-2 expression increases in high-renin states (salt restriction, angiotensin-converting enzyme inhibition, renovascular hypertension), and selective COX-2 inhibitors significantly decrease plasma renin levels, renal renin activity, and mRNA expression. There is evidence for negative regulation of MD/cTALH COX-2 by angiotensin II and by glucocorticoids and mineralocorticoids. Conversely, nitric oxide generated by neuronal nitric oxide synthase is a positive modulator of COX-2 expression. Decreased extracellular chloride increases COX-2 expression in cultured cTALH, an effect mediated by increased p38 mitogen-activated protein kinase activity, and, in vivo, a sodium-deficient diet increases expression of activated p38 in MD/cTALH. In contrast to COX-2 in MD/cTALH, COX-2 expression increases in MICs in response to a high-salt diet as well as water deprivation. Studies in cultured MICs have confirmed that expression is increased in response to hypertonicity and is mediated, at least in part, by nuclear factor-kappaB activation. COX-2 inhibition leads to apoptosis of MICs in response to hypertonicity in vitro and after water deprivation in vivo. In addition, COX-2 metabolites appear to be important mediators of medullary blood flow and renal salt handling. Therefore, there is increasing evidence that COX-2 is an important physiological mediator of kidney function.

Chen JK, Zimpelmann J, Harris RC, Burns KD. Angiotensin IV induces tyrosine phosphorylation of focal adhesion kinase and paxillin in proximal tubule cells. Am J Physiol Renal Physiol (2001) 280:F980-8
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Angiotensin IV (ANG IV), the COOH-terminal hexapeptide fragment of angiotensin II (ANG II), binds to specific sites in the kidney, distinct from type 1 (AT(1)) and type 2 (AT(2)) receptors and designated type 4 (AT(4)) receptors. We determined signaling pathways for ANG IV in a proximal tubular cell line, LLC-PK(1)/Cl(4). In these cells, we found no specific binding of [(125)I]-ANG II. In contrast, ANG IV dose dependently competed for [(125)I]-labeled ANG IV binding, with no displacement by either ANG II, the AT(1) receptor antagonist losartan, or the AT(2) antagonist PD-123319. Saturation binding indicated the presence of AT(4) receptors of high affinity [dissociation constant (K(d)) = 1.4 nM]. ANG IV did not affect cAMP or cGMP production and did not increase cytosolic calcium concentration in these cells. In contrast, immunoprecipitation and immunoblotting studies revealed that ANG IV caused dose-dependent tyrosine phosphorylation of p125-focal adhesion kinase (p125-FAK) and p68-paxillin within 2 min, with maximal stimulation at 30 min. ANG IV-stimulated tyrosine phosphorylation of p125-FAK and paxillin was not affected by pretreatment with either losartan or PD-123319, and ANG II (10(-7) M) did not induce protein tyrosine phosphorylation. Our results indicate that LLC-PK(1)/Cl(4) cells express ANG IV receptors, which we demonstrate for the first time are linked to tyrosine phosphorylation of focal adhesion-associated proteins. This suggests that ANG IV, a product of ANG II metabolism, may regulate function of the focal adhesion complex in proximal tubule cells.

Takemura T, Hino S, Kuwajima H, Yanagida H, Okada M, Nagata M, Sasaki S, Barasch J, Harris RC, Yoshioka K. Induction of collecting duct morphogenesis in vitro by heparin-binding epidermal growth factor-like growth factor. J Am Soc Nephrol (2001) 12:964-72
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Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a member of the epidermal growth factor family of growth factors, is synthesized as a membrane-an-chored precursor (proHB-EGF) that is capable of stimulating adjacent cells in a juxtacrine manner. ProHB-EGF is cleaved in a protein kinase C-dependent process, to yield the soluble form. It was observed that HB-EGF acts as a morphogen for the collecting duct system in developing kidneys. HB-EGF protein was expressed in the ureteric bud of embryonic kidneys. Cultured mouse ureteric bud cells (UBC) produced HB-EGF via protein kinase C activation. After stimulation with phorbol ester (12-O-tetradecanoylphorbol-13-acetate) or recombinant soluble HB-EGF, UBC cultured in three-dimensional collagen gels formed short tubules with varied abundant branches. When proHB-EGF-transfected UBC were stimulated with 12-O-tetradecanoylphorbol-13-acetate and cultured in collagen gels, they exhibited linear growth, forming long tubular structures with few branches at the time of appearance of proHB-EGF on the cell surface. The structures exhibited a strong resemblance to the early branching ureteric bud of embryonic kidneys. When UBC were cultured in the presence of transforming growth factor-beta and soluble HB-EGF, they formed long tubules and few branches, similar to the structures observed in proHB-EGF-transfected UBC. These cells exhibited apical-basolateral polarization and expression of the water channel aquaporin-2. These findings indicate that soluble HB-EGF and proHB-EGF induce branching tubulogenesis in UBC in different ways. Juxtacrine activation by proHB-EGF or the synergic action of soluble HB-EGF with transforming growth factor-beta is important for well balanced morphogenesis of the collecting duct system.

Breyer MD, Harris RC. Cyclooxygenase 2 and the kidney. Curr Opin Nephrol Hypertens (2001) 10:89-98
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Cyclooxygenase metabolizes arachidonic acid to a family of bioactive fatty acids designated prostaglandins. Two isoforms of cyclooxygenase exist, designated COX1 and COX2. These isoforms are expressed in distinct but important areas of the kidney. COX1 predominates in vascular smooth muscle and collecting ducts, whereas COX2 predominates in the macula densa and nearby cells in the cortical thick ascending limb. COX2 is also highly expressed in medullary interstitial cells. Whereas COX1 expression does not exhibit dynamic regulation, COX2 expression is subject to regulation by several environmental conditions, including salt intake, water intake, medullary tonicity, growth factors, cytokines, and adrenal steroids. Recently, COX2-selective non-steroidal anti-inflammatory drugs have become widely available. Many of the renal effects of non-selective non-steroidal anti-inflammatory drugs (including sodium retention, decreased glomerular filtration rate, and effects on renin-angiotensin levels) appear to be mediated by the inhibition of COX2 rather than COX1. Therefore, in contrast to the gastrointestinal-sparing effects of COX2-selective non-steroidal anti-inflammatory drugs, when considering the kidney, the same caution must be applied when using COX2-selective inhibitors as has been used with traditional non-selective non-steroidal anti-inflammatory drugs.

Cheng HF, Wang JL, Zhang MZ, Wang SW, McKanna JA, Harris RC. Genetic deletion of COX-2 prevents increased renin expression in response to ACE inhibition. Am J Physiol Renal Physiol (2001) 280:F449-56
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Cyclooxygenase-2 (COX-2) is expressed in macula densa (MD) and surrounding cortical thick ascending limb of the loop of Henle (cTALH) and is involved in regulation of renin production. We and others have previously found that selective COX-2 inhibitors can inhibit renal renin production (Cheng HF, Wang JL, Zhang MZ, Miyazaki Y, Ichikawa I, McKanna JA, and Harris RC. J Clin Invest 103: 953-961, 1999; Harding P, Sigmon DH, Alfie ME, Huang PL, Fishman MC, Beierwaltes WH, and Carretero OA. Hypertension 29: 297-302, 1997; Traynor TR, Smart A, Briggs JP, and Schnermann J. Am J Physiol Renal Physiol 277: F706-F710, 1999; Wang JL, Cheng HF, and Harris RC. Hypertension 34: 96-101, 1999). In the present studies, we utilized mice with genetic deletions of the COX-2 gene in order to investigate further the potential role of COX-2 in mediation of the renin-angiotensin system (RAS). Age-matched wild-type (+/+), heterozygotes (+/-), and homozygous null mice (-/-) were administered the angiotensin-converting enzyme inhibitor (ACEI), captopril, for 7 days. ACEI failed to significantly increase plasma renin activity, renal renin mRNA expression, and renal renin activity in (-/-) mice. ACEI increased the number of cells expressing immunoreactive renin in the (+/+) mice both by inducing more juxtaglomerular cells to express immunoreactive renin and by recruiting additional renin-expressing cells in the more proximal afferent arteriole. In contrast, there was minimal recruitment of renin-expressing cells in the more proximal afferent arteriole of the -/mice. In summary, these results indicate that ACEI-mediated increases in renal renin production were defective in COX-2 knockout (K/O) mice and provide further indication that MD COX-2 is an important mediator of the renin-angiotensin system.

Harris RC. Cyclooxygenase-2 in the kidney. J Am Soc Nephrol (2000) 11:2387-94
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Harris RC. Macula densa signalling--a potential role of cyclooxygenase-2 (COX-2)? Nephrol Dial Transplant (2000) 15:1504-6
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Cheng HF, Wang JL, Zhang MZ, McKanna JA, Harris RC. Role of p38 in the regulation of renal cortical cyclooxygenase-2 expression by extracellular chloride. J Clin Invest (2000) 106:681-8
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We have previously shown that in renal cortex, COX-2 expression is localized to macula densa and surrounding cortical thick ascending limb of Henle (cTALH). Dietary salt restriction increases local expression of COX-2, which mediates renin production and secretion. Given that decreased luminal chloride [Cl(-)] at the level of the macula densa increases renin production and secretion, we investigated the role of extracellular ion concentration on COX-2 expression. Quiescent rabbit cTALH cells were incubated in a physiological salt solution containing high or low levels of NaCl. Immunoreactive COX-2 expression increased significantly in the low NaCl solution. COX-2 expression also increased after administration of the Na(+)/K(+)/2Cl(-) cotransport inhibitor, bumetanide. Selective substitution of chloride led to increased COX-2 expression, whereas selective substitution of sodium had no effect. The p38 MAP kinase inhibitor PD169316 decreased low NaCl-induced COX-2 expression. Low-salt or low-chloride medium induced cultured cTALH to accumulate >/= 3-fold higher levels of pp38, the activated (phosphorylated) form of p38; low-salt medium also increased pJNK and pERK levels. Feeding rats a low-salt diet for 14 days induced a significant increase in renal cortical pp38 expression, predominantly in the macula densa and cTALH. These results suggest that reduced extracellular chloride leads to increased COX-2 expression, which may be mediated by activation of a p38-dependent signaling pathway.

Harris RC. EGF receptor activation by G-protein coupled receptors. Kidney Int (2000) 58:898-9
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Cheng HF, Wang JL, Zhang MZ, McKanna JA, Harris RC. Nitric oxide regulates renal cortical cyclooxygenase-2 expression. Am J Physiol Renal Physiol (2000) 279:F122-9
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We have previously shown that cyclooxygenase-2 (COX-2) is localized to the cortical thick ascending limb of the loop of Henle (cTALH)/macula densa of the rat kidney, and expression increases in response to low-salt diet and/or angiotensin-converting enzyme (ACE) inhibition. Because of the localization of neuronal nitric oxide synthase (nNOS) to macula densa and surrounding cTALH, the present study investigated the role of nitric oxide (NO) in the regulation of COX-2 expression. For in vivo studies, rats were fed a normal diet, low-salt diet or low-salt diet combined with the ACE inhibitor captopril. In each group, one-half of them were treated with the nNOS inhibitors 7-nitroinidazole (7-NI) or S-methyl-thiocitrulline. Both of these NOS inhibitors inhibited increases in COX-2 mRNA and immunoreactive protein in response to low salt and low salt+captopril. For in vitro studies, COX-2 expression was studied in primary cultures of rabbit cTALH cells immunodisssected with Tamm-Horsfall antibody. Basal COX-2 immunoreactivity expression was stimulated by S-nitroso-N-acetyl-penicillamine (SNAP), an NO donor, and intracellular cGMP concentration. The cultured cells expressed immunoreactive nNOS, and 7-NI inhibited basal COX-2 immunoreactivity expression, which could be partially overcome by cGMP. In summary, these studies indicate that NO is a mediator of increased renal cortical COX-2 expression seen in volume depletion and suggest important interactions between the NO and COX-2 systems in the regulation of arteriolar tone and the renin-angiotensin system by the macula densa.

Wang JL, Cheng HF, Shappell S, Harris RC. A selective cyclooxygenase-2 inhibitor decreases proteinuria and retards progressive renal injury in rats. Kidney Int (2000) 57:2334-42
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BACKGROUND: We have previously shown that cyclooxygenase-2 (COX-2) expression is low in the renal cortex of adult rats, but is increased in macula densa/cortical thick ascending limb and in glomerular podocytes after subtotal renal ablation. METHODS: To evaluate the functional consequences of this increased COX-2 expression, male rats were subjected to subtotal renal ablation and divided into four groups: (1) treatment with the selective COX-2 inhibitor SC58236, (2) treatment with vehicle, (3) treatment with the angiotensin-converting enzyme inhibitor enalapril, and (4) treatment with enalapril + SC58236. The administration of drugs was begun on the third day after ablation and continued for 6 to 10 weeks. RESULTS: Within one week after ablation, vehicle-treated rats developed hypertension. Although enalapril led to significant reductions in blood pressure, either alone or in combination with the COX-2 inhibitor, SC58236 alone did not significantly alter ablation-induced hypertension. However, the SC58236-treated animals exhibited levels of proteinuria at six weeks after ablation that were comparable to those seen with enalapril (vehicle, 47 +/4; enalapril, 27 +/2; SC58236, 30 +/2 mg/day; N = 7, P < 0.01, each group compared with vehicle), and continued SC58236 treatment led to persistent reductions in proteinuria at 10 weeks after renal ablation (vehicle, 77 +/4; SC58236, 50 +/4 mg/day; N = 6, P < 0. 01). SC58236 treatment also significantly reduced the percentage of glomeruli exhibiting segmental or global sclerosis at 10 weeks (32.6 +/7.8% vs. 10.9 +/2.8%, N = 6, P < 0.03). Furthermore, SC58236 treatment partially inhibited increases in transforming growth factor-beta1 mRNA expression and increases in collagen III and collagen IV mRNA expression. CONCLUSIONS: These studies indicate that chro

Chen JK, Capdevila J, Harris RC. Overexpression of C-terminal Src kinase blocks 14, 15-epoxyeicosatrienoic acid-induced tyrosine phosphorylation and mitogenesis. J Biol Chem (2000) 275:13789-92
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We have previously reported that 14,15-epoxyeicosatrienoic acid (14, 15-EET) is a potent mitogen for the renal epithelial cell line, LLCPKcl4. This mitogenic effect is dependent upon activation of a protein-tyrosine kinase cascade that results in activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase. Because of suggestive evidence that 14,15-EET also activated Src in these cells, we stably transfected LLCPKcl4 with an expression construct of the C-terminal Src kinase (CSK), which inhibits Src family kinase activity. In vitro Src kinase activity assays confirmed that in empty vector-transfected cells (Vector cells), 14, 15-EET increased Src kinase activity, while in clones overexpressing CSK mRNA and immunoreactive protein (CSK cells), 14,15-EET-induced activation of Src was almost completely blocked (94% inhibition). Of interest, epidermal growth factor (EGF) and fetal bovine serum (FBS) also increased Src activity in Vector cells, but not in CSK cells, further confirming the ability of CSK overexpression to prevent Src activation. CSK cells failed to increase [(3)H]thymidine incorporation in response to exogenous 14,15-EET. In contrast, both EGF and FBS significantly increased [(3)H]thymidine incorporation in CSK cells. Immunoprecipitation with anti-phosphotyrosine antibodies and immunoblotting with an antibody against extracellular signal-regulated kinase (ERK) indicated that in CSK cells, 14,15-EET failed to activate ERK1 and ERK2; however, EGFand FBS-induced activation of ERKs was not different from that seen in Vector cells. In Vector cells, the 14,15-EET-stimulated tyrosine phosphorylation of ERKs was blocked by pretreatment with 1 microm PP2, a selective inhibitor of Src kinases. The present study demonstrates that 14, 15-EET exerts its mitogenic effects predom

Gibbs SR, Goins RA, Belvin EL, Dimari SJ, Merriam AP, Bowling-Brown S, Harris RC, Haralson MA. Characterization of the collagen phenotype of rabbit proximal tubule cells in culture. Connect Tissue Res (1999) 40:173-88
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Studies were performed to characterize the collagen phenotype of cultured rabbit proximal tubule (RPT) epithelial cells grown on plastic and on the reconstituted basement membrane preparation, Matrigel. When grown on a plastic substratum, RPT cells display a cobblestone appearance characteristic of glomerular epithelial cells. While initially forming an interlocking network of cells after subculture on Matrigel, this pattern of culture morphology rapidly develops into one characterized by isolated, organized groups of cells. Notwithstanding the effects of Matrigel on culture morphology, total cellular proliferation was reduced only 25% when RPT cells were grown on this substrate. Greater than 90% of the collagen synthesized by RPT cells grown on plastic was secreted into the culture medium. Qualitative analysis by SDS-PAGE revealed components exhibiting electrophoretic mobilities corresponding to the chains present in type IV and type I collagens. Quantitative analysis by CM-Trisacryl chromatography established that approximately 2/3 of the total collagen synthesized by RPT cells grown on plastic was type IV and approximately 1/3 type I. Quantitative analysis of the collagens produced by RPT cells grown on Matrigel again indicated the synthesis of only type IV and type I molecules but in a slightly more equal ratio of both collagen types and in the ratio of secreted to cell-associated molecules. However, the total amount of collagen synthesized by RPT cells grown on Matrigel was reduced to approximately 1% of the level synthesized by the cells grown on plastic. On plastic, approximately 3/4 of the type I collagen produced was recovered as the type I homotrimer, but on Matrigel type I homotrimers represented only approximately 55% of the total type I collagen synthesized. On Matrigel, the majori

Harris RC, Cheng H, Wang J, Zhang M, McKanna JA. Interactions of the renin-angiotensin system and neuronal nitric oxide synthase in regulation of cyclooxygenase-2 in the macula densa. Acta Physiol Scand (2000) 168:47-51
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Cyclooxygenase-2 (COX-2) expression in rat kidney is localized to the macula densa and the immediately proximal cTALH and increases after salt restriction. Either ACE inhibitors or AT1 receptor blockers increase COX-2 expression in both control and salt-restricted animals, suggesting that the RAS activation feedback inhibits renal cortical COX-2 expression. To determine whether increased COX-2 expression in response to ACE inhibition mediated increases in renin production, rats were treated with Captopril for 1 week with or without the specific COX-2 inhibitor, SC58236. Plasma renin activity increased significantly in the Captopril group. This increase was partially reversed by simultaneous treatment with SC58236. Kidney renin activity also increased in the Captopril group compared with control, which was also significantly inhibited by SC58236 treatment. Because of the localization of bNOS to MD and surrounding cTALH, the current study investigated the role of NO in the regulation of COX-2 expression. Rats were fed a normal diet, low salt diet or low salt diet combined with captopril and half of them were treated with the neuronal NOS inhibitor, 7-NI, and half with vehicle. After 7 days, mRNA was extracted and the microsome proteins purified from renal cortex. COX-2 mRNA expression was measured by Northern-blot and normalized with GAPDH. 7-NI treatment decreased COX-2 mRNA and immunoreactive COX-2 expression in each group. In summary, these studies indicate that COX-2 from macula densa/cTALH is a regulator of renin production and release. Angiotensin II may be a negative regulator of cTALH/macula densa COX-2 expression, and NO may mediate increased renal cortical COX-2 expression seen in volume depletion. These studies suggest important interactions between the NO and COX-2 systems in the regu

Haralson MA, DiMari SJ, Hoover RL, Harris RC. Effects of epidermal growth factor on collagen expression by rat kidney mesangial cells in culture. Matrix Biol (2000) 19:47-59
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Increased collagen production by mesangial cells plays a key role in the development and progression of glomerular sclerosis. These changes reflect in part the impact of growth factors on mesangial cells. Since mesangial cells possess receptors for epidermal growth factor (EGF) and since previous studies have documented that EGF affects collagen synthesis in other cell types, we have examined the effects of EGF on collagen biosynthesis by rat kidney mesangial (RKM) cells in culture. Exposure for 24 h to EGF did not substantially affect the growth rate of RKM cells. While the types of collagen produced by RKM cells (types I, III, IV and V) were unaltered by exposure to EGF, total collagen production was reduced ( approximately 50%). This decrease in collagen expression was not uniform for each collagen type. Type I collagen production was inhibited by approximately 50%, both type III and type IV expression were each reduced by approximately 30%, but type V collagen production was suppressed by only approximately 15%. The reduction in type I collagen synthesis was accounted for mainly by a decrease in type I homotrimer production. Since type I molecules represent approximately 95% of the total collagen produced, the decrease in overall collagen expression reflects a specific suppression by EGF on type I homotrimer production in mesangial cells. As EGF exposure resulted in a decrease in collagen production, these results suggest that the increases in synthesis and deposition of collagen observed in several glomerular diseases likely do not reflect the short-term effects of EGF on mesangial cells. Rather, these findings suggest the possibility that EGF or EGF-like growth factors may ameliorate the effects of other soluble factors that cause enhanced matrix production and deposition in renal disease

Capdevila JH, Falck JR, Harris RC. Cytochrome P450 and arachidonic acid bioactivation. Molecular and functional properties of the arachidonate monooxygenase. J Lipid Res (2000) 41:163-81
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The demonstration of in vivo arachidonic acid epoxidation and omega-hydroxylation established the cytochrome P450 epoxygenase and omega/omega-1 hydroxylase as formal metabolic pathways and as members of the arachidonate metabolic cascade. The characterization of the potent biological activities associated with several of the cytochrome P450-derived eicosanoids suggested new and important functional roles for these enzymes in cellular, organ, and body physiology, including the control of vascular reactivity and systemic blood pressures. Past and current advances in cytochrome P450 biochemistry and molecular biology facilitate the characterization of cytochrome P450 isoforms responsible for tissue/organ specific arachidonic acid epoxidation and omega/omega-1 hydroxylation, and thus, the analysis of cDNA and/or gene specific functional phenotypes. The combined application of physiological, biochemical, molecular, and genetic approaches is beginning to provide new insights into the physiological and/or pathophysiological significance of these enzymes, their endogenous substrates, and products.

Smoyer WE, Ransom R, Harris RC, Welsh MJ, Lutsch G, Benndorf R. Ischemic acute renal failure induces differential expression of small heat shock proteins. J Am Soc Nephrol (2000) 11:211-21
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AlphaB-crystallin and heat shock protein (hsp) 25 are structurally and functionally related small stress proteins induced by a variety of insults, including heat and ischemia. Cytoprotection by these two hsp is thought to result from molecular chaperoning and/or cytoskeletal stabilization. Because renal ischemia is characterized by disruption of the renal tubular cell actin cytoskeleton, this study was conducted to determine the localization and quantify the expression and phosphorylation of both hsp in renal cortex, isolated glomeruli, outer medulla, and inner medulla of rats after bilateral renal ischemia. Sham-operated kidneys had similarly small amounts of hsp25 and alphaB-crystallin in cortex and glomeruli, with substantially greater amounts of alphaB-crystallin versus hsp25 in outer and inner medulla. Ischemia resulted in significantly increased hsp25 (and hsp70i) but variable alphaB-crystallin levels in cortex and outer medulla, and progressively decreased glomerular hsp25 phosphorylation. In sham-operated kidneys, hsp25 localized to glomeruli, vessels, and collecting ducts, with alphaB-crystallin primarily in medullary thin limbs and collecting ducts. After ischemia, hsp25 accumulated in proximal tubules in cortex and outer medulla, while alphaB-crystallin labeling became nonhomogeneous in outer medulla, and increased in Bowman's capsule. It is concluded that: (1) There is striking differential expression of hsp25 and alphaB-crystallin in various renal compartments; and (2) Renal ischemia results in differential accumulation of hsp25 and alphaB-crystallin, with hsp25 part of a generalized stress response in renal proximal tubular cells, which may play a role in recovery from ischemia-induced actin filament disruption.

Komhoff M, Wang JL, Cheng HF, Langenbach R, McKanna JA, Harris RC, Breyer MD. Cyclooxygenase-2-selective inhibitors impair glomerulogenesis and renal cortical development. Kidney Int (2000) 57:414-22
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BACKGROUND: Antenatal exposure to nonsteroidal anti-inflammatory drugs (NSAIDs) has been associated with renal dysgenesis in humans. METHODS: These studies characterized cyclooxygenase-2 (COX-2) versus COX-1-selective inhibition on nephrogenesis in the rodent using histomorphometry, immunohistology, and in situ hybridization. RESULTS: Administration of a COX-2-selective inhibitor (SC58236), started during pregnancy until weaning, significantly impaired development of the renal cortex and reduced glomerular diameter in both mice and rats. An identical phenotype was demonstrated in COX-2 -/mice. In contrast to its effects on the developing kidney, a COX-2 inhibitor had no effect on glomerular volume in adult mice. This effect was specific for COX-2 because maternal administration of a COX-1-selective inhibitor (SC58560) did not affect renal development despite significantly inhibiting gastric mucosal prostaglandin E2 (PGE2) synthesis in pups. The expression of COX-2 immunoreactivity peaked in the first postnatal week and was localized to S-shaped bodies and the macula densa in the cortex. Treatment with a COX-2 inhibitor during this period (from postnatal day 0 to day 21) severely reduced glomerular diameter, whereas treatment limited to pregnancy did not affect glomerular size. CONCLUSION: These data demonstrate an important role for COX-2 activity in nephrogenesis in the rodent, and define a specific time period of susceptibility to these effects.

Harris RC. Potential mechanisms and physiologic actions of intracellular angiotensin II. Am J Med Sci (1999) 318:374-9
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As with other G protein-coupled receptors, type I angiotensin II receptors (AT1R) are plasma membrane-associated receptors that bind an extracellular agonist and initiate signal transduction pathways that mediate the physiologic actions elicited by the peptide. After agonist binding, AT1R undergo endocytosis and, after intracellular ligand dissociation, the receptors recycle to the plasma membrane. There are data from a variety of cell types suggesting that internalization of AT1R may be involved in activating a subset of angiotensin II-mediated signaling pathways. The evidence for active cytoplasmic and nuclear angiotensin II receptors is also reviewed. These studies suggest that internalization of peptide and receptor may be a necessary step for the transmission of the full range of cellular responses produced by angiotensin II.

Zhang MZ, Harris RC, McKanna JA. Regulation of cyclooxygenase-2 (COX-2) in rat renal cortex by adrenal glucocorticoids and mineralocorticoids. Proc Natl Acad Sci U S A (1999) 96:15280-5
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Production of prostaglandins involved in renal salt and water homeostasis is modulated by regulated expression of the inducible form of cyclooxygenase-2 (COX-2) at restricted sites in the rat renal cortex. Because inflammatory COX-2 is suppressed by glucocorticoids, and prostaglandin levels in the kidney are sensitive to steroids, the sensitivity of COX expression to adrenalectomy (ADX) was investigated. By 2 weeks after ADX in mature rats, cortical COX-2 immunoreactivity increased 10-fold in the cortical thick ascending limb and macula densa. The constitutive isoform, COX-1, was unchanged. The magnitude of the changes and specificity of COX-2 immunoreactivity were validated by in situ hybridization histochemistry of COX-2 mRNA and Western blot analysis. Increased COX-2 activity (>5-fold) was documented by using a specific COX-2 inhibitor. The COX-2 up-regulation in ADX rats was reversed by replacement therapy with either corticosterone or deoxycorticosterone acetate. In normal rats, inhibition of glucocorticoid receptors with RU486 or mineralocorticoid receptors with spironolactone caused up-regulation of renal cortical COX-2. These results indicate that COX-2 expression in situ is tonically inhibited by adrenal steroids, and COX-2 is regulated by mineralocorticoids as well as glucocorticoids.

Becker BN, Kondo S, Chen JK, Harris RC. Tyrosine kinase inhibition affects type 1 angiotensin II receptor internalization. J Recept Signal Transduct Res (1999) 19:975-93
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Growth factor receptors activate tyrosine kinases and undergo endocytosis. Recent data suggest that tyrosine kinase inhibition can affect growth factor receptor internalization. The type 1 angiotensin II receptor (AT1R) which is a G-protein-coupled receptor, also activates tyrosine kinases and undergoes endocytosis. Thus, we examined whether tyrosine kinase inhibition affected AT1R internalization. To verify protein tyrosine phosphorylation, both LLCPKCl4 cells expressing rabbit AT1R (LLCPKAT1R) and cultured rat mesangial cells (MSC) were treated with angiotensin II (Ang II) [1-100 nM] then solubilized and immunoprecipitated with antiphosphotyrosine antisera. Immunoblots of these samples demonstrated that Ang II stimulated protein tyrosine phosphorylation in both cell types. Losartan [1 microM], an AT1R antagonist, inhibited Ang II-stimulated protein tyrosine phosphorylation. LLCPKAT1R cells displayed specific 125I-Ang II binding at apical (AP) and basolateral (BL) membranes, and both AP and BL AT1R activated tyrosine phosphorylation. LLCPKAT1R cells, incubated with genistein (Gen) [200 microM] or tyrphostin B-48 (TB-48) [50 microM], were assayed for acid-resistant specific 125I-Ang II binding, a measure of Ang II internalization. Both Gen (n = 7) and TB-48 (n = 3) inhibited AP 125I-Ang II internalization (80+/-7% inhibition; p<0.025 vs. control). Neither compound affected BL internalization. TB-1, a non-tyrosine kinase-inhibiting tyrphostin, did not affect AP 125I-Ang II endocytosis (n = 3), suggesting that the TB-48 effect was specific for tyrosine kinase inhibition. Incubating MSC with Gen (n = 5) or herbimycin A [150 ng/ml] (n = 4) also inhibited MSC 125I-Ang II internalization (82+/-11% inhibition; p<0.005 vs. control). Thus, tyrosine kinase inhibition prevented Ang II internalization in M

Harris RC. Molecular basis of injury and progression in focal glomerulosclerosis. Nephron (1999) 82:289-99
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Wang JL, Cheng HF, Harris RC. Cyclooxygenase-2 inhibition decreases renin content and lowers blood pressure in a model of renovascular hypertension. Hypertension (1999) 34:96-101
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It has been proposed that the macula densa participates in the regulation of increased renin expression in renovascular hypertension (RVH) and that prostaglandins may be among the mediators of macula densa function. We have previously shown that in renal cortex, cyclooxygenase-2 (COX-2) expression is localized to the macula densa and surrounding cortical thick ascending limb and increases in high-renin states, such as salt restriction and angiotensin-converting enzyme inhibition. In the present studies, we examined the effect of the selective COX-2 inhibitor SC58236 on plasma renin activity (PRA) and renal renin expression in RVH in rats. The aorta was coarcted between right and left renal arteries, and animals received either SC58236 or vehicle for 1 week. At day 8, vehicle-treated coarcted rats were hypertensive (mean carotid arterial blood pressure: 138+/-3 versus 87+/-2 mm Hg in sham-operated controls; n=9 to 11; P<0.001) and exhibited a disparity of kidney size (ratio left/right kidney: 0.78+/-0.04 versus 1.02+/-0.02; n=9 to 10; P<0.001). PRA increased significantly (84.6+/-6.5 versus 9.0+/-1.4 ng angiotensin I [Ang I] per milliliter per hour; n=8 to 9; P<0.01). In the coarcted rats, neither renin mRNA expression nor renin activity of the right kidney was altered (renin/GAPDH mRNA: 1.12+/-0.05-fold levels in control rats; n=6; P=NS; renin activity: 23.4+/-1.8 versus 27.1+/-3.4 ng Ang I per hour per milligram protein; n=8 to 9; P=NS). However, the renin mRNA of the left kidney increased to 3.0+/-0.6-fold of control (n=6), and the renin activity increased to 189.0+/-28.6 ng Ang I per hour per milligram protein (n=8; P<0.01). Expression of COX-2 mRNA and immunoreactive protein increased in the affected left kidney but was not different from control in the unaffected right kidney. SC58236 trea

Cheng HF, Wang JL, Zhang MZ, Miyazaki Y, Ichikawa I, McKanna JA, Harris RC. Angiotensin II attenuates renal cortical cyclooxygenase-2 expression. J Clin Invest (1999) 103:953-61
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We have previously shown that in rat renal cortex, cyclooxygenase-2 (COX-2) expression is localized to cTALH cells in the region of the macula densa, and that dietary salt restriction increases COX-2 expression. Administration of the angiotensin converting inhibitor, captopril, further increased COX-2 mRNA and renal cortical COX-2 immunoreactivity, with the most pronounced expression in the macula densa. Administration of an AT1 receptor antagonist, losartan, also significantly increased cortical COX-2 mRNA expression and COX-2 immunoreactivity. Mutant mice homozygous for both Agtr1a and Agtr1b null mutations (Agtr1a-/-,Agtr1b-/-) demonstrated large increases in immunoreactive COX-2 expression inthe cTALH/macula densa. To determine whether increased COX-2expression in response to ACE inhibition mediated increases in renin production, rats were treated with captopril for one week with or without the specific COX-2 inhibitor, SC58236. Plasma renin activity increased significantly in the captropril group, and this increase was significantly inhibited by simultaneous treatment with SC58236. Thus, these studies indicated that angiotensin II inhibitors augment upregulation of renal cortical COX-2 in states of volume depletion, suggesting that negative feedback by the renin-angiotensin system modulates renal cortical COX-2 expression and that COX-2 is a mediator of increased renin production in response to inhibition of angiotension II production.

Yasuda T, Kondo S, Owada S, Ishida M, Harris RC. Integrins and the cytoskeleton: focal adhesion kinase and paxillin. Nephrol Dial Transplant (1999) 14 Suppl 1:58-60
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Chen JK, Wang DW, Falck JR, Capdevila J, Harris RC. Transfection of an active cytochrome P450 arachidonic acid epoxygenase indicates that 14,15-epoxyeicosatrienoic acid functions as an intracellular second messenger in response to epidermal growth factor. J Biol Chem (1999) 274:4764-9
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A common feature of most isolated cell systems is low or undetectable levels of bioactive cytochrome P450. We therefore developed stable transfectants of the renal epithelial cell line, LLCPKcl4, that expressed an active regioand enantioselective arachidonic acid (AA) epoxygenase. Site-specific mutagenesis was used to convert bacterial P450 BM-3 into an active regioand stereoselective 14S,15R-epoxygenase (F87V BM-3). In clones expressing F87V BM-3 (F87V BM-3 cells), exogenous AA induced significant 14S,15R-epoxyeicosatrienoic acid (EET) production (241. 82 ng/10(8) cells, >97% of total EETs), whereas no detectable EETs were seen in cells transfected with vector alone. In F87V BM-3 cells, AA stimulated [3H]thymidine incorporation and increased cell proliferation, which was blocked by the tyrosine kinase inhibitor, genistein, by the phosphatidylinositol 3 (PI-3) kinase inhibitors, wortmannin and LY294002, and by the mitogen-activated protein kinase kinase inhibitor, PD98059. AA also induced tyrosine phosphorylation of extracellular signal-regulated kinase (ERK) and PI-3 kinase that was inhibited by the cytochrome P450 BM-3 inhibitor, 17-ODYA. Epidermal growth factor (EGF) increased EET production in F87V BM-3 cells, which was completely abolished by pretreatment with either 17-ODYA or the phospholipase A2 (PLA2) inhibitor, quinacrine. Compared with vector-transfected cells, F87 BM-3 transfected cells demonstrated marked increases in both the extent and sensitivity of DNA synthesis in response to EGF. These changes occurred in the absence of significant differences in EGF receptor expression. As seen with exogenous AA, EGF increased ERK tyrosine phosphorylation to a significantly greater extent in F87V BM-3 cells than in vector-transfected cells. Furthermore, in these control cells, neither 17-ODY

Takemura T, Hino S, Murata Y, Yanagida H, Okada M, Yoshioka K, Harris RC. Coexpression of CD9 augments the ability of membrane-bound heparin-binding epidermal growth factor-like growth factor (proHB-EGF) to preserve renal epithelial cell viability. Kidney Int (1999) 55:71-81
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BACKGROUND: Transfection of renal epithelial cells (NRK 52E) with membrane-associated heparin-binding epidermal growth factor-like growth factor (proHB-EGF) increased renal epithelial cell survival by promoting cell-cell and cell-extracellular matrix interactions. ProHB-EGF has been shown to form a complex in the plasma membrane with the tetraspanin CD9, an interaction that significantly increases the effectiveness of proHB-EGF as a juxtacrine mitogenic agent. METHODS: We examined whether the coexpression of proHB-EGF and CD9 would increase renal epithelial cell survival. CD9 was stably transfected into NRK 52E cells, either alone (NRKCD9) or together with proHB-EGF (NRKboth). RESULTS: Juxtacrine mitogenic activity of NRKCD9 was no different than in cells transfected with vector alone (NRKvector), but was increased by NRKboth; juxtacrine mitogenic activity by NRKboth was twofold greater than when proHB-EGF was transfected alone (NRKproHB-EGF). When grown in 10% fetal calf serum, growth rates were similar among all transfectants. However, in 1% fetal calf serum, NRKproHB-EGF grew 50% faster than NRKvector or NRKCD9, and NRKboth grew 20% to 50% faster than NRKproHB-EGF at one, two, and three days of culture. NRKproHB-EGF attachment to plastic substratum at one, two, and three hours was 250% greater than that of NRKvector, and NRKboth was 20% to 30% greater than that of NRKproHB-EGF. Coating plates with either poly 2-hydroxyethyl methacrylate or the GRGDTP peptide prevented normal cell-extracellular matrix attachment, and NRKvector or NRKCD9 failed to attach or form cell-cell attachments. NRKproHB-EGF exhibited 300% and NRKboth exhibited 600% greater cell viability under these conditions. Expression of type I and type III collagen mRNA was enhanced similarly in NRKproHB-EGF and NRKboth, but the ex

Miyazaki Y, Tsuchida S, Nishimura H, Pope JC 4th, Harris RC, McKanna JM, Inagami T, Hogan BL, Fogo A, Ichikawa I. Angiotensin induces the urinary peristaltic machinery during the perinatal period. J Clin Invest (1998) 102:1489-97
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The embryonic development of mammalian kidneys is completed during the perinatal period with a dramatic increase in urine production, as the burden of eliminating nitrogenous metabolic waste shifts from the placenta to the kidney. This urine is normally removed by peristaltic contraction of the renal pelvis, a smooth muscle structure unique to placental mammals. Mutant mice completely lacking angiotensin type 1 receptor genes do not develop a renal pelvis, resulting in the buildup of urine and progressive kidney damage. In mutants the ureteral smooth muscle layer is hypoplastic and lacks peristaltic movements. We show that angiotensin can induce the ureteral smooth muscles in organ cultures of wild-type, but not mutant, ureteral tissues and that, in wild-type mice, expression of both renal angiotensin and the receptor are transiently upregulated at the renal outlet at birth. These results reveal a new role for angiotensin in the unique cellular adaptations of the mammalian kidney to the physiological stresses of postnatal life.

Chen JK, Falck JR, Reddy KM, Capdevila J, Harris RC. Epoxyeicosatrienoic acids and their sulfonimide derivatives stimulate tyrosine phosphorylation and induce mitogenesis in renal epithelial cells. J Biol Chem (1998) 273:29254-61
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In our present studies utilizing a well characterized proximal tubule cell line, LLCPKcl4, we determined that all four EET regioisomers (5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET) stimulated [3H]thymidine incorporation, with 14,15-EET being the most potent. In contrast, no mitogenic effects were seen with arachidonic acid, other cP450 arachidonate metabolites (12R-hydroxyeicosatetraenoic acid (12R-HETE), 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), or 20-HETE), or lipoxygenase metabolites (5S-HETE, leukotriene B4, or lipoxin A4). We found that their metabolically more stable sulfonimide (SI) analogs (11,12-EET-SI and 14,15-EET-SI) were also potent mitogens. In addition 14,15-EET-SI also increased cell proliferation as well as expression of both c-fos and egr-1 mRNA. The protein kinase C and A inhibitors, H-7 and H-8, or the cyclooxygenase inhibitor, indomethacin, had no effect upon 14, 15-EET-induced [3H]thymidine incorporation, but the selective tyrosine kinase inhibitor, genistein, significantly inhibited it. Immunoprecipitation and immunoblotting demonstrated increased tyrosine phosphorylation of PI3-kinase and epidermal growth factor receptor (EGFR) within 1 min of EET administration. EETs also stimulated association of PI3-kinase with EGFR. PI3-kinase inhibitors, wortmannin and LY 294002, markedly inhibited 14, 15-EET-SI-stimulated [3H]thymidine incorporation. In addition, 14, 15-EET-SI administration stimulated tyrosine phosphorylation of src homologous and collagen-like protein (SHC) and association of SHC with both growth factor receptor-binding protein (GRB2) and EGFR. Mitogen-activated protein kinase was also activated within 5 min. Pretreatment of the cells with the mitogen-activated protein kinase kinase inhibitor, PD98059, inhibited the 14,15-EET-SI-stimulated [3H]thymi

Wang JL, Cheng HF, Zhang MZ, McKanna JA, Harris RC. Selective increase of cyclooxygenase-2 expression in a model of renal ablation. Am J Physiol (1998) 275:F613-22
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Previous studies have suggested a possible role for prostaglandins (PGs) in mediating alterations in nephron structure and function ensuing after renal ablation. Two isoforms of cyclooxygenase (COX) have been described: constitutive (COX-1) and inducible (COX-2). We examined expression of these isoforms following subtotal renal ablation (5/6 ablation, RA) in rats. In renal cortex, COX-2 mRNA and immunoreactive protein (IP) increased progressively compared with sham-operated littermates. In contrast, there were no significant changes in COX-1 mRNA expression. In normal kidney, cortical COX-1 IP was immunolocalized predominantly to mesangial cells and collecting tubules, whereas COX-2 IP was found in a subset of cortical thick ascending limb of Henle's loop (CTAL) cells in the region of the macula densa (MD). Following RA, significantly increased COX-2 IP was detected in the MD and surrounding CTAL cells. In addition, fainter immunoreactive COX-2 was detected in scattered visceral epithelial cells and mesangial cells of the glomerulus. Immunoblotting of isolated glomeruli demonstrated a selective increase of glomerular immunoreactive COX-2 expression following RA. No change of COX-1 expression was seen. To determine COX activity, isolated glomeruli were incubated with arachidonic acid and PGE2 measured by enzyme immunoassay (EIA). Compared with sham, glomeruli from 2 wk RA produced significantly more PGs. SC-58560, a selective COX-1 inhibitor, did not inhibit PG production in the remnant glomeruli at concentrations up to 10(-4) M, whereas SC-58236, a relatively selective COX-2 inhibitor, significantly inhibited PG production by RA glomeruli. In preliminary studies, to define mechanisms of altered expression of glomerular COX-2, rat mesangial cells were incubated with serum from sham or 2 wk RA. T

Harris RC, Wang JL, Cheng HF, Zhang MZ, McKanna JA. Prostaglandins in macula densa function. Kidney Int Suppl (1998) 67:S49-52
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Cyclooxygenase (COX)-2 mRNA and immunoreactive protein localize to the macula densa and adjacent cortical thick ascending limb in renal cortex, and chronic NaCl restriction increases expression of this enzyme. These findings suggest an integral role for eicosanoids generated by macula densa-associated COX-2 in mediating renin release. As selective inhibitors of COX-2 become available, it will be important to assess their effects on the renin-angiotensin system and glomerular hemodynamics.

McKanna JA, Zhang MZ, Wang JL, Cheng H, Harris RC. Constitutive expression of cyclooxygenase-2 in rat vas deferens. Am J Physiol (1998) 275:R227-33
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Prostaglandins, lipoid substances discovered in human semen as modulators of uterine muscle contractility, are known to play significant roles in virtually all mammalian organ systems, but their male reproductive functions are unclear. Cyclooxygenase, the rate-limiting enzyme in prostaglandin synthesis, occurs in two isoforms distinguished on the basis of constitutive (COX-1) or inducible (COX-2) expression patterns in mammalian tissues. However, in the adult rat male reproductive system, immunohistochemistry and Western and Northern analysis showed that COX-2 is the predominant isoform and is heavily localized to the epithelium of the distal vas deferens, where constitutive expression is manyfold greater than in any other organs of the body. COX-2 is not detected in the proximal one-half of the vas nor in the testis, epididymis, seminal vesicles, or prostate. Elimination of luminal sperm by vasectomy does not affect COX-2 levels, whereas castration severely depletes COX-2 and androgen replacement after castration restores COX-2, indicating that COX-2 expression in the vas is androgen dependent. Because the distal vas also comprises an extensive submucosal venous plexus connected to the penile corpora cavernosa, prostaglandins from the vas may play a role in erection.

Becker BN, Yasuda T, Kondo S, Vaikunth S, Homma T, Harris RC. Mechanical stretch/relaxation stimulates a cellular renin-angiotensin system in cultured rat mesangial cells. Exp Nephrol (1998) 6:57-66
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Angiotensin II (Ang II) may play a significant role mediating intraglomerular hypertension and glomerular sclerosis. Therefore, we investigated whether a model of pressure-induced stress, mechanical stretch/relaxation, affected the renin-angiotensin system (RAS) in cultured rat mesangial cells. Type 1 Ang II receptor (AT1R) expression was assessed by 125I-Ang II binding and quantitative reverse-transcription polymerase chain reaction. Stretch/relaxation increased steady-state AT1R mRNA levels as well as specific [125I]Ang II binding. Increased AT1R expression was associated with altered AT1R signaling. Ang II (100 nM) increased total phosphoinositide hydrolysis in control cells (186 +/25%, n = 6; p < 0.025 vs. no treatment). However, stretch/relaxation for 48 h further augmented AT1R-mediated PI hydrolysis (293 +/38%, n = 6; p < 0.025 vs. Ang II treatment alone). We examined other RAS components in mesangial cells subjected to stretch/relaxation. Angiotensinogen, determined by radioimmunoassay of Ang I generation in conditioned media, increased with stretch/relaxation, and reverse-transcription polymerase chain reaction demonstrated increased angiotensinogen gene expression in stretch/relaxation-treated cells. However, renin activity and angiotensin-converting-enzyme-like activity were unaffected by stretch/relaxation. Thus, mesangial cells maintain a local RAS similar to those described in other tissues, and AT1R expression and angiotensinogen production in this cellular RAS are increased by stretch/relaxation. It is likely that mesangial cells in vivo, exposed to variations in intraglomerular pressure, may regulate their responses via a local RAS.

Cheng HF, Wang JL, Vinson GP, Harris RC. Young SHR express increased type 1 angiotensin II receptors in renal proximal tubule. Am J Physiol (1998) 274:F10-7
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A potential role for the renin-angiotensin system (RAS) in the development and/or maintenance of hypertension in the genetic model of rat hypertension, spontaneously hypertensive rats (SHR), has been suggested by studies indicating that treatment of immature animals with angiotensin-converting enzyme (ACE) inhibitors prevents subsequent development of hypertension. Because young SHR also demonstrate RAS-dependent increased sodium retention, we examined proximal tubule type 1 angiotensin II receptor (AT1R) mRNA expression in young (4 wk) or adult (14 wk) SHR compared with age-matched Wistar-Kyoto (WKY) rats. Proximal tubules were isolated by Percoll gradient centrifugation, and AT1R mRNA expression was measured by quantitative reverse transcription-polymerase chain reaction (RT-PCR). At 14 wk, when SHR had established hypertension [mean arterial blood pressure (MAP) of SHR vs. WKY: 145 +/6 vs. 85 +/5 mmHg, n = 14-15], there were no differences in proximal tubule AT1R mRNA levels [SHR vs. WKY: 79 +/14 vs. 72 +/14 counts/min (cpm) per cpm mutant AT1R per cpm beta-actin x 10(-6), n = 6; not significant (NS)]. In contrast, in 4 wk SHR, at a time of minimal elevations in blood pressure (MAP: 70 +/8 vs. 63 +/3), SHR proximal tubule AT1R mRNA levels were 263 +/30% that of WKY (143 +/18 vs. 60 +/11 cpm per cpm of mutant AT1R per cpm beta-actin x 10(-6), n = 8; P < 0.005). We have recently shown that chronic ACE inhibition decreases proximal tubule AT1R expression and have also shown that chronic L-3,4-dihydroxyphenylalamine (L-DOPA) administration inhibits AT1R expression in adult Sprague-Dawley proximal tubule and cultured proximal tubule, and this inhibition is mediated via Gs-coupled DA1 receptors. When 3-wk-old animals were given L-DOPA or captopril for 1 wk, MAP was not altered (70 +/8 vs. 60 +/4

Zhang MZ, Wang JL, Cheng HF, Harris RC, McKanna JA. Cyclooxygenase-2 in rat nephron development. Am J Physiol (1997) 273:F994-1002
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The inducible second isoform of cyclooxygenase (COX-2) that mediates inflammation also is expressed at low levels in normal adult rat kidneys and is upregulated in response to noninflammatory stimuli (R. C. Harris, J. A. McKanna, Y. Akai, H. R. Jacobson, R. N. DuBois, and M. D. Breyer, J. Clin. Invest. 94: 2504-2510, 1994). Roles in morphogenesis are indicated by reported teratogenicity of COX inhibitors and renal dysgenesis in COX-2 knockout mice (J. E. Dinchuk, B. D. Car, R. J. Focht, J. J. Johnston, B. D. Jaffee, M. B. Covington, N. R. Contel, V. M. Eng, R. J. Collins, P. M. Czerniak, A. G. Stewart, and J. M. Trzaskos, Nature 378: 406-409, 1995; S. G. Morham, R. Lagenbach, C. D. Loftin, H. F. Tiano, N. Vouloumanos, J. C. Jennette, J. F. Mahler, K. D. Kluckman, A. Ledford, C. A. Lee, and O. Smithies. Cell 83: 473-482, 1995). Blots from developing rat kidneys demonstrated that COX-2 mRNA and immunoreactive protein were present in neonates, peaked in the 2nd and 3rd postnatal weeks and declined to adult levels by the 3rd month. Immunolocalization and in situ hybridization detected intense COX-2 immunoreactivity and mRNA in a subset of thick ascending limb epithelial cells near the macula densa in each developing nephron; after 2 wk the COX-2 gradually waned. These data demonstrate that COX-2 expression is subject to normal developmental regulation and can be sustained over extended periods; they also support the conclusion that metabolites of COX-2 play important roles in the differentiation and early functions of mammalian nephrons.

Takemura T, Kondo S, Homma T, Sakai M, Harris RC. The membrane-bound form of heparin-binding epidermal growth factor-like growth factor promotes survival of cultured renal epithelial cells. J Biol Chem (1997) 272:31036-42
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To understand whether expression of membrane-anchored heparin binding epidermal growth factor (proHB-EGF) is involved in renal epithelial cell survival, rat membrane-bound HB-EGF precursor was stably transfected into a renal epithelial cell line, NRK 52E cells (NRKproHB-EGF). When exposed to 10% fetal calf serum (FCS), there were no differences in growth rates among wild-type (WT), vector-transfected (NRKvector), and NRKproHB-EGF. However, when cells were grown in the presence of 1% FCS, the growth rate of NRKproHB-EGF was 65% faster. When confluent cell monolayers were exposed to H2O2 or etoposide, WT or NRKvector exhibited significant apoptotic bodies and DNA laddering; in contrast, NRKproHB-EGF were resistant to both stimuli, as indicated by increased cell viability and marked decrease of apoptotic bodies and DNA laddering. When plated at high density onto plastic dishes without FCS, WT and NRKvector formed few attachments, did not proliferate, and underwent apoptosis. By day 3, no cells survived. Addition of exogenous recombinant HB-EGF (10(-8) M) to WT or NRKvector increased cell survival by <10% and incubation with conditioned media of NRKproHB-EGF had no effect. In contrast, NRKproHB-EGF attached and formed epithelial colonies, although they did not proliferate. After 3 days, cell viability was 84% of the initial cell number plated, and no evidence of apoptosis was present. When plated in 10% FCS, NRKproHB-EGF attachment to plastic substratum at 1, 2, and 3 h was 250% greater than that of WT or NRKvector. Addition of exogenous recombinant human HB-EGF to WT or NRKvector increased attachment by <50%. When grown on poly(2-hydroxyethyl methacrylate) or in the presence of the integrin receptor-blocking peptide GRGDTP, neither WT nor NRKvector attached to the substratum or formed cell-cell at

Becker BN, Cheng HF, Harris RC. Apical ANG II-stimulated PLA2 activity and Na+ flux: a potential role for Ca2+-independent PLA2. Am J Physiol (1997) 273:F554-62
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Type 1 angiotensin II (ANG II) receptors (AT1R), which mediate proximal tubule (PT) salt and water reabsorption, undergo endocytosis and recycling. Prior studies in a PT-like model (LLC-PKcl4 cells expressing rabbit AT1R) (LLC-PK-AT1R cells) determined that quinacrine, a nonspecific phospholipase A2 (PLA2) inhibitor, and the haloenol lactone suicide substrate (HELSS), a Ca2+-independent PLA2 inhibitor, attenuated apical (AP) AT1R recycling. Further studies were undertaken to examine the association between AT1R endocytotic movement and PLA2 activity in this model. AP ANG II (100 nM) increased [3H]arachidonic acid ([3H]AA) release 4.4 +/0.38-fold in LLC-PK-AT1R cells cultured on permeable supports. Basolateral (BL) ANG II had no significant effect. Reversed-phase high-performance liquid chromatography confirmed that AP ANG II stimulated free [3H]AA release. Quinacrine, HELSS, and palmitoyl trifluoromethyl ketone, another Ca2+-independent PLA2 inhibitor, inhibited AP ANG II-stimulated [3H]AA release, as did inhibiting AP AT1R internalization with phenylarsine oxide. The role of HELSS-inhibitable AA release in ANG II-mediated 22Na flux was examined, given the effects of AT1R-mediated PLA2 activity on salt and water reabsorption. AP ANG II (100 nM) stimulated 22Na flux (AP--> BL), a response inhibited by HELSS. Thus, in this model, AP AT1R activated PLA2 with concomitant 22Na flux (AP --> BL), suggesting a link between AP AT1R endocytotic movement, AT1R-stimulated PLA2 activity, and 22Na flux in this model. The effects of HELSS suggest that Ca2+-independent PLA2 activity may be involved in this AP ANG II response.

Harris RC, Kondo S, Yasuda T. Mechanical stress-cell function relationships in renal cells. Exp Nephrol (1997) 5:263-70
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Becker BN, Kondo S, Cheng HF, Harris RC. Effect of glucose, pyruvate, and insulin on type 1 angiotensin II receptor expression in SV40-immortalized rabbit proximal tubule epithelial cells. Kidney Int (1997) 52:87-92
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Ambient glucose concentrations alter type 1 angiotensin II receptor (AT1R) expression in renal tissues. The direction of change in AT1R density may depend on the specific cell type and the capacity for that cell type to use glucose as an energy substrate. Given the effects of angiotensin II (Ang II) in proximal tubule epithelia, glucose-mediated fluctuations in AT1R expression could significantly alter tubular Na(+)-H+ exchange and volume reabsorption. To determine if glucose influenced AT1R expression in cultured proximal tubule epithelial cells, SV40-immortalized rabbit proximal tubule epithelial cells (RPTEC) were exposed to 25 mmol (hi-glc) or 5 mmol glucose-containing serum-free medium (lo-glc) for seven to nine days, with or without an alternative energy substrate, pyruvate. AT1R expression, assessed by quantitative reverse-transcription polymerase chain reaction and specific 125I-Ang II binding, decreased in lo-glc medium (% reduction AT1R mRNA expression: 52 +/8%; N = 6; P < 0.005 vs. hi-glc; % reduction specific 125I-Ang II binding: 48 +/12%; N = 12; P < 0.03 vs. hi-glc). AT1R mRNA expression and specific 125I-Ang II binding recovered to hi-glc levels following the addition of pyruvate [60 mmol] to lo-glc cells. To ascertain if a growth factor that increases glucose uptake in vivo also altered AT1R expression, RPTEC were cultured in hi-glc medium with or without exogenous insulin [100 nM]. Insulin addition increased AT1R mRNA expression and specific 125I-Ang II binding in a concentration-dependent manner. However, insulin (100 nM) addition to lo-glc cells did not significantly increase specific 125I-Ang II binding. These results suggest that AT1R expression in SV40-immortalized rabbit proximal tubule cells is significantly affected by the availability of energy substrate. Ultimately, c

Sakai M, Zhang M, Homma T, Garrick B, Abraham JA, McKanna JA, Harris RC. Production of heparin binding epidermal growth factor-like growth factor in the early phase of regeneration after acute renal injury. Isolation and localization of bioactive molecules. J Clin Invest (1997) 99:2128-38
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We have recently reported that heparin-binding epidermal growth factor-like growth factor (HB-EGF) mRNA is induced in the rat kidney after acute ischemic injury. The present studies were designed to investigate whether bioactive HB-EGF protein is also produced in response to renal injury induced by either ischemia/reperfusion or aminoglycosides. Heparin-binding proteins were purified from kidney homogenates by heparin affinity column chromatography using elution with a 0.2-2.0 M gradient of NaCl. A single peak of proteins that eluted at 1.0-1.2 M NaCl was detected in the postischemic kidney within 6 h of injury. This eluate fraction stimulated DNA synthesis in quiescent Balb/c3T3, RIE, and NRK-52E cell lines, all of which are responsive to the epidermal growth factor family of mitogenic proteins. The EGF receptor of A431 cells was also tyrosine phosphorylated by this eluate peak. Furthermore, immunoblotting with a polyclonal antibody against rat HB-EGF indicated that the eluate peak contained immunoreactive proteins of 22 and 29 kD mol wt, consistent with the reported sizes of the secreted form and membrane anchored form of HB-EGF, respectively. Immunohistochemical studies revealed that HB-EGF was produced predominantly in distal tubules in kidneys injured either by ischemia/reperfusion or aminoglycoside administration. We also found that during metanephric development immunoreactive HB-EGF was detected in the ureteric bud as early as E14.5 and persisted in structures arising from the ureteric bud throughout embryogenesis. These results suggest that in response to acute injury, HB-EGF is produced predominantly in distal tubules and that endogenous HB-EGF may be an important growth factor involved in renal epithelial cell repair, proliferation, and regeneration in the early stages of recovery af

Harris RC. Growth factors and cytokines in acute renal failure. Adv Ren Replace Ther (1997) 4:43-53
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The mammalian kidney is susceptible to injury by ischemia/reperfusion and toxins, and regeneration after injury is characterized by hyperplasia and recovery of the damaged epithelial cells that line the tubules. Locally produced growth factors may serve as mediators of nephrogenesis and differentiation during renal development and of renal regeneration after acute injury. In cultured cells, administration of one or a mixture of growth factors to quiescent cells will initiate progression through the cell cycle and cell division. In the adult kidney, cell division normally is very low, but will increase up to 10-fold after acute injury. In addition to proliferation after lethal injury, there also is cellular repair in cells that have undergone sublethal injury. Recent studies indicate that growth factors inhibit programmed cell death in response to acute injury. Growth factors also may initiate or promote protein and lipid biosynthesis and provide an intracellular milieu that promotes cellular repair. In addition to cellular repair, growth factors also may be involved in the re-establishment of cell-extracellular matrix and cell-cell integrity. Finally, growth factors may limit injury by decreasing the factors that induce damage. Increased local renal expression of growth factors in response to acute injury include heparin binding epidermal growth factor (HB-EGF), hepatocyte growth factor (HGF), insulin-like growth factor-I (IGF-I), transforming growth factor-beta, parathyroid hormone-related peptide, and acidic fibroblast growth factor. In a number of experimental models of acute renal injury, administration of exogenous growth factors has been shown to accelerate both structural and functional recovery. Specifically, EGF, IGF-1, and HGF all have been shown to be effective in this regard. These

Harris RC, Becker BN, Cheng HF. Acute and chronic mechanisms for regulating proximal tubule angiotensin II receptor expression. J Am Soc Nephrol (1997) 8:306-13
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Harris RC, Cheng HF. The intrarenal renin-angiotensin system: a paracrine system for the local control of renal function separate from the systemic axis. Exp Nephrol (1996) 4 Suppl 1:2-7
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The kidney serves an integral role in the systemic renin-angiotensin system (RAS) both as an effector and as a target organ. Increasing evidence also accumulates that local intrarenal RAS serve as important regulators of renal function. Evidence is reviewed concerning the expression and potential role of components of the RAS in glomeruli, proximal tubule and renal interstitium. In addition to regulating normal renal function, local renal RAS may be triggered in response to renal injury and may contribute to structural and functional abnormalities, including increased glomerulosclerosis and tubulointerstitial fibrosis.

Becker BN, Harris RC. A potential mechanism for proximal tubule angiotensin II-mediated sodium flux associated with receptor-mediated endocytosis and arachidonic acid release. Kidney Int Suppl (1996) 57:S66-72
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Angiotensin II (Ang II) receptors in the proximal nephron significantly affect renal salt and water reabsorption. The predominant tubular-epithelial cell Ang II receptor, type 1 Ang II receptors (AT1R), is a member of the superfamily of G-protein-coupled receptors. Tubular cell AT1R are unusual as they are polarized to apical (AP) and basolateral (BL) cell membranes. In a proximal tubule cell model that mimics the in vivo polar distribution of AT1R (LLC-PKC14 cells transfected with rabbit AT1R), we have determined that AP and BL AT1R display differential rates of endocytosis and recycling. In addition, AP AT1R recycling was selectively inhibited by quinacrine, a non-specific inhibitor of phospholipase A2 (PLA2) activity and HELSS, a mechanism-based inhibitor of calcium-independent PLA2 activity. PLA2 catalyzes the hydroysis of sn-2 fatty acyl bonds, liberating free fatty acids and lysophosphoslipids, and proximal tubule AT1R are known to couple to PLA2 as a signaling pathway. In this model, AP Ang II treatment selectively stimulated [3H]-arachidonic acid ([3H]-AA) release. Furthermore, inhibiting AP AT1R endocytosis+recycling with either phenylarsine oxide (PAO) or HELSS, decreased AP Ang II-stimulated [3H]-AA release. In other studies, Ang II internalization also has been linked to vectorial sodium (Na+) flux. Preliminary studies in our model also suggested that HELSS inhibited AP Ang II-stimulated Na+ flux (AP --> BL), raising the possibility that AP AT1R targeting in proximal tubular epithelium, the endocytic profile of these receptors, and AP AT1R-mediated arachidonic acid release combine to serve as additional contributors to Ang II-mediated Na+ flux in the nephron.

Yasuda T, Kondo S, Homma T, Harris RC. Regulation of extracellular matrix by mechanical stress in rat glomerular mesangial cells. J Clin Invest (1996) 98:1991-2000
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Increases in intraglomerular pressure are known to predispose to the development of glomerular sclerosis, which is characterized by accumulation of extracellular matrix within the glomerulus. Glomerular mesangial cells are exposed to pulsatile capillary pressures and are a potential target for mechanical stress. In the present studies, we subjected cultured rat mesangial cells to continuous cycles of stretching and relaxation (stretch/relaxation) and examined alterations in extracellular matrix gene expression. After 48 h of stretch/relaxation, immunofluorescent localization of matrix accumulation indicated increases in types I, III, and IV collagens, fibronectin, and laminin, with the greatest increases seen at the periphery of the culture dish, at the point of the greatest deformation. Northern blot analysis of total RNA revealed time-dependent induction of alpha1(I) collagen, alpha1(III) collagen, alpha1(IV) collagen, fibronectin, and laminin by stretch/relaxation, with maximal increases occurring between 12 and 24 h. Transient transfection of reporter gene constructs of the 5' flanking region of alpha1(I) collagen gene indicated that stimulation of gene transcription was involved in the increased expression of matrix mRNA. Gelatinolytic activity in conditioned media was decreased at 24 and 48 h of stretch/relaxation, in association with a significant decrease in levels of mRNA for matrix metalloproteinase-2 (68-72 kD type IV collagenase) occurring within 6 h of stretch/relaxation. In contrast, expression of tissue inhibitor of metalloproteinase-2 was increased within 12 h of stretch/relaxation. Stretch/relaxation increased immunoreactive TGF-beta at 48 but not 12 h. TGF-beta1 mRNA levels remained unchanged during the initial 12 h of stretch/relaxation, but were significantly elevated at 48

Harris RC. The macula densa: recent developments. J Hypertens (1996) 14:815-22
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Yasuda T, Akai Y, Kondo S, Becker BN, Homma T, Owada S, Ishida M, Harris RC. Alteration of cellular function in rat mesangial cells in response to mechanical stretch relaxation. Contrib Nephrol (1996) 118:222-8
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Cheng HF, Becker BN, Harris RC. Dopamine decreases expression of type-1 angiotensin II receptors in renal proximal tubule. J Clin Invest (1996) 97:2745-52
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Systemic and/or locally produced angiotensin II stimulates salt and water reabsorption in the renal proximal tubule. In vivo, dopamine (DA) may serve as a counterregulatory hormone to angiotensin II's acute actions on the proximal tubule. We examined whether dopamine modulates AT1 receptor expression in cultured proximal tubule cells (RPTC) expressing DA1 receptors. Dopamine decreased basal RPTC AT1 receptor mRNA levels by 67 +/7% (n = 10; P < 0.005) and decreased 125I-angiotensin II binding by 41 +/7% (n = 4; P < 0.05). The DA1-specific agonist, SKF38393 decreased basal AT1 receptor mRNA levels (65 +/5% inhibition; n = 5; P < 0.025), and the DA1-specific antagonist, SCH23390 reversed dopamine's inhibition of AT1 receptor mRNA expression (24 +/10% inhibition; n = 8; NS) and angiotensin II binding (5 +/15%; n = 4; NS). DA2-specific antagonists were ineffective. In rats given L-DOPA in the drinking water for 5 d, there were decreases in both proximal tubule AT1 receptor mRNA expression (80 +/5%; n = 6; P < 0.005) and specific [125I] Ang II binding (control: 0.74 +/0.13 fmol/mg pro vs. 0.40 +/0.63 fmol/mg pro; n = 5; P < 0.05). In summary, dopamine, acting through DA1 receptors, decreased AT1 receptor expression in proximal tubule, an effect likely mediated by increased intracellular cAMP levels. Local dopamine production also led to decreased AT1 receptor expression, suggesting dopamine may reset sensitivity of the proximal tubule to angiotensin II.

Homma T, Zhang JY, Shimizu T, Prakash C, Blair IA, Harris RC. Cyclooxygenase-derived metabolites of 8,9-epoxyeicosatrienoic acid are potent mitogens for cultured rat glomerular mesangial cells. Biochem Biophys Res Commun (1993) 191:282-8
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The mitogenic effects of 11(R)-hydroxy-8,9-epoxyeicosatrienoic acid (EET) enantiomers were investigated in cultured rat glomerular mesangial cells. Both 11(R)-hydroxylated 8(R),9(S)and 8(S),9(R)-EET at 1 microM stimulated [3H]-thymidine incorporation to 300% and 280%, with 50% maximal effect occurring at 8 x 10(-9) M and 1 x 10(-8) M, respectively. Similar concentration-dependent effects were observed in stimulating induction of the immediate early gene, c-fos. Mitogenic activity of the 11(R)-hydroxylated enantiomers was not affected by prior downregulation of protein kinase C, suggesting involvement of protein kinase C-independent mechanisms. These findings suggest that either transor intracellular metabolism of 8,9-EET by cyclooxygenase occurs during inflammatory glomerular diseases and that the resulting metabolites are involved in mesangial cell proliferation.

Burns KD, Homma T, Harris RC. The intrarenal renin-angiotensin system. Semin Nephrol (1993) 13:13-30
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In this article, we have discussed the localization of components of the renal renin-angiotensin system, as well as the existing information on the regulation of this axis and the effects of Ang II on renal function. All the components of the renin-angiotensin system are present in both fetal and adult kidney. In the adult kidney, renin is principally localized to jg cells of the distal afferent arteriole, where release is stimulated by increases in intracellular cAMP and inhibited by increases in cytosolic calcium. Four distinct stimuli mediating renin release are (1) NaCl sensed at the macula densa, (2) the sympathetic nervous system, (3) humoral factors, with Ang II, vasopressin, endothelin, and adenosine inhibiting renin release, and (4) changes in intrarenal blood pressure. Alterations in renal renin gene expression have been reported in pathophysiological states, such as salt depletion, diabetes mellitus, ureteral obstruction, Bartter's syndrome, and with high protein feeding. The highest renal concentrations of mRNA for the renin substrate angiotensinogen are found in the PT, where the protein is localized to subapical granules. Both salt depletion and androgens upregulate renal angiotensinogen mRNA. Of interest, renal angiotensinogen mRNA levels are lower in SHR than in normotensive WKY rats. As with angiotensinogen, renal ACE is mainly localized to the PT, with highest concentration on the brush border. The mechanisms of regulation of both renal angiotensinogen and ACE require further study. Using recently developed specific nonpeptide Ang II receptor antagonists, it appears that adult renal Ang II receptors are principally of the AT1 class, whereas fetal kidney Ang II receptors are of the AT2 subtype. By binding to AT1 receptors, Ang II exerts constrictive effects on both afferent and

Takahashi K, Harris RC, Capdevila JH, Karara A, Makita K, Jacobson HR, Munger KA, Badr KF. Induction of renal arachidonate cytochrome P-450 epoxygenase after uninephrectomy: counterregulation of hyperfiltration. J Am Soc Nephrol (1993) 3:1496-500
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After unilateral nephrectomy (UNx) in the rat, cytochrome P-450 (cP-450)-linked arachidonate enzymatic activity was markedly and specifically induced in microsomal fractions from the remaining kidney. The enzymatic activity reached 200% at 1 wk and 285% at 2 wk post-UNx as compared with non-UNx controls. Mean baseline values for GFR and RPF rate in the remaining kidney 2 wk after UNx were 1.56 +/0.10 and 6.47 +/- 0.35 mL/min, respectively. In these rats, the administration of ketoconazole, a cP-450 inhibitor, led to 75% inhibition of renal cP-450 arachidonate metabolism and was associated with acute augmentations in both GFR and RPF to 1.82 +/0.18 (P < 0.05 versus baseline) and 7.54 +/- 0.37 mL/min (P < 0.05 versus baseline), respectively. Because vasoconstrictor arachidonate epoxygenase products are endogenously generated in the rat kidney, these findings suggest that the stimulation of renal cP-450-mediated oxygenation of arachidonic acid may subserve an important counterregulatory function in mitigating the renal hyperperfusion and hyperfiltration that follow reductions in renal mass.

Harris RC, Burns KD, Alattar M, Homma T, Nakamura T. Hepatocyte growth factor stimulates phosphoinositide hydrolysis and mitogenesis in cultured renal epithelial cells. Life Sci (1993) 52:1091-100
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Hepatocyte growth factor (HGF), a novel heparin-binding peptide growth factor of MW 97-kDa, is a potent mitogen for parenchymal hepatocytes. HGF is present in normal serum and increases following liver injury or partial hepatectomy. In addition to liver, HGF mRNA has been detected in kidney. In cultured rabbit proximal tubule cells, recombinant human HGF (10(-10) M) increased DNA synthesis, measured as [3H] thymidine incorporation, from 1345 +/213 to 2931 +/636 cpm/10(6) cells; n = 9; p < 0.005). HGF was found to exert mitogenic effects at lower concentrations than epidermal growth factor (EGF), with half maximal effects seen at 6 x 10(-11) M compared to 7 x 10(-10) M for EGF. HGF was additive with EGF in stimulating [3H] thymidine incorporation. In addition to rabbit proximal tubule cells, HGF increased proliferation in a cultured mouse proximal tubule cell line, MCT, and in rat glomerular epithelial cells. In contrast, HGF did not stimulate proliferation of either rat mesangial cells or a rat aortic smooth muscle cell line, A7r5. The HGF receptor is the product of the c-met proto-oncogene. C-met mRNA was detected in total kidney and in cultured proximal tubule cells but was not detected in cultured mesangial cells. In contrast, HGF mRNA was detected in mesangial cells but not in cultured proximal tubule cells. Preincubation of rabbit proximal tubule cells with the tyrosine kinase inhibitor, genistein (50 microM), prevented HGF-stimulation of [3H] thymidine incorporation. In LiCl pretreated rabbit proximal tubule cells loaded with [3H] myoinositol, HGF increased total inositol phosphate release, measured by anion exchange chromatography (control: 2181 +/414 vs HGF: 2609 +/478 cpm/10(6) cells; n = 6; p < 0.05). Although genistein did not affect baseline phosphoinositide hydrolysis, it inhibited

Harris RC, Akai Y, Yasuda T, Homma T. The role of physical forces in alterations of mesangial cell function. Kidney Int Suppl (1994) 45:S17-21
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Harris RC, Ismail N. Extrarenal complications of the nephrotic syndrome. Am J Kidney Dis (1994) 23:477-97
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The systemic complications of nephrotic syndrome are responsible for much of the morbidity and mortality seen with this condition. This review discusses the causes for the hypoalbuminemia and the associated metabolic abnormalities of the nephrotic syndrome. No unifying hypothesis exists for the induction, maintenance, and resolution of nephrotic edema. In view of the wide spectrum of renal diseases leading to the nephrotic syndrome, more than a single mechanism may be responsible for the renal salt retention in these diverse conditions. Although hypoalbuminemia may be important, especially when plasma oncotic pressure is very low (serum albumin < 1.5 to 2.0 g/dL), primary impairment of salt and water excretion by the nephrotic kidney appears to be a major factor in pathogenesis of the edema. However, the decreased serum albumin and/or oncotic pressure seen with nephrotic syndrome is a major contributing factor to the development of the hyperlipidemia of nephrotic syndrome. Patients with unremitting nephrotic syndrome should be considered for combined dietary and lipid-lowering drug therapy. Urinary losses of binding proteins lead to the observed abnormalities in the endocrine system and in trace metals, and urinary losses of coagulation factors contribute to the hypercoagulable state. At present, selective renal venography is recommended when the suspicion of renal vein thrombosis is justified by clinical presentation. The impact on renal function caused by treating asymptomatic chronic renal vein thrombosis is undetermined, but anticoagulation for chronic renal vein thrombosis is associated with relatively few complications.

Harris RC, Akai Y, Yasuda T, Homma T. Role of physical factors in the regulation of mesangial cell growth and the interaction with vasoactive agents. Exp Nephrol (1994) 2:104
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Akai Y, Homma T, Burns KD, Yasuda T, Badr KF, Harris RC. Mechanical stretch/relaxation of cultured rat mesangial cells induces protooncogenes and cyclooxygenase. Am J Physiol (1994) 267:C482-90
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In cultured rat glomerular mesangial cells, continuous cycles of stretching and relaxation (stretch/relaxation) stimulate cell proliferation, protein synthesis, and prostaglandin production. We examined regulation of gene expression that may underlie these alterations in cell functions. Stretch/relaxation caused time-dependent induction of the immediate early genes, c-fos and zif 268/egr-1, with maximal increases occurring between 15 and 30 min. The mitogen-inducible prostaglandin G2/H2 synthase (PGH2S-2) gene was also induced within 30 min of stretch/relaxation, with concomitant increases in the immunoreactive PGH2S-2 protein. These gene inductions were preceded by transient translocation of protein kinase C activity from cytosol to membrane as well as by increases in 45Ca2+ uptake and total cellular calcium content. The stretch/relaxation-induced expression was suppressed by protein kinase C inhibition, whereas less profound inhibition was observed with inhibition of calcium influx in low (100 nM) calcium buffer. These findings indicate that in mesangial cells mechanical stress induces expression of the protooncogenes and the mitogen-inducible cyclooxygenase primarily through protein kinase C-dependent mechanisms.

Harris RC, McKanna JA, Akai Y, Jacobson HR, Dubois RN, Breyer MD. Cyclooxygenase-2 is associated with the macula densa of rat kidney and increases with salt restriction. J Clin Invest (1994) 94:2504-10
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The kidney is a rich source of prostaglandins. These eicosanoids, formed by cyclooxygenase-dependent metabolism of arachidonic acid, are important physiologic mediators of renal glomerular hemodynamics and tubular sodium and water reabsorption. Two separate isoforms of cyclooxygenase (COX) have now been identified: constitutive COX-1, encoded by a 2.8-kb mRNA, and mitogen-activated COX-2, encoded by a 4.0-4.5-kb mRNA. COX-2 expression increases during development and inflammation, but, except for brain, constitutive expression is low. It has been generally accepted that physiologic renal production of prostaglandins is mediated by COX-1. However, in the absence of inflammation, low levels of COX-2 mRNA are also detectable in the kidney. To examine the role of COX-2 in the kidney and determine its intrarenal localization, we used a 1.3-kb cDNA probe specific for the 3' untranslated region of rat COX-2 and COX-2-specific antiserum. The COX-2-specific cDNA probe hybridized with a 4.4-kb transcript in total RNA from adult rat kidney. Immunoblots of microsomes isolated from kidney cortex and papilla indicated immunoreactive COX-2 in both locations. In situ hybridization and immunohistochemistry indicated that renal cortical COX-2 expression was localized to the macula densa of the juxtaglomerular apparatus and to adjacent epithelial cells of the cortical thick ascending limb of Henle. In addition, COX-2 immunoreactivity was detected in interstitial cells in the papilla. No COX-2 message or immunoreactive protein was detected in arterioles, glomeruli, or cortical or medullary collecting ducts. When animals were chronically sodium restricted, the level of COX-2 in the region of the macula densa increased threefold (from 0.86 +/0.08 to 2.52 +/0.43/mm2) and the total area of the COX-2 immunoreactive cel

Burns KD, Inagami T, Harris RC. Cloning of a rabbit kidney cortex AT1 angiotensin II receptor that is present in proximal tubule epithelium. Am J Physiol (1993) 264:F645-54
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The rabbit proximal tubule (PT) has been widely utilized to study the direct effects of angiotensin II (ANG II) on PT function. The purpose of the present study was to characterize the binding properties of PT ANG II receptors, using nonpeptide antagonists, and to clone a rabbit PT ANG II receptor. In rat and rabbit kidney cortical brush-border and basolateral membranes, specific binding of 125I-ANG II was inhibited by the AT1 ANG II-receptor antagonist DuP 753, but not by the AT2 antagonist PD 123319. Using a rabbit kidney cortex cDNA library, we isolated cDNA encoding an ANG II receptor, with an open-reading frame sharing a high degree of sequence homology to previously cloned AT1 ANG II receptors. In transfected COS-1 cells, this rabbit ANG II receptor had properties of the AT1 class. Northern analysis revealed high levels of mRNA expression for this receptor in rabbit kidney cortex and adrenal gland. Within the kidney, message was detected in primary cultures of rabbit PT cells, as well as in freshly isolated rabbit PT segments. Message was also present in cells of the mouse PT line, MCT, and in rat glomerular mesangial cells. Utilizing polymerase chain reaction (PCR) with primers derived from the 1st and 4th transmembrane domains of the rat AT1A ANG II receptor, a 279-bp DNA fragment was amplified from reverse-transcribed RNA from rabbit PT cells. This DNA encoded an amino acid sequence identical to that encoded by the rabbit kidney cDNA clone in the corresponding region and differed by a single base substitution. Southern analysis of rabbit genomic DNA restriction digests with the rabbit ANG II receptor probe revealed hybridization to a single band in each lane. These results indicate that an AT1 ANG II receptor is present in the PT and that a single gene codes for the AT1 receptor in rab

Cheng HF, Becker BN, Burns KD, Harris RC. Angiotensin II upregulates type-1 angiotensin II receptors in renal proximal tubule. J Clin Invest (1995) 95:2012-9
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Angiotensin II (Ang II) is an important regulator of proximal tubule salt and water reabsorption. Recent studies indicate that rabbit proximal tubule angiotensin II receptors are the type-1 (AT1R) subtype. We studied the effect of Ang II on proximal tubule receptor expression. Rabbits were treated with either angiotensin converting enzyme inhibitors or a low salt diet to modulate endogenous Ang II levels. In captopril-treated rabbits, liver and glomerular AT1R mRNA levels increased 242 +/125 and 141 +/- 60%, respectively (n = 6-7; P < 0.05), as determined by quantitative PCR. In contrast, proximal tubule AT1R mRNA levels decreased 40 +/11% (n = 6; P < 0.05). Binding of 125I Ang II to renal cortical basolateral membranes of captopril-treated rabbits decreased from 2.9 +/0.55 to 1.4 +/0.17 fmol/mg protein (n = 6; P < 0.025). In rabbits fed a sodium chloride-deficient diet for 4 wk, AT1R mRNA levels decreased 52 +/11% in liver and 43 +/7% in glomeruli (n = 4-5; P < 0.05), whereas they increased 141 +/85% (n = 5; P < 0.05) in proximal tubule. In basolateral membranes from rabbits on the sodium chloride-deficient diet, specific binding of 125I Ang II increased from 2.1 +/0.2 to 4.3 +/1.1 fmol/mg protein (n = 7; P < 0.05). To determine whether Ang II directly regulates expression of proximal tubule AT1 receptors, further studies were performed in cultured proximal tubule cells grown from microdissected S1 segments of rabbit proximal tubules and immortalized by transfection with a replication-defective SV40 vector. Incubation of these cells with Ang II (10(-11) to 10(-7) M) led to concentration-dependent increases in both AT1R mRNA levels and specific 125I Ang II binding. Pretreatment with pertussis toxin inhibited Ang II stimulation of AT1R mRNA. AT1R mRNA expression was decreased by either forskolin

Burns KD, Harris RC. Signaling and growth responses of LLC-PK1/Cl4 cells transfected with the rabbit AT1 ANG II receptor. Am J Physiol (1995) 268:C925-35
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Angiotensin II (ANG II) receptors of the AT1 subtype are present on the apical and basolateral membranes of renal proximal tubule cells. Cells of the proximal tubulelike cell line, LLC-PK1/Cl4, were transfected with an expression plasmid containing cDNA encoding the rabbit AT1 ANG II receptor. In transfected cells, specific binding of 125I-ANG II was detected on both apical and basolateral membranes; wild-type LLC-PK1/Cl4 cells did not express ANG II receptors. In transfected cells, apical or basolateral ANG II increased both S6 kinase activity and incorporation of [3H]leucine. In cells pretreated with pertussis toxin, the stimulatory effect of apical or basolateral ANG II on [3H]leucine incorporation was abolished. In contrast, ANG II did not affect mitogenesis, determined by [3H]thymidine incorporation. Apical or basolateral ANG II (10(-6) M) stimulated phosphoinositide turnover by 13.4 +/4.4% (n = 8) and 16.3 +/- 4.2% (n = 9), respectively. The activity of protein kinase C, determined by phosphorylation of a specific protein kinase C peptide substrate, was also stimulated by ANG II in transfected cells. Apical or basolateral ANG II had no significant effect on cellular adenosine 3',5'-cyclic monophosphate levels. In permeabilized transfected cells, apical ANG II (10(-6) M) inhibited the phosphorylation of a specific peptide substrate of protein kinase A; lower apical concentrations or basolateral ANG II were without significant effect. These results indicate that AT1 ANG II receptors sort to both apical and basolateral membranes in renal epithelial cells and are coupled to activation of phospholipase C. ANG II stimulates protein synthesis by binding to either apical or basolateral receptors; this effect requires coupling to G proteins and may be mediated by activation of S6 kinase. Because h

Cheng HF, Burns KD, Harris RC. Reduced proximal tubule angiotensin II receptor expression in streptozotocin-induced diabetes mellitus. Kidney Int (1994) 46:1603-10
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Diabetes mellitus is characterized by alterations in the intrarenal renin-angiotensin system, including decreases in glomerular angiotensin II (Ang II) receptor density. Since Ang II regulates proximal tubule transport function, the present studies examined whether diabetes altered expression of proximal tubule receptors. In basolateral membranes from 14 day streptozotocin-induced diabetic rats, specific binding of 125I Ang II was decreased to 53 +/8% of control (3.2 +/0.5 vs. 1.5 +/0.2 fmol/mg protein; N = 7; P < 0.02). Similarly, in proximal tubule brush border membranes from diabetic animals, specific binding was decreased to 63 +/- 11% of control (1.1 +/0.2 vs 0.6 +/0.1 fmol/mg protein; N = 9; P < 0.05). Concomitant insulin treatment reversed the decrease in specific binding of 125I Ang II to basolateral membranes (109 +/26% of control; N = 3) and to brush border membranes (85 +/17% of control; N = 6). In order to determine if changes in expression of type-1 Ang II receptors (AT1R) accompanied the changes in binding, quantitative polymerase chain reaction of AT1R mRNA was performed and expressed as the ratio of the amplified AT1R to that of an Msc1/Msc1 internal deletion mutant and normalized to that of beta-actin. In total RNA from proximal tubule suspensions of diabetic animals, AT1R mRNA expression decreased by 38% (21 +/3 vs. 13 +/2 cpm AT1R/cpm deletion mutant/cpm beta actin/10(6); N = 4; P < 0.0025). Insulin treatment reverted AT1R mRNA expression to control levels (22 +/3 cpm AT1R/cpm deletion mutant/cpm beta actin/10(6); P < 0.001 compared to the untreated group).(ABSTRACT TRUNCATED AT 250 WORDS)

Homma T, Sakai M, Cheng HF, Yasuda T, Coffey RJ Jr, Harris RC. Induction of heparin-binding epidermal growth factor-like growth factor mRNA in rat kidney after acute injury. J Clin Invest (1995) 96:1018-25
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Previous studies have suggested that EGF or other members of the EGF family of mitogenic proteins are involved in proliferation of renal tubular epithelial cells occurring during recovery from injury to the kidney. The present studies examined whether expression of mRNA for the recently identified heparin-binding EGF-like growth factor (HB-EGF) is regulated in response to renal injury induced by either ischemia/reperfusion or mercuric chloride. Increased expression of HB-EGF mRNA was demonstrated in the post-ischemic kidney within 45 min of unilateral ischemia/reperfusion in the rat. Induction of HB-EGF mRNA occurred only when ischemia was followed by reperfusion, and was not eliminated by removal of blood cells from the post-ischemic kidney by saline perfusion. In situ hybridization with 35S-labeled antisense riboprobes of HB-EGF indicated that compared with control, there was increased HB-EGF mRNA expression in the 6 h post-ischemic kidney in the inner cortex and outer medulla in a patchy distribution, with the greatest expression in the inner stripe of the outer medulla. Expression occurred primarily in tubular epithelial cells. Recombinant human HB-EGF stimulated [3H]-thymidine incorporation in both primary cultures of rabbit proximal tubule cells and NRK 52E normal rat kidney epithelial cells, with potency similar to that of EGF. Induction of HB-EGF mRNA was observed in tubules freshly isolated from rat renal cortex or outer medulla when the tubules were subjected to reoxygenation after incubation in anoxic conditions. The nephrotoxin, mercuric chloride, also caused induction of HB-EGF mRNA both in vivo and in isolated rat cortical tubules. The anoxia/reoxygenation-induced expression of HB-EGF mRNA in isolated tubules was inhibited by the free radical scavengers, diand tetra-methylt

Threadgill DW, Dlugosz AA, Hansen LA, Tennenbaum T, Lichti U, Yee D, LaMantia C, Mourton T, Herrup K, Harris RC, et al.. Targeted disruption of mouse EGF receptor: effect of genetic background on mutant phenotype. Science (1995) 269:230-4
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Gene targeting was used to create a null allele at the epidermal growth factor receptor locus (Egfr). The phenotype was dependent on genetic background. EGFR deficiency on a CF-1 background resulted in peri-implantation death due to degeneration of the inner cell mass. On a 129/Sv background, homozygous mutants died at mid-gestation due to placental defects; on a CD-1 background, the mutants lived for up to 3 weeks and showed abnormalities in skin, kidney, brain, liver, and gastrointestinal tract. The multiple abnormalities associated with EGFR deficiency indicate that the receptor is involved in a wide range of cellular activities.

Harris RC, Martinez-Maldonado M. Angiotensin II-mediated renal injury. Miner Electrolyte Metab (1995) 21:328-35
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During the past decade, experimental and clinical evidence has indicated an important role for the renin-angiotensin system in the progressive destruction of nephrons in a wide variety of chronic renal diseases. Studies have indicated that in the subtotally nephrectomized rat model of progressive glomerulosclerosis, in experimental diabetes mellitus, in the chronic phase of puromycin aminonucleoside-induced nephrotic syndrome and in Heymann's nephritis, angiotensin-converting enzyme (ACE) inhibitors dramatically preserve both nephron structure and function. Clinical studies have similarly noted that chronic administration of ACE inhibitors inhibits progression of renal failure in type I diabetes and type II diabetes as well as primary glomerulopathies, sickle cell nephropathy, systemic lupus erythematosis, chronic pyelonephritis and adult polycystic kidney disease. Current evidence suggests that the beneficial effect of ACE inhibitors is primarily due to inhibition of angiotensin II production, and there is strong suggestive evidence for increases in local intrarenal activation of the renin-angiotensin system in these conditions. In obstructive uropathy, activation of the renin-angiotensin system has also been shown to be an important aspect of the early functional changes and may be of importance in the subsequent generation of interstitial fibrosis. In the obstructed kidney, renin and angiotensinogen production increase and type I angiotensin receptors decrease. Inhibitors of angiotensin II production and angiotensin II action partially reverse the vasoconstriction and the reduced renal blood flow, and abolish the changes in expression of AT1 MRNA induced by obstruction. Studies suggest that the angiotensin-mediated increases in tubulointerstitial fibrosis may be mediated by increased product

Burns KD, Capdevila J, Wei S, Breyer MD, Homma T, Harris RC. Role of cytochrome P-450 epoxygenase metabolites in EGF signaling in renal proximal tubule. Am J Physiol (1995) 269:C831-40
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Epidermal growth factor (EGF) is a potent epithelial cell mitogen and induces eicosanoid production in many cell types. The present study examined signaling mechanisms for EGF in the renal proximal tubule, where high concentrations of cytochrome P-450 epoxygenase have been reported. In primary cultures of rabbit proximal tubule cells, EGF (30 nM) increased endogenous epoxyeicosatrienoic acid (EET) levels 5.3 +/1.4-fold within 10 min (n = 6). In these cells EGF-stimulated [3H]thymidine incorporation was significantly inhibited by the cytochrome P-450 inhibitors ketoconazole or clotrimazole but not by the cyclooxygenase inhibitor indomethacin. In fura 2-loaded proximal tubule cells, EGF caused a concentration-dependent increase in cytosolic Ca2+ concentration ([Ca2+]i), due to Ca2+ influx, which was inhibited by either ketoconazole or SKF-525A but not by indomethacin. Addition of 5,6-EET (0.5 microM) also induced Ca2+ influx in proximal tubule cells, whereas 8,9-11,12-, or 14,15-EET did not. In cells treated with bis(2-amino-5-methylphenoxy)ethane N,N,N',N'-tetraacetic acid tetraacetoxy-methyl ester to chelate [Ca2+]i, EGF-stimulated [3H]thymidine incorporation. These results indicate that EGF increases EET levels in proximal tubule and suggest that 5,6-EET or its metabolites may be a modulator of EGF-induced [Ca2+]i increases and involved in mitogenesis.

Becker BN, Cheng HF, Burns KD, Harris RC. Polarized rabbit type 1 angiotensin II receptors manifest differential rates of endocytosis and recycling. Am J Physiol (1995) 269:C1048-56
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Receptor-mediated endocytosis and recycling have been described for extrarenal angiotensin II (ANG II) receptors. In proximal tubule (PT) epithelia expressing polarized ANG II receptors, these processes have not been examined as thoroughly. We utilized a PT cell model, LLC-PKCl4 cells stably transfected with rabbit type 1 ANG II receptor (AT1R) cDNA, to investigate these properties. LLC-PK-AT1R cells expressed the rabbit AT1R transcript and displayed losartan-inhibitable specific 125I-labeled ANG II binding at apical (AP) and basolateral (BL) membranes when grown on permeable supports. AP AT1R internalized 125I-ANG II more rapidly than BL AT1R, and phenylarsine oxide treatment inhibited AP AT1R internalization without significantly affecting BL AT1R endocytosis. Pertussis toxin had no effect on AP or BL AT1R endocytosis. In addition, AP AT1R recovered specific 125I-ANG II binding after ANG II treatment (a measure of recycling). BL AT1R displayed minimal recovery of 125I-ANG II binding after ANG II pretreatment. These data suggested that AP AT1R enter endocytic/endosomal pathways. Phospholipase A2 (PLA2) activity has been linked to endosomal fusion in other systems, and PT brush-border membrane AT1R also have been associated with PLA2 activity. LLC-PK-AT1R cells were therefore treated with quinacrine, a nonspecific PLA2 inhibitor, or Compound I (CI), a selective Ca(2+)-independent PLA2 inhibitor, to determine if PLA2 activity was linked to AT1R recycling. Both quinacrine and CI decreased AP AT1R recycling without affecting BL AT1R recycling. Polarized AT1R in LLC-PKCl4 cells thus display differential rates of endocytosis and recycling.(ABSTRACT TRUNCATED AT 250 WORDS)

Harris RC, Seifter JL, Brenner BM. Adaptation of Na+-H+ exchange in renal microvillus membrane vesicles. Role of dietary protein and uninephrectomy. J Clin Invest (1984) 74:1979-87
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Harris RC, Hoover RL, Jacobson HR, Badr KF. Evidence for glomerular actions of epidermal growth factor in the rat. J Clin Invest (1988) 82:1028-39
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Epidermal growth factor (EGF), an endogenous mitogenic peptide, has recently been shown to be a potent vasoconstrictor of vascular smooth muscle. In view of its potential role in proliferative and inflammatory renal glomerular diseases, we examined the effects of EGF both on cultured rat mesangial cells and on in vivo glomerular hemodynamics. Mesangial cells possess specific, saturable EGF receptors of differing affinities, with Kd's of 0.1 and 1.7 nM, respectively. EGF produced a rapid increase in intracellular pH of 0.12 +/0.01 pH U, which was sodium dependent and amiloride inhibitable. The addition of EGF to mesangial cells cultured on either glass or dimethylpolysiloxane substratum induced reproducible cell contraction. Intrarenal EGF infusion did not affect systemic blood pressure or hematocrit but reversibly decreased GFR and renal blood flow from 4.19 +/0.33 to 3.33 +/0.26 and from 1.17 +/0.09 to 0.69 +/- 0.07 ml/min, respectively. Glomerular micropuncture confirmed decreases in single nephron plasma flow and in single nephron GFR (from 142 +/9 to 98 +/8 and from 51.6 +/11.7 to 28.5 +/3.5 nl/min, respectively) which were due to significant increases in both preand postglomerular arteriolar resistances (from 1.97 +/0.31 to 2.65 +/0.36 and from 1.19 +/0.11 to 2.00 +/0.15 10(10) dyn.s.cm-5 respectively) and to a significant decrease in the ultrafiltration coefficient, Kf, which fell from 0.100 +/0.019 to 0.031 +/0.007 nl/(s.mmHg). These studies demonstrate that mesangial cells possess specific receptors for EGF, and exposure of these cells to physiologic concentrations of EGF results in an in vitro functional response characterized by activation of Na+/H+ exchange and by resultant intracellular alkalinization, as well as by cell contraction. EGF administration in vivo significantly reduces

Harris RC, Brenner BM, Seifter JL. Sodium-hydrogen exchange and glucose transport in renal microvillus membrane vesicles from rats with diabetes mellitus. J Clin Invest (1986) 77:724-33
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Harris RC, Daniel TO. Epidermal growth factor binding, stimulation of phosphorylation, and inhibition of gluconeogenesis in rat proximal tubule. J Cell Physiol (1989) 139:383-91
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Epidermal growth factor and insulin share many biological activities, including stimulation of cell proliferation, ion flux, glycolysis, fatty acid and glycogen synthesis, and activation of receptor-linked tyrosine kinase activity. In the kidney, insulin has been shown to regulate transport processes and inhibit gluconeogenesis in the proximal tubule. Since the kidney represents a major source of EGF, the present studies investigated whether proximal tubule contained EGF receptors, whether EGF receptors were localized to apical or basolateral membranes, and whether EGF receptor activation participated in the regulation of an important proximal tubule function, gluconeogenesis. Specific EGF receptors were demonstrated in the basolateral membrane of proximal tubule. Following incubation with 125I EGF, basolateral membranes demonstrated equilibrium binding at 4 degrees C and 23 degrees C. There was 78 +/2% specific binding (n = 13). The dissociation constant (Kd) was 1.5 x 10(-9) M and maximal binding was 44 fmol/mg protein. There was ninefold more specific binding to proximal tubule basolateral membrane than to brush border membrane. In basolateral, but not brush border membranes, EGF induced phosphorylation of the tyrosine residues of intrinsic membrane proteins, including a 170 kDa protein, corresponding to the EGF receptor. In the presence of the gluconeogenic substrates, alanine, lactate, and succinate, proximal tubule suspensions synthesized glucose. EGF inhibited glucose production in a concentration-dependent manner over a concentration range of 3 x 10(-11) to 3 x 10(-9) M. In addition, EGF inhibited angiotensin II-stimulated glucose production in the proximal tubule suspensions. EGF did not significantly increase net glucose metabolism nor decrease cellular ATP concentrations. Therefore,

Badr KF, Mong S, Hoover RL, Schwartzberg M, Ebert J, Jacobson HR, Harris RC. Leukotriene D4 binding and signal transduction in rat glomerular mesangial cells. Am J Physiol (1989) 257:F280-7
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We examined the characteristics of [3H]leukotriene D4 (LTD4) binding to mesangial cells in culture. Binding is stereoselective, specific, saturable, and rapidly reversible. Two binding sites are recognized with dissociation constants and binding site densities at equilibrium of 2.2 and 16.8 nM and 1.1 x 10(4) and 3 x 10(4) binding sites per cell. LTD4, LTE4, (5R,6S)LTD4, LTB4, and the LTD4-receptor antagonist, SKF 104353, competitively inhibit radioligand binding in the following rank order of potency: LTD4 greater than LTE4 = SKF 104353 greater than (5R,6S)LTD4 greater than LTB4. LTD4 also induces timeand concentration-dependent phosphoinositide hydrolysis in mesangial cells. Formation of inositol 1,4,5-trisphosphate (IP3) is maximal at 5 s, followed by a time-dependent increase in inositol monophosphate generation, and inhibited by 100-fold excess concentration of SKF 104353. Addition of LTD4 to mesangial cells is associated with an increase in intracellular pH and dose-dependent stimulation of [3H]thymidine incorporation and mitogenesis. Thus rat mesangial cells possess specific binding sites for LTD4, the activation of which stimulates IP3 formation and induces cellular alkalinization and mitogenic responses. These studies provide insight into the cellular basis for LTD4-mesangial cell interactions, which are of potential pathophysiological relevance during acute glomerular inflammatory injury.

Harris RC. Response of rat inner medullary collecting duct to epidermal growth factor. Am J Physiol (1989) 256:F1117-24
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Urine is an abundant source of epidermal growth factor (EGF) and prepro-EGF has been localized to the thick ascending limb and distal convoluted tubule of the kidney. However, the functional role of EGF in the kidney is poorly understood. Determination of EGF receptors and functional responses to EGF in intrarenal structures distal to the site of renal EGF production may prove critical to our understanding of the role of this peptide. These studies were designed to investigate the response to EGF of rat inner medullary collecting duct cells in culture and in freshly isolated suspensions. Primary cultures of inner medullary collecting duct cells demonstrated equilibrium binding of 125I-labeled EGF at 4 and 23 degrees C. At 23 degrees C, there was 89 +/1% specific binding (n = 30). Scatchard analysis of 125I-EGF binding suggested the presence of both high-affinity binding with a dissociation constant (Kd) of 5 X 10(-10) M and maximal binding sites (Ro) of 2.7 X 10(3) binding sites/cell and low-affinity binding, with Kd of 8.3 X 10(-9) M and Ro of 1.8 X 10(4) binding sites/cell. Bound EGF, 68 +/3%, was internalized by 45 min. EGF binding was not inhibited by antidiuretic hormone, atrial natriuretic peptide or bradykinin at 23 degrees C, but there was concentration-dependent inhibition of binding by transforming growth factor-alpha. Incubation with phorbol myristate acetate decreased 125I-EGF binding in a concentration-dependent manner. 125I-EGF binding was also demonstrated in freshly isolated suspensions of rat inner medullary collecting duct cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Badr KF, Murray JJ, Breyer MD, Takahashi K, Inagami T, Harris RC. Mesangial cell, glomerular and renal vascular responses to endothelin in the rat kidney. Elucidation of signal transduction pathways. J Clin Invest (1989) 83:336-42
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We investigated the actions of endothelin in anesthetized rats and cultured mesangial cells. Intravenous infusion of endothelin (10 pmol/min) decreased renal blood flow by 44% at 20 min without changing arterial pressure, which subsequently rose significantly from 124 +/3 to 133 +/- 4 mmHg over 60 min. Micropuncture during the nonhypertensive period revealed increases in afferent (65%) and efferent (82%) arteriolar resistances, thereby reducing nephron plasma flow rate. The glomerular ultrafiltration coefficient (Kf) fell from 0.097 +/0.035 to 0.031 +/- 0.011 nl/(s.mmHg) as did single nephron filtration rate (41 +/3 to 19 +/3 nl/min). Addition of 5 nM endothelin to mesangial cells plated on a silicone rubber substrate increased the intensity and number of tension-generated wrinkles, and caused their reappearance in forskolin prerelaxed cells. 20-30 s following exposure of fura-2 loaded mesangial cells to 10 nM endothelin, single cell intracellular calcium concentration ([Ca]i) increased from a mean baseline value of 66 +/11 (SE) to a peak of 684 +/250 nM (P less than 0.05) followed by a sustained elevation at 145 +/42 nM. Anion exchange HPLC revealed rapid (15 s) and dose-dependent stimulation of inositol 1,4,5-trisphosphate (IP3) generation following exposure of [3H]myoinositol preloaded mesangial cells to 10-100 nM endothelin. Endothelin also led to intracellular alkalinization of 2'7'-bis(2-carboxy-ethyl)-5(and-6)carboxyfluorescein (BCECF)-loaded mesangial cells and its addition was associated with dramatic augmentation of mitogenic activity. Thus, endothelin exerts potent constrictor effects on renal arterioles which precede its systemic hypertensive action. It lowers Kf and contracts mesangial cells, likely through stimulation of IP3 generation and elevation of [Ca]i. It is a potent

Harris RC, Seifter JL, Lechene C. Coupling of Na-H exchange and Na-K pump activity in cultured rat proximal tubule cells. Am J Physiol (1986) 251:C815-24
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Safirstein R, Price PM, Saggi SJ, Harris RC. Changes in gene expression after temporary renal ischemia. Kidney Int (1990) 37:1515-21
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Temporary renal ischemia is followed by increased DNA synthesis and cell division as the kidney restores the continuity of the renal epithelium. We sought to characterize some of the changes in proto-oncogene and growth factor expression during this proliferative response. Northern analysis of polyadenylated RNAs of kidney cortical and outer stripe of outer medullary tissue from male Sprague-Dawley rats was performed following release of renal hilar clamping of 50 minutes duration. Ischemia produced an increase in c-fos mRNA that reached a peak at one hour and declined rapidly to control levels by four hours after release of the clamp. A similar rapid increase and decrease in early growth response 1 (Egr 1) mRNA was noted. The response of these immediate early genes was typical of their response to mitogens, suggesting that they served a similar role in renal cell regeneration. Levels of c-Ki-ras and glyceraldehyde phosphate dehydrogenase mRNA were unchanged. Renal preproEGF mRNA decreased at two hours, was virtually absent by 24 hours and remained low for at least four days after ischemia. Urinary excretion of EGF fell immediately after release of ischemia and before the decline in preproEGF mRNA or SNGFR, suggesting post-transcriptional affects of ischemia on renal EGF production. EGF excretion returned to only 50% of control by day 21. Specific 125I-EGF binding increased in membrane fractions of cortex, outer medulla and inner medulla as early as 24 hours after release of the clamp. Cortical 125I-EGF binding increased in the proximal tubule but not in the glomerulus.(ABSTRACT TRUNCATED AT 250 WORDS)

Homma T, Hoover RL, Harris RC. Loop diuretic-sensitive potassium flux pathways of rat glomerular mesangial cells. Am J Physiol (1990) 258:C862-70
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Glomerular mesangial cells are smooth muscle-like contractile cells that mediate hormonal regulation of glomerular filtration. To gain better understanding of ionic events that accompany contraction/relaxation of these cells, flux pathways of K+ and their regulation by vasoactive agents were investigated in cultured rat mesangial cells using 86Rb+ as a tracer. Of total 86Rb+ influx (16.6 +/0.6 nmol x mg protein -1 x min-1), 46% was inhibited by 2 mM ouabain. Loop diuretics inhibited 43% of ouabain-insensitive 86Rb+ influx (3.9 +/0.2 nmol x mg protein-1 x min-1). Half-maximum inhibition was observed at 2 and 0.4 microM for furosemide and bumetanide, respectively. Loop diuretic-sensitive 86Rb+ influx was dependent on extracellular Na+ and Cl-; a hyperbolic dependency on extracellular Na+ was noted with apparent Michaels constant of 39 mM while a sigmoidal dependency on extracellular Clwas present, which, assuming 1:1:2 stoichiometry for Na(+)-K(+)-Cl-, produced an apparent mean affinity constant of 64 mM. Moreover, a fraction of amiloride-insensitive 22Na+ influx was found to be sensitive to furosemide and dependent on extracellular K+ and Cl-, further indicating the presence of Na(+)-K(+)-Clcotransport. Efflux of 86Rb+ followed first-order kinetics, of which 60% was inhibitable by furosemide. Manipulations of extracellular osmolarity revealed that these furosemide-sensitive 86Rb+ flux pathways were coordinately regulated in response to osmotic stress. Concentration-dependent stimulation of Na(+)-K(+)-Clcotransport-mediated 86Rb+ influx was induced by two vasoconstrictors, angiotensin II (ANG II) and arginine vasopressin (AVP), and by a vasodilator, atrial natriuretic peptide (ANP).(ABSTRACT TRUNCATED AT 250 WORDS)

Homma T, Burns KD, Harris RC. Agonist stimulation of Na+/K+/Clcotransport in rat glomerular mesangial cells. Evidence for protein kinase C-dependent and Ca2+/calmodulin-dependent pathways. J Biol Chem (1990) 265:17613-20
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Studies were performed to investigate regulatory pathways of loop diuretic-sensitive Na+/K+/Clcotransport in cultured rat glomerular mesangial cells. Angiotensin II, alpha-thrombin, and epidermal growth factor (EGF) all stimulated Na+/K+/Clcotransport in a concentration-dependent manner. Pertussis toxin pretreatment reduced the effects of angiotensin II and alpha-thrombin but not that of EGF. Addition of the protein kinase C inhibitor staurosporine or down-regulation of protein kinase C by prolonged incubation with phorbol 12-myristate 13-acetate partially reduced the effects of angiotensin II and alpha-thrombin and completely blunted the phorbol 12-myristate 13-acetate-induced stimulation of Na+/K+/Clcotransport but did not affect EGF-induced stimulation. Exposure of cells to a calcium ionophore, A23187, resulted in a concentration-dependent stimulation of Na+/K+/Cl- cotransport, which was not significantly inhibited by down-regulation of protein kinase C but was completely inhibited by the calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). Stimulation of the cotransport by angiotensin II or alpha-thrombin was also partially inhibited by W-7. Inhibitory effects of protein kinase C down-regulation and W-7 were additive and, when combined, produced a complete inhibition of angiotensin II-induced stimulation of Na+/K+/Cl- cotransport. In saponin-permeabilized mesangial cells, phosphorylation of a synthetic decapeptide substrate for Ca2+/calmodulin-dependent kinase II, Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ser-Ser-NH3, was demonstrated. Maximal activation of the decapeptide substrate phosphorylation required the presence of Ca2+ and calmodulin and was dependent on Ca2+ concentration. These findings indicate that stimulation of Na+/K+/Clcotransport by angiotensin II a

Harris RC, Homma T, Jacobson HR, Capdevila J. Epoxyeicosatrienoic acids activate Na+/H+ exchange and are mitogenic in cultured rat glomerular mesangial cells. J Cell Physiol (1990) 144:429-37
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The present study examined responses of cultured rat glomerular mesangial cells to exogenous exposure of epoxyeicosatrienoic acids (EET's), products of cytochrome P450 epoxygenase. One day after administration of 8,9or 14,15-EET, cultured rat mesangial cells demonstrated significant increases in [3H]thymidine incorporation (10(-7) M 14,15-EET: 120 +/7% of control; n = 6; P less than 0.025; 10(-6) M 14,15-EET: 145 +/10%; n = 20; P less than 0.0005; 10(-6) M 8,9-EET: 167 +/31%; n = 9; P less than 0.05), which was not affected by addition of the cyclooxygenase inhibitor indomethacin. In addition to stimulation of [3H]thymidine incorporation, the epoxides stimulated mesangial cell proliferation. 14,15-EET administration induced intracellular alkalinization of 0.2-0.3 pH units, which was prevented by extracellular Na+ removal and blunted by amiloride (0.5 mM). Following intracellular acidification with NH4Cl addition and removal, greater than 85% of 3 mM 22Na uptake into mesangial cells was inhibited by 1 mM amiloride, indicating Na+/H+ exchange. Under these conditions, 14,15-EET stimulated Na+/H+ exchange by 42% and 8,9-EET stimulated Na+/H+ exchange by 59%. Neither protein kinase C depletion nor addition of the protein kinase C inhibitor, staurosporine, affected this stimulation. In [3H]myo-inositol loaded mesangial cells, no significant stimulation of phosphoinositide hydrolysis was detected in response to administration of 14,15-EET. Twenty-four hours after addition of [14C]14,15-EET, greater than 90% was preferentially esterified to cellular lipids, with predominant incorporation into phosphatidylinositol, phosphatidylethanolamine, and diacylglycerol. Thus, these results demonstrate epoxyeicosatrienoic acids stimulate Na+/H+ exchange and mitogenesis in mesangial cells. These effects do not appe

Kon V, Harris RC, Ichikawa I. A regulatory role for large vessels in organ circulation. Endothelial cells of the main renal artery modulate intrarenal hemodynamics in the rat. J Clin Invest (1990) 85:1728-33
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After arterial denudation by external rubbing of the left main renal artery, we assessed renal plasma flow rate (RPF) and glomerular filtration rate (GFR) in left and right kidneys of Munich-Wistar rats before and after intravenous infusion of acetylcholine (ACH), atrial natriuretic peptide (ANP), or nitroprusside (NP). In the right kidney RPF and/or GFR increased in response to both endothelium-derived relaxing factor (EDRF)-dependent (i.e., ACH) and -independent vasodilators (i.e., ANP and NP); on average, RPF rose by 22 +/4% (P less than 0.005), 19 +/10% (P less than 0.005), and 37 +/12% (P greater than 0.05), respectively. By contrast, in the left kidney RPF failed to increase after ACH (falling by 23 +/10%, P less than 0.001) and rose only in response to ANP and NP. To further evaluate the main renal artery's contribution to renal vasodilation, ACH and another EDRF-dependent agent, histamine, were infused through a micropipette into either the proximal or distal portions of the endothelium-intact renal artery. Proximal infusion of ACH led to increases in RPF and GFR, on average by 8 +/2% (P less than 0.025) and 10 +/3% (P less than 0.01), while bypassing the arterial endothelium by distal infusion failed to increase RPF and GFR, which fell by 24 +/6% (P less than 0.025) and 22 +/6% (P less than 0.005), respectively. Similarly, proximal infusion of histamine increased RPF by 12 +/3% (P less than 0.05), while distal infusion was virtually without effects on plasma flow. Micropuncture study during intravenous ACH infusion revealed significantly higher afferent and efferent arteriolar resistances and lower ultrafiltration coefficients in denuded versus nondenuded kidneys. These data indicate that the main renal artery is a major regulator of renal blood flow and vascular resistances. Similar t

Harris RC, Munger KA, Badr KF, Takahashi K. Mediation of renal vascular effects of epidermal growth factor by arachidonate metabolites. FASEB J (1990) 4:1654-60
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In the rat, intrarenal infusion of epidermal growth factor decreases renal blood flow and glomerular filtration rate, and epidermal growth factor (EGF) induces contraction of cultured rat mesangial cells. The present studies examined the role of arachidonic acid metabolites in this response. Intrarenal EGF infusion increased urinary iPGF2 alpha by 300%, and in isolated glomeruli EGF stimulated iPGF2 alpha by 38%, but did not affect thromboxane B2 production. Furthermore, the thromboxane A2 receptor antagonist, SQ29548, did not block EGF's vasoconstrictive effects. After selective cyclooxygenase inhibition with ibuprofen, intrarenal EGF infusion no longer produced local vasoconstriction but instead led to systemic vasodilation (SBP: 117 +/10 vs. 98 +/7; n = 5; P less than 0.05) that was accompanied by significant increases in RPF (3.8 +/0.4 vs. 5.6 +/0.2; P less than 0.01) and glomerular filtration rate (0.9 +/- 0.1 vs. 1.1 +/0.1; P less than 0.05). When total arachidonate metabolism was inhibited by the additional administration of 5,8,11,14-eicosatetraynoic acid, the EGF-induced vasodilation observed during cyclooxygenase inhibition alone was abolished, and vasoconstrictor responses to EGF were again noted. Similar effects were noted with concomitant administration of the c-P450 inhibitor ketoconazole. EGF's vasoconstrictive effects were unaltered by the simultaneous administration of the angiotensin II antagonist saralasin. Thus, the renal hemodynamic responses to EGF are mediated in part by arachidonic acid metabolites. Cyclooxygenase inhibition unmasks a potent renal and systemic vasodilator action of EGF owing to its stimulation of systemic release of noncyclooxygenase arachidonate metabolites.

Harris RC, Badr KF. Recovery of prostaglandin synthesis in rat glomerular mesangial cells after aspirin inhibition: induction of cyclooxygenase activity by serum and epidermal growth factor. Prostaglandins (1990) 39:213-22
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We assessed cyclooxygenase activity in cultured rat mesangial cells by measuring prostaglandin production with reverse phase HPLC upon addition of exogenous 14C arachidonic acid. The profile of prostaglandins produced was PGE2 greater than PGF2a much greater than 6-keto PGF1a much greater than thromboxane and PGD2. In quiescent mesangial cells, exposure to 300 microM aspirin for 30 minutes irreversibly inhibited cyclooxygenase activity; after 5 hours, cyclooxygenase activity was only 19 +/3% of control. Addition of 10% fetal bovine serum after aspirin inactivation stimulated time-dependent recovery of cyclooxygenase activity to 118 +/- 30% of control by 5 hours. Serum induced-recovery was significantly inhibited by the simultaneous administration of the protein kinase C inhibitor, staurosporine. Phorbol myristate acetate also induced recovery of cyclooxygenase activity, suggesting that protein kinase C may be involved in the signaling process. In addition to serum, epidermal growth factor was also found to lead to partial recovery of cyclooxygenase activity. The serum and EGF-induced recoveries were inhibitable by cycloheximide and actinomycin D. These results suggest that recovery of cyclooxygenase activity in mesangial cells is stimulated by EGF and other components of serum, is dependent upon new protein synthesis and appears to be transcriptionally regulated.

Harris RC. Potential physiologic roles for epidermal growth factor in the kidney. Am J Kidney Dis (1991) 17:627-30
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Epidermal growth factor (EGF) is a 53-amino acid polypeptide that is known to produce a number of biologic effects both in vitro and in vivo. High concentrations of EGF are found in urine, and high concentrations of prepro-EGF mRNA have been detected in kidney, localized to thick ascending limb of Henle (TALH) and distal convoluted tubule. Specific high-affinity EGF receptors have been demonstrated in mesangial cells, proximal tubule, and cortical and inner medullary collecting duct, as well as in medullary interstitial cells. In the proximal tubule, EGF binding and EGF receptor-associated tyrosine kinase activity are localized to basolateral membrane, and functional responses in collecting duct are observed only with basolateral administration of EGF. A number of renal responses to administration of EGF have recently been described, including modulation of glomerular hemodynamics, renal metabolism, tubular transport functions, and eicosanoid synthesis. In addition, EGF has been shown to be a potent mitogen in vitro for a variety of cell types in the kidney and may be an important mediator of renal repair following injury.

Homma T, Harris RC. Time-dependent biphasic regulation of Na+/K+/Clcotransport in rat glomerular mesangial cells. J Biol Chem (1991) 266:13553-9
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Time-dependent regulation of loop diuretic-sensitive Na+/K+/Cl- cotransport and [3H]bumetanide binding was investigated in cultured rat glomerular mesangial cells. Angiotensin II or epidermal growth factor induced stimulation of Na+/K+/Clcotransport within 5 min, with a return to the control values by 30 min. Treatment of cells with phorbol 12-myristate 13-acetate (0.1 microM) (PMA), the calcium ionophore A23187 (1 microM), or the combination of 5 mM NaF and 10 microM AlCl3 produced a transient stimulation of Na+/K+/Clcotransport in 5-10 min to 148, 135, and 163% of control, respectively, which was followed by a progressive decrease to 34, 64, and 20% of the base-line activity, respectively, by 60 min. Exposure to cyclic 8-bromo-AMP (0.1 mM) or to forskolin (1 microM) and isobutylmethylxanthine (0.1 mM) caused a maximal inhibition of the cotransport in 5 min to 79 and 60% of control, respectively, with a subsequent gradual increase to 137 and 164% of the base-line activity, respectively, by 60 min. The effects of PMA, forskolin, and cyclic 8-bromo-AMP were concentration-dependent. In order to characterize further the alterations in the cotransport activity, binding of [3H]bumetanide was determined. Saturation binding analyses showed that the late inhibition of the cotransport by PMA and stimulation by forskolin were associated with a significant decrease and increase, respectively, in Bmax, with no significant changes in binding affinity. Correlations between changes in the cotransport activity and [3H]bumetanide binding were also observed in cells treated with cyclic 8-bromo-AMP or with NaF and AlCl3. Incubation of cells in Clor Na+ free solution greater than or equal to 60 min resulted in an increase in both the cotransport activity and [3H]bumetanide binding. These observations indicate that

Ichikawi I, Harris RC. Angiotensin actions in the kidney: renewed insight into the old hormone. Kidney Int (1991) 40:583-96
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Burns KD, Homma T, Breyer MD, Harris RC. Cytosolic acidification stimulates a calcium influx that activates Na(+)-H+ exchange in LLC-PK1. Am J Physiol (1991) 261:F617-25
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We have previously shown that cytosolic acidification-stimulated Na(+)-H+ exchange in LLC-PK1 cells is inhibited by calmodulin antagonists. To investigate further the role of Ca(2+)-calmodulin-dependent processes in intracellular pH (pHi) regulation in these cells, we studied the effects of pHi changes on cytosolic Ca2+ concentration ([Ca2+]i). In fura-2/acetoxymethylester (fura-2/AM)-loaded cells maintained in isotonic Na(+)-free buffer containing 1.8 mM CaCl2, [Ca2+]i was 168 +/59 nM (n = 5). After NH4Cl-induced alkalinization, [Ca2+]i decreased to 83 +/28 nM and partially recovered to 126 +/42 nM. Cytosolic acidification, after NH4Cl washout, caused an increase in [Ca2+]i to 481 +/166 nM (P less than 0.05; n = 5) that was dependent on extracellular Ca2+. An increase in [Ca2+]i was also observed in cells acidified with KCl-nigericin, with a return of [Ca2+]i to baseline with cell alkalinization. No increase in 45Ca2+ efflux occurred in association with initial NH4Cl-induced [Ca2+]i decrease, suggesting Ca2+ flux into an intracellular store during alkalinization. Membrane depolarization did not alter [Ca2+]i. The acidification-induced [Ca2+]i increase was inhibited by preincubation with verapamil or the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). Na(+)-dependent pHi recovery in 2,'7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethylester (BCECF/AM)-loaded LLC-PK1 cells and cytosolic acidification-stimulated basolateral Na(+)-H+ exchange activity in LLC-PK1/CL4 cells were both attenuated in the absence of extracellular Ca2+. The results indicate that cytosolic acidification activates an influx of extracellular Ca2+ in LLC-PK1 cells. Furthermore, in the absence of Ca2+ influx, Na(+)-H+ exchange is inhibited.(ABSTRACT TRUNCATED AT 250 WORDS)

Burns KD, Homma T, Harris RC. Regulation of Na(+)-H+ exchange by ATP depletion and calmodulin antagonism in renal epithelial cells. Am J Physiol (1991) 261:F607-16
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The present studies examined effects of ATP depletion and calmodulin antagonism on stimulation of Na(+)-H+ exchange by cytosolic acidification in renal epithelial cells (LLC-PK1). ATP depletion significantly inhibited both amiloride-sensitive 22Na+ uptake (P less than 0.001; n = 12) and Na(+)-dependent intracellular pH (pHi) recovery in 2',7'-bis (carboxyethyl)-5(6)-carboxyfluorescein acetoxymethylester (BCECF/AM)-loaded cells. Calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7) and calmidazolium, both caused a concentration-dependent inhibition of Na(+)-H+ exchange activity. The W-7-induced inhibition of Na(+)-H+ exchange occurred in cells incubated for 24 h with phorbol 12-myristate 13-acetate, indicating that the effect of W-7 was not mediated by protein kinase C inhibition. Both W-7 and ATP depletion shifted the pHi dependence of the antiporter, and ATP depletion also reduced the maximal activity. In LLC-PK1/CL4 cells grown on permeable filters, W-7 inhibited the cytosolic acidification-stimulated basolateral exchanger by 54 +/5% (P less than 0.005; n = 7) and, in contrast, stimulated the apical exchanger by 28 +/- 13% (P less than 0.05; n = 6). ATP depletion significantly inhibited apical Na(+)-H+ exchange. These results suggest that an ATP-Ca(2+)-calmodulin-dependent process is involved in regulation of Na(+)-H+ exchange in LLC-PK1 cells. A Ca(2+)-calmodulin-dependent process activated the amiloride-sensitive basolateral Na(+)-H+ exchanger and inhibited the amiloride-resistant apical antiporter. Phosphorylation of these two Na(+)-H+ exchangers or regulatory proteins by a Ca(2+)-calmodulin-dependent protein kinase may mediate this differential regulation.

Awazu M, Kon V, Harris RC, Imada T, Inagami T, Ichikawa I. Renal sympathetic nerves modulate glomerular ANP receptors and filtration. Am J Physiol (1991) 261:F29-35
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We examined characteristics of atrial natriuretic peptide (ANP) receptors in glomeruli isolated from subacutely (3-5 days) denervated (DNX) and contralateral nondenervated (non-DNX) kidneys of normal rats (NL) and rats subjected to water deprivation for 48 h (WD). Total ANP receptor density in DNX kidneys of WD rats, measured by competitive inhibition binding between 125I-labeled ANP and ANP, was twofold higher than non-DNX kidneys (726 +/96 vs. 384 +/32 fmol/mg protein, P less than 0.05). Equilibrium association constant (Ka) was not significantly different (2.33 +/0.43 vs. 3.34 +/0.78 x 10(9) M-1). In NL rats, there was no difference in ANP receptor density between DNX and non-DNX kidneys (244 +/20 and 264 +/- 16 fmol/mg protein). Production of guanosine 3',5'-cyclic monophosphate (cGMP), a putative second messenger of ANP, in response to ANP (10(-7) M) in glomeruli isolated from DNX was significantly larger than non-DNX kidneys of WD rats. To determine whether these changes in ANP receptors have functional consequences in vivo, glomerular capillary ultrafiltration coefficient (Kf) was assessed by micropuncture technique in WD Munich-Wistar rats. In DNX kidneys, ANP infusion (4 micrograms.kg-1.h-1) significantly increased whole kidney glomerular filtration rate (GFR) and single-nephron (SN) GFR (0.64 +/0.06 to 0.89 +/0.17 ml/min and 25 +/- 2 to 33 +/2 nl/min, respectively; n = 7) and Kf (1.26 +/0.29 to 2.18 +/0.41 nl.min-1.mmHg-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Harris RC. Regulation of S6 kinase activity in renal proximal tubule. Am J Physiol (1992) 263:F127-34
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The proximal tubule undergoes hypertrophy in response to loss of functioning renal mass and hyperplasia following injury by ischemia or nephrotoxins. Both hypertrophic growth and cell proliferation are characterized by increases in the rate of protein synthesis. To investigate regulation of protein synthesis in mammalian proximal tubule cells, potential peptide mediators of proximal tubule growth, epidermal growth factor (EGF) and angiotensin II, were studied in cultured rabbit proximal tubule cells. Although only EGF stimulated DNA synthesis, both agonists stimulated protein synthesis. One potential regulatory mechanism of eukaryotic protein synthesis involves phosphorylation of ribosomal protein S6 by activation of a specific serine/threonine kinase (S6 kinase). Both EGF and angiotensin II stimulated S6 kinase activity and S6 phosphorylation. Phorbol 12-myristate 13-acetate was also found to activate S6 kinase, and 24 h of pretreatment to deplete protein kinase C inhibited subsequent S6 kinase activation by a high concentration (10(-6) M) of angiotensin II. To determine whether S6 kinase was also activated in the kidney in vivo, S6 kinase activity was examined after ablation of renal mass. Within 1 h after contralateral nephrectomy, S6 kinase activity increased in rat renal cortex. In summary, both EGF and angiotensin II stimulated protein synthesis and S6 kinase activity in cultured proximal tubule cells, and S6 kinase activity also increased in renal cortex after contralateral nephrectomy.

Harris RC, Haralson MA, Badr KF. Continuous stretch-relaxation in culture alters rat mesangial cell morphology, growth characteristics, and metabolic activity. Lab Invest (1992) 66:548-54
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Mesangial cells serve many functions in the glomerulus, including regulation of glomerular ultrafiltration coefficient, matrix production, and eicosanoid generation. The glomerulus is a vascular bed, and the mesangial cell is continually exposed to rhythmic alterations in intraglomerular pressure. Since increased intraglomerular pressure has been implicated as a potential causative agent in the ultimate development of nephrosclerosis, we sought to determine the effect of continuous stretch-relaxation upon parameters of mesangial cell growth and function. Early passage (2-4) cultured rat mesangial cells were plated onto either rigid-bottom or flexible-bottom culture plates coated with type I collagen. After cell attachment, the cells on flexible supports were exposed to continuous stretch-relaxation for 72 to 96 hours at a rate of 100 cycles/minutes at an applied pressure of 7 to 8 KPa (53 to 61 mm Hg). Cellular morphology was altered by continuous stretch-relaxation, with the majority of mesangial cells presenting stellate or straplike morphology. Fluorescein isothiocyanate-labeled phalloidin staining indicated an increase in density of actin filaments running the long axis of the cell. Stretch-relaxation resulted in an approximately 50% increase in cell number. Prostaglandin production, assessed as irPGE2 production, was increased by stretching in mesangial cells from 28 +/1 to 49 +/4 pg/10(6) cells (N = 12; p less than 0.005). Mechanical stretch/relaxation increased the percentage of protein representing collagenous proteins from 47 +/6% to 70 +/4%, as assessed by collagenase susceptibility (p less than 0.025). Analysis of pepsin-resistant proteins synthesized indicated that stretch/relaxation resulted in increases in the relative amounts of types I and III collagens produced/cell. Additional

McKanna JA, Chuncharunee A, Munger KA, Breyer JA, Cohen S, Harris RC. Localization of p35 (annexin I, lipocortin I) in normal adult rat kidney and during recovery from ischemia. J Cell Physiol (1992) 153:467-76
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The 35-kDa protein (p35, lipocortin I, annexin I), originally discovered as a Ca(++)-dependent substrate for the EGF receptor tyrosine kinase, binds Ca++ and phospholipids, is developmentally regulated in embryos and has restricted expression in adults. Immunohistochemistry of normal rat kidney shows that p35 is enriched in epithelia of Bowman's capsule, the macula densa, and medullary/papillary collecting ducts, suggesting that p35 is related to specialized renal functions. Light staining is observed in the thick ascending limb; elsewhere, immunoreactivity is nil. Since renal recovery from ischemia involves both hyperplasia and hypertrophy and reportedly is accelerated by EGF, we examined p35 distribution during this process. After 48 hours of recovery, both the distribution and amount of renal p35 are altered. Immunoblots show p35 levels increased at least threefold in whole-kidney homogenates. The expression of p35 is still highly restricted in recovering kidneys; however, the thick ascending limb now stains heavily. This is the first documentation of alterations in annexin levels during a pathophysiologic response. However, our attempts to discern effects of exogenous EGF on the recovery from ischemia were negative for both mitotic index and renal function assays.

Homma T, Akai Y, Burns KD, Harris RC. Activation of S6 kinase by repeated cycles of stretching and relaxation in rat glomerular mesangial cells. Evidence for involvement of protein kinase C. J Biol Chem (1992) 267:23129-35
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Quiescent rat glomerular mesangial cells were exposed to repeated cycles of stretching and relaxation, and the effects on the rate of collagen production, proliferation, and S6 kinase activity were investigated. Stretch/relaxation induced increases in production of both collagen and non-collagenous proteins. Proliferation of mesangial cells was stimulated by stretch/relaxation and epidermal growth factor, but not by angiotensin II; however, administration of angiotensin II augmented stretch/relaxation-induced cell proliferation. Cytosolic S6 kinase activity was stimulated by stretch/relaxation, angiotensin II, epidermal growth factor, or phorbol 12-myristate 13-acetate. The increased S6 kinase activity was detectable within 30 min after initiation of stretch/relaxation and was blocked by either inhibitors of protein kinase C or prior down-regulation of protein kinase C following prolonged incubation with phorbol 12-myristate 13-acetate. Both translocation of protein kinase C from the cytosolic to the membrane fraction and phosphorylation of an endogenous 80-kDa protein were observed within 5 min of initiation of stretch/relaxation. These results demonstrate that in mesangial cells, mechanical factors alone can induce increases in production of collagen and non-collagenous proteins and in cell proliferation. The observation that stretch/relaxation induced stimulation of S6 kinase activity through protein kinase C-dependent mechanisms suggests that activation of protein kinase C may be a key event in initiating adaptive responses of mesangial cells to increased workload.

Rudman D, Kutner MH, Fleming GA, Harris RC, Kennedy EE, Bethel RA, Priest JH. Effect of 10-day courses of human growth hormone on height of short children. J Clin Endocrinol Metab (1978) 46:28-35
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Zhang, M.

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