Effects of xanthine oxidase inhibition with febuxostat on the development of nephropathy in experimental type 2 diabetes.

BACKGROUND AND PURPOSE: Elevated serum uric acid (UA) is a risk factor for the development of kidney disease. Inhibitors of xanthine oxidase (XOi), an enzyme involved in UA synthesis, have protective effects at early stages of experimental diabetic nephropathy (DN). However, long-term effects of XOi in models of DN remain to be determined. EXPERIMENTAL APPROACH: The development of albuminuria, renal structure and molecular markers of DN were studied in type 2 diabetic Zucker obese (ZO) rats treated for 18 weeks with the XOi febuxostat and compared with vehicle-treated ZO rats, ZO rats treated with enalapril or a combination of both agents, and lean Zucker rats without metabolic defects. RESULTS: Febuxostat normalized serum UA and attenuated the development of albuminuria, renal structural changes, with no significant effects on BP, metabolic control or systemic markers of oxidative stress (OS). Most of these actions were comparable with those of enalapril. Combination treatment induced marked decreases in BP and was more effective in ameliorating structural changes, expression of profibrotic genes and systemic OS than either monotherapy. Febuxostat attenuated renal protein expression of TGF-ß, CTGF, collagen 4, mesenchymal markers (FSP1 and vimentin) and a tissue marker of OS nitrotyrosine. Moreover, febuxostat attenuated TGF-ß- and S100B-induced increased expression of fibrogenic molecules in renal tubular cells in vitro in UA-free media in an Akt kinase-dependent manner. CONCLUSIONS AND IMPLICATIONS: Febuxostat is protective and enhances the actions of enalapril in experimental DN. Multiple mechanisms might be involved, such as a reduction of UA, renal OS and inhibition of profibrotic signalling.

Epigenetic changes in renal genes dysregulated in mouse and rat models of type 1 diabetes.

Epigenetic processes are increasingly being recognized as factors in the pathophysiology of diabetes complications, but few chromatin studies have been done in diabetic nephropathy (DN). We hypothesized that changes in mRNA expression of DN-related genes are associated with epigenetic alterations and aberrant expression of histone-modifying enzymes. RT-PCR and a matrix-chromatin immunoprecipitation platform were used to examine renal mRNA expression, RNA polymerase II (Pol II) recruitment, and epigenetic marks at DN-related genes in the mouse (OVE26) and streptozotocin-induced rat models of type 1 diabetes. Diabetes induced renal expression of Cox2, S100A4/FSP-1, and vimentin genes in both the mouse and the rat models of DN. Mcp-1 and laminin γ1 (Lamc1) expression were increased in diabetic mice but not in rats. Comparison of mRNA and Pol II levels suggested that the diabetes-induced expression of these transcripts is mediated by transcriptional and posttranscriptional processes. Decreases in histone H3 lysine 27 tri-methylation (H3K27m3, silencing mark) and increases in H3 lysine 4 di-methylation (H3K4m2, activating mark) levels were the most consistent epigenetic alterations in the tested genes. In agreement with these results, immunoblot analysis showed increased protein abundance of renal H3K27m2/3 demethylase KDM6A, but no changes in cognate methyltransferase Ezh2 in kidneys of the OVE26 mice compared with controls. In diabetic rats, Ezh2 expression was higher without changes in KDM6A, demonstrating that mechanisms of DN-induced H3K27m3 loss could be species specific. In summary, we show that altered mRNA expression of some DN-related genes is associated with changes in Pol II recruitment and a corresponding decrease in repressive H3K27m3 at the selected loci, and at least in mice with equivalent changes in renal expression of cognate histone-modifying enzymes. This pattern could contribute to diabetes-mediated transitions in chromatin that facilitate transcriptional changes in the diabetic kidney.

Enhanced phosphorylation of Na(+)-Cl- co-transporter in experimental metabolic syndrome: role of insulin.

In the present study, we investigated the activity of the thiazide-sensitive NCC (Na(+)-Cl(-) co-transporter) in experimental metabolic syndrome and the role of insulin in NCC activation. Renal responses to the NCC inhibitor HCTZ (hydrochlorothiazide), as a measure of NCC activity in vivo, were studied in 12-week-old ZO (Zucker obese) rats, a model of the metabolic syndrome, and in ZL (Zucker lean) control animals, together with renal NCC expression and molecular markers of NCC activity, such as localization and phosphorylation. Effects of insulin were studied further in mammalian cell lines with inducible and endogenous expression of this molecule. ZO rats displayed marked hyperinsulinaemia, but no differences in plasma aldosterone, compared with ZL rats. In ZO rats, natriuretic and diuretic responses to NCC inhibition with HCTZ were enhanced compared with ZL rats, and were associated with a decrease in BP (blood pressure). ZO rats displayed enhanced Thr(53) NCC phosphorylation and predominant membrane localization of both total and phosphorylated NCC, together with a different profile in expression of SPAK (Ste20-related proline/alanine-rich kinase) isoforms, and lower expression of WNK4. In vitro, insulin induced NCC phosphorylation, which was blocked by a PI3K (phosphoinositide 3-kinase) inhibitor. Insulin-induced reduction in WNK4 expression was also observed, but delayed compared with the time course of NCC phosphorylation. In summary, we report increased NCC activity in hyperinsulinaemic rodents in conjunction with the SPAK expression profile consistent with NCC activation and reduced WNK4, as well as an ability of insulin to induce NCC stimulatory phosphorylation in vitro. Together, these findings indicate that hyperinsulinaemia is an important driving force of NCC activity in the metabolic syndrome with possible consequences for BP regulation.

2-Hydroxyestradiol slows progression of experimental polycystic kidney disease.

Male gender is a risk factor for progression of polycystic kidney disease (PKD). 17ß-Estradiol (E2) protects experimentally, but clinical use is limited by adverse effects. Novel E2 metabolites provide many benefits of E2 without stimulating the estrogen receptor, and thus may be safer. We hypothesized that E2 metabolites are protective in a model of PKD. Studies were performed in male control Han:SPRD rats, and in cystic males treated with orchiectomy, 2-methoxyestradiol, 2-hydroxyestradiol (2-OHE), or vehicle, from age 3 to 12 wk. Cystic rats exhibited renal functional impairment (∼50% decrease in glomerular filtration and renal plasma flow rates, P < 0.05) and substantial cyst development (20.5 ± 2.0% of cortex area). 2-OHE was the most effective in limiting cysts (6.0 ± 0.7% of cortex area, P < 0.05 vs. vehicle-treated cystic rats) and preserving function, in association with suppression of proliferation, apoptosis, and angiogenesis markers. Downregulation of p21 expression and increased expression of Akt, the mammalian target of rapamycin (mTOR), and some of its downstream effectors were significantly reversed by 2-OHE. Thus, 2-OHE limits disease progression in a cystic rodent model. Mechanisms include reduced renal cell proliferation, apoptosis, and angiogenesis. These effects may be mediated, at least in part, by preservation of p21 and suppression of Akt and mTOR. Estradiol metabolites may represent a novel, safe intervention to slow progression of PKD.

Rho kinase inhibition protects kidneys from diabetic nephropathy without reducing blood pressure.

Rho-associated kinases (ROCK) are activated in the kidney as well as in cultured cells of diabetic models and have been implicated in renal pathophysiology. To explore whether inhibition of ROCK is protective, we studied its role in a model of accelerated diabetic nephropathy where uninephrectomized rats were made diabetic by streptozotocin. After establishing diabetes, rats were treated with the ROCK inhibitor fasudil continuously or for the final 6 weeks of an 18-week experimental period. The results were compared to similar rats given losartan, an established treatment of clinical and experimental diabetic nephropathy, or a combination of both agents. Vehicle-treated diabetic and non-diabetic uninephrectomized rats served as controls. Diabetes resulted in a rapid development of albuminuria, higher glomerulosclerosis and interstitial fibrosis scores, lower glomerular filtration rates, and increased expression of several molecular markers of diabetic nephropathy. Eighteen weeks of fasudil treatment reduced renal ROCK activity, and ameliorated diabetes-induced structural changes in the kidney and expression of the molecular markers in association with a modest anti-proteinuric effect but no change in blood pressure. Late intervention with fasudil reduced glomerulosclerosis, but did not influence proteinuria. Most effects of fasudil were comparable to those of losartan, although losartan lowered blood pressure and further lowered proteinuria. The combination of both treatments was no different than losartan alone. Thus, ROCK inhibition protected the kidney from diabetic nephropathy even though it did not reduce the blood pressure.

Effect of orchiectomy on renal function in control and diabetic rats with chronic inhibition of nitric oxide.

1. Male gender is associated with higher blood pressure (BP) and more rapid loss of renal function in a spectrum of clinical and experimental renal diseases, including diabetic nephropathy. Consequently, modulation of testosterone levels could exert beneficial effects in the diabetic kidney. 2. The aim of the present study was to determine whether testosterone deficiency (orchiectomy) could influence BP and renal function in streptozotocin-diabetic rats, with or without accelerated endothelial dysfunction achieved by chronic inhibition of nitric oxide (NO) synthesis using N(G)-nitro-L-arginine methyl ester (l-NAME; 40-100 mg/L in the drinking water for 2 weeks), as well as in age-matched non-diabetic rats subjected to the same interventions. 3. Orchiectomy did not affect L-NAME-induced increases in BP in non-diabetic or diabetic rats. In non-diabetic rats, orchiectomy prevented L-NAME-induced increases in proteinuria. These effects on proteinuria were not observed in diabetic rats. In non-diabetic rats, orchiectomy had no effect on renal haemodynamics in animals receiving vehicle and did not affect L-NAME-induced changes in renal haemodynamics, characterized by reductions in renal plasma flow (RPF) and higher filtration fractions (FF). In intact diabetic rats, L-NAME treatment resulted in lower RPF. This difference was not observed in diabetic rats subjected to orchiectomy, although L-NAME-treated diabetic orchiectomized rats had lower RPF and higher FF compared with vehicle-treated intact diabetic rats. 4. In conclusion, we report modest beneficial effects of orchiectomy on proteinuria in normal, but not in diabetic, rats with inhibition of NO production. This suggests that testosterone reduction does not attenuate the deleterious impact of the diabetic metabolic milieu in the kidney.

Effects of p38 mitogen-activated protein kinase inhibition on blood pressure, renal hemodynamics, and renal vascular reactivity in normal and diabetic rats.

p38 mitogen-activated protein kinase (p38) has been implicated in mediating vascular smooth muscle and mesangial cell contraction in response to several vasoactive factors, including angiotensin II. Early stages of diabetic nephropathy are associated with renal hemodynamic changes that are, at least in part, attributable to the dysbalance of vasoactive factors that control afferent and efferent arteriolar tone resulting in increased glomerular capillary pressure. Vascular and renal p38 have been found to be activated in diabetes. Therefore, p38 may be involved in the control of systemic and renal hemodynamics in diabetes. To address this issue, mean arterial blood pressure (MAP), glomerular filtration rate (GFR, inulin clearance), renal plasma flow (RPF, PAH clearance), metabolic parameters, and plasma renin concentrations (PRC) were determined in streptozotocin-diabetic rats (DM), and in age-matched non-diabetic controls (C), administered with the p38 inhibitor SB 239063 (SB, 50 mg/bwt, p.o.) or with vehicle. Furthermore, renal vascular responses to p38 inhibition (SB 202190, 25 microM) before and after stimulation with the endothelium-dependent vasodilator acetylcholine (ACh) were studied in vitro in tertiary branches of the renal artery from separate groups of DM and C rats, using a fixed support and a force transducer in a myograph system. SB treatment was associated with marked reductions in MAP and GFR in both C and DM rats, whereas RPF remained unchanged, as compared with vehicle-treated animals. Observed differences in MAP and renal hemodynamics were not associated with changes in urinary sodium excretion or PRC. Incubation of KCl-contracted renal arteries from both C and DM rats with the p38 inhibitor resulted in progressive and significant vasorelaxation. Also, vessels from control and diabetic rats treated with the p38 inhibitor exhibited enhancement of ACh-induced vasorelaxation. These data indicate the role of p38 in the control of systemic and renal hemodynamics both in normal and in diabetic rats. The observed effects of p38 inhibition could be mediated at least in part by enhancement of endothelium-dependent vasodilation.

Functional effects of nonsynonymous polymorphisms in the human TRPV1 gene.

The prototypical member of the vanilloid-responsive-like subfamily of transient receptor potential (TRP) channels is TRPV1. TRPV1 mediates aspects of nociception and neurogenic inflammation; however, new roles are emerging in sensation of both luminal stretch and systemic tonicity. Although at least six nonsynonymous polymorphisms in the human TRPV1 gene have been identified, there has been no systematic investigation into their functional consequences. When heterologously expressed in HEK293 cells, all variants exhibited equivalent EC(50) for the classic agonist capsaicin. This agonist elicited a greater maximal response in TRPV1(I315M) and TRPV1(P91S) variants (relative to TRPV1(WT)), as did a second agonist, anandamide. Expression of these two variants in whole-cell lysates and at the cell surface was markedly greater than that of wild-type TRPV1, whereas expression at the mRNA level was either unchanged (TRPV1(P91S)) or only very modestly increased (TRPV1(I315M)). Incorporation of multiple nonsynonymous SNPs, informed by the population-specific haplotype block structure of the TRPV1 gene, did not lead to variant channels with unique features vis-à-vis capsaicin responsiveness. Recently, polymorphisms/mutations were identified in two highly conserved TRPV1 residues in the nonobese diabetic (NOD) murine model. Incorporation of these changes into human TRPV1 gave rise to a channel with a normal EC(50) for capsaicin, but with a markedly elevated Hill slope such that the variant channel was hyporesponsive to capsaicin at low doses (<10 nM) and hyperresponsive at high doses (>10 nM). In aggregate, these data underscore expression-level and functional differences among naturally occurring TRPV1 variants; the implications with respect to human physiology are considered.

p21 is decreased in polycystic kidney disease and leads to increased epithelial cell cycle progression: roscovitine augments p21 levels.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disease with few treatment options other than renal replacement therapy. p21, a cyclin kinase inhibitor which has pleiotropic effects on the cell cycle, in many cases acts to suppress cell cycle progression and to prevent apoptosis. Because defects in cell cycle arrest and apoptosis of renal tubular epithelial cells occur in PKD, and in light of earlier reports that polycystin-1 upregulates p21 and that the cyclin-dependent kinase inhibitor roscovitine arrests progression in a mouse model, we asked whether (1) p21 deficiency might underlie ADPKD and (2) the mechanism of the salutary roscovitine effect on PKD involves p21. METHODS: p21 levels in human and animal tissue samples as well as cell lines were examined by immunoblotting and/or immunohistochemisty. Apoptosis was assessed by PARP cleavage. p21 expression was attenuated in a renal tubular epithelial cell line by antisense methods, and proliferation in response to p21 attenuation and to roscovitine was assessed by the MTT assay. RESULTS: We show that p21 is decreased in human as well as a non-transgenic rat model of ADPKD. In addition, hepatocyte growth factor, which induces transition from a cystic to a tubular phenotype, increases p21 levels. Furthermore, attenuation of p21 results in augmentation of cell cycle transit in vitro. Thus, levels of p21 are inversely correlated with renal tubular epithelial cell proliferation. Roscovitine, which has been shown to arrest progression in a murine model of PKD, increases p21 levels and decreases renal tubular epithelial cell proliferation, with no affect on apoptosis. CONCLUSION: The novelty of our study is the demonstration in vivo in humans and rat models of a decrement of p21 in cystic kidneys as compared to non-cystic kidneys. Validation of a potential pathogenetic model of increased cyst formation due to enhanced epithelial proliferation and apoptosis mediated by p21 suggests a mechanism for the salutary effect of roscovitine in ADPKD and supports further investigation of p21 as a target for future therapy.

Renal p38 MAP kinase activity in experimental diabetes.

Renal cell activity of p38 mitogen-activated protein kinase (p38) is increased in the diabetic milieu. p38 mediates signals relevant for the development of diabetic nephropathy (DN). However, renal p38 in Type 1 diabetes in vivo, particularly in conditions reflecting the differences in metabolic control, and its activity in advanced stages of DN, has received less attention. We examined the p38 pathway in renal cortex of rats with streptozotocin diabetes (4 weeks) with poor (DS), moderate (DM), and intensive (DII) metabolic control, achieved by varying doses of insulin therapy. Renal p38 was also studied in 12-month diabetic rats with established nephropathy (DM12) and compared with age-matched controls. p38 activity (in vitro kinase assay and expression of phosphorylated (active) p38 (P-p38)) was increased in DM and DS rats, as compared with non-diabetic controls, and attenuated by intensive insulin treatment. In all groups, P-p38 was predominantly localized in macula densa cells. Diabetic rats also demonstrated P-p38 immunoreactivity in the distal tubule and glomeruli. Enhanced p38 activity in DS and DM rats was not associated with increases in expression of active mitogen-activated protein kinase 3/6, an activator of p38, but paralleled with increased expression of scaffolding protein transforming growth factor-beta-activated protein kinase 1-binding protein 1. Expression of mitogen-activated protein phosphatase-1 (MKP-1), one of the phosphatases involved in inactivation of mitogen-activated protein kinase signaling, was increased in all diabetic groups, irrespective of metabolic control. Renal p38 activation was also detectable in D12 rats with established albuminuria and glomerulosclerosis. In summary, renal cortical p38 activity was increased in diabetic rats at early and advanced stages of nephropathy, as compared with non-diabetic animals, and attenuated by improved metabolic control. p38 activation in diabetes is likely to occur via multiple pathways and cannot be explained by downregulation of MKP-1.

Cyclo-oxygenase-2 inhibition attenuates the progression of nephropathy in uninephrectomized diabetic rats.

1. Cyclo-oxygenase (COX)-2 is involved in constitutive production of prostanoids in the kidney and plays a role in the control of renal function and morphology. Renal cortical COX-2 expression and function is increased in experimental models of diabetes (DM). However, pathophysiological roles of this phenomenon in the diabetic kidney have not been fully elucidated. To address this issue, we studied the nephroprotective potential of long-term (16 weeks) COX-2 inhibition in uninephrectomized streptozotocin-diabetic rats (D). 2. Diabetic rats received either a low or high dose of the selective COX-2 inhibitor MF-tricyclic (MF; 1 or 5 mg/kg per day in chow). Another group of D rats received high-dose MF as late intervention starting at 8 weeks of DM (D-MFlate). The effects of treatments were compared with age-matched uninephrectomized diabetic and non-diabetic rats receiving drug-free chow (D-VE and C-VE, respectively). 3. No differences in blood pressure and metabolic control were observed between groups of D rats throughout the study. The D-VE group developed progressive albuminuria and glomerulosclerosis, associated with increased excretion of the thromboxane (TX) A(2) metabolite TxB(2). Treatment with MF attenuated albuminuria in diabetic rats with late intervention, but not in D rats treated with MF from the onset of DM. Moreover, D-MFlate rats demonstrated a significant reduction in the development of glomerulosclerosis. These effects coincided with prevention of diabetes-induced rise in urinary TxB(2) excretion. 4. In conclusion, long-term COX-2 inhibition is associated with modest nephroprotection in uninephrectomized diabetic rats when administered as late intervention. These effects are independent of metabolic control and blood pressure.

Altered endothelial nitric oxide synthase targeting and conformation and caveolin-1 expression in the diabetic kidney.

Experimental diabetes is associated with complex changes in renal nitric oxide (NO) bioavailability. We explored the effect of diabetes on renal cortical protein expression of endothelial NO synthase (eNOS) with respect to several determinants of its enzymatic function, such as eNOS expression, membrane localization, phosphorylation, and dimerization, in moderately hyperglycemic streptozotocin-induced diabetic rats compared with nondiabetic control rats and diabetic rats with intensive insulin treatment to achieve near-normal metabolic control. We studied renal cortical expression and localization of caveolin-1 (CAV-1), an endogenous modulator of eNOS function. Despite similar whole-cell eNOS expression in all groups, eNOS monomer and dimer in membrane fractions were reduced in moderately hyperglycemic diabetic rats compared with control rats; the opposite trend was apparent in the cytosol. Stimulatory phosphorylation of eNOS (Ser1177) was also reduced in moderately hyperglycemic diabetic rats. eNOS colocalized and interacted with CAV-1 in endothelial cells throughout the renal vascular tree both in control and moderately hyperglycemic diabetic rats. However, the abundance of membrane-localized CAV-1 was decreased in diabetic kidneys. Intensive insulin treatment reversed the effects of diabetes on each of these parameters. In summary, we observed diabetes-mediated alterations in eNOS and CAV-1 expression that are consistent with the view of decreased bioavailability of renal eNOS-derived NO.

Gender hormones and the progression of experimental polycystic kidney disease.

BACKGROUND: Male gender is a risk factor for progression of autosomal-dominant polycystic kidney disease (ADPKD), clinically and in the Han:SPRD rat model. Orchiectomy limits progression, but mechanisms of the detrimental effect of androgen, and/or beneficial effects of estrogen, are not known. This protocol tested the hypothesis that male gender (intact androgen status) promotes progression, while female gender (intact estrogen status) is protective; and that these disease-modifying effects are due to changes in expression of known fibrotic mediators. METHODS: Studies were performed in male and female noncystic control (+/+) and cystic (+/-) rats subjected to orchiectomy, ovariectomy, or sham operation. At 12 weeks of age, renal function was measured. Blood and kidneys were taken for measurement of plasma and renal renin, endothelin (ET-1), endothelial nitric oxide synthase (eNOS), and vascular endothelial growth factor (VEGF), using biochemical, protein expression, and immunohistochemical methods. RESULTS: Cystic male rats exhibited significantly reduced glomerular filtration (GFR) and effective renal plasma flow (ERPF) rates, with suppression of plasma and renal renin, up-regulation of renal ET-1 and eNOS, and down-regulation of renal VEGF expression. Orchiectomy attenuated the fall in GFR and ERPF, while numerically limiting changes in eNOS and VEGF. Female rats exhibited less cystic growth, with normal renin status, lesser elevation of renal ET-1, and proportionately lesser changes in VEGF and eNOS. Ovariectomy led to higher blood pressure and reduced GFR and ERPF, with a trend toward upregulation of ET-1, and significant down-regulation of VEGF and eNOS. CONCLUSION: Female gender is protective, but ovariectomy attenuates the protective effect of female gender, in association with changes in renal expression of ET-1, VEGF, and eNOS. The accelerated disease in male rats can be attenuated by orchiectomy and consequent changes in expression of disease mediators.

EphA2: expression in the renal medulla and regulation by hypertonicity and urea stress in vitro and in vivo.

EphA2, a member of the large family of Eph receptor tyrosine kinases, is highly expressed in epithelial tissue and has been implicated in cell-cell and cell-matrix interactions, as well as cell growth and survival. Expression of EphA2 mRNA and protein was markedly upregulated by both hypertonic stress and by elevated urea concentrations in cells derived from the murine inner medullary collecting duct. This upregulation likely required transactivation of the epidermal growth factor (EGF) receptor tyrosine kinase and metalloproteinase-dependent ectodomain cleavage of an EGF receptor ligand, based on pharmacological inhibitor studies. A human EphA2 promoter fragment spanning nucleotides -4030 to +21 relative to the putative EphA2 transcriptional start site was responsive to tonicity but insensitive to urea. A promoter fragment spanning -1890 to +128 recapitulated both tonicity- and urea-dependent upregulation of expression, consistent with transcriptional activation. Neither the bona fide p53 response element at approximately -1.5 kb nor a pair of putative TonE elements at approximately -3 kb conferred the tonicity responsiveness. EphA2 mRNA and protein were expressed at low levels in rat renal cortex but at high levels in the collecting ducts of the renal medulla and papilla. Water deprivation in rats increased EphA2 expression in renal papilla, whereas dietary supplementation with 20% urea increased EphA2 expression in outer medulla. These data indicate that transcription and expression of the EphA2 receptor tyrosine kinase are regulated by tonicity and urea in vitro and suggest that this phenomenon is also operative in vivo. Renal medullary EphA2 expression may represent an adaptive response to medullary hypertonicity or urea exposure.

Effects of long-term inhibition of neuronal nitric oxide synthase (NOS1) in uninephrectomized diabetic rats.

Nitric oxide (NO) has been implicated in the pathogenesis of renal hemodynamic changes in diabetes mellitus (DM). However, the role of NO in the pathophysiology of diabetic nephropathy remains controversial. Renal hemodynamic changes in experimental DM can be acutely normalized by selective inhibition of neuronal NO synthase (nNOS). This observation suggests a nephroprotective potential of nNOS inhibition in DM. To explore this issue we assessed the long-term effects (12 weeks) of selective nNOS inhibition with the specific inhibitor S-methyl-L-thiocitrulline (SMTC) in uninephrectomized control and streptozotocin-diabetic rats. No beneficial effects of SMTC were observed in nondiabetic controls. In contrast, SMTC delayed the development of proteinuria (32+/-8 vs. 53+/-9 mg/24h, week 8, p < 0.05) and glomerulosclerosis (GS, 0.30+/-0.08 vs. 0.57+/-0.05, p < 0.05) in diabetic rats. These effects coincided with early effects of treatment on the glomerular filtration rate, and were associated with lower renal expression of nNOS. Furthermore, SMTC-treated diabetic rats demonstrated reduced weight gain and urinary sodium excretion as compared to vehicle-treated counterparts, despite similar metabolic control and blood pressure. In summary, long-term nNOS inhibition had modest nephroprotective effects in uninephrectomized diabetic rats. These effects may be mediated by renal hemodynamic mechanisms, as well as by lower food (protein) intake.

Renal expression of osmotically responsive cation channel TRPV4 is restricted to water-impermeant nephron segments.

TRPV4, a nonselective cation channel of the transient receptor potential (TRP) family, is gated by hypotonicity. Expression of TRPV4 mRNA has been detected in the circumventricular organs of the brain responsible for sensing systemic tonicity and in the kidney distal convoluted tubule (DCT), among other sites. No analysis of TRPV4 expression at the protein level has been undertaken and no systematic analysis of expression of this channel has been reported in the kidney. Via RNAse protection assay and immunoblotting, abundant expression of TRPV4 was detected in the cortex, medulla, and papilla. The expression pattern of TRPV4 was characterized in both rat and mouse kidney, which revealed similar patterns of immunoreactivity. TRPV4 expression was absent from the proximal tubule (PT) and descending thin limb (DTL), whereas the strongest expression was observed in the ascending thin limb (ATL). The thick ascending limb (TAL) was strongly positive as was the DCT and connecting tubule. Importantly, the water-permeant cells of the macula densa were unstained. Moderate TRPV4 expression was noted in all collecting duct portions and in papillary epithelium; intercalated cells (type A) exhibited a particularly strong signal. In all positive segments, TRPV4 expression was concentrated at the basolateral membrane. Therefore, TRPV4 is expressed in only those nephron segments that are constitutively (i.e., ATL, TAL, and DCT) or conditionally (i.e., collecting duct) water impermeant and where generation of a substantial transcellular osmotic gradient could be expected. TRPV4 expression is absent from nephron segments exhibiting constitutive water permeability and unregulated apical aquaporin expression (i.e., PT and DTL). These data, although circumstantial, are consistent with a role for TRPV4 in the response to anisotonicity in the mammalian kidney.

Endothelial-derived vasoactive mediators in polycystic kidney disease.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is characterized by hypertension and renal vasoconstriction. Mediators of these hemodynamic changes are not well understood, but evidence suggests that endothelial-derived mediators may participate. METHODS: Baseline measurements of blood pressure, proteinuria, and urinary nitrite/nitrate excretion were performed in control and cystic male Han:SPRD rats (6 weeks of age). They were then treated with the nitric oxide (NO), nitric oxide synthase (NOS) inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), or vehicle, for 6 weeks. After repeat systemic measurements, renal function was determined using inulin and para-aminohippurate (PAH) clearances. Levels of renal endothelin-1 (ET-1) and renal endothelial NOS (eNOS) proteins were determined, and immunohistochemistry localized renal eNOS and neuronal NOS (nNOS). RESULTS: Administration of L-NAME aggravated systemic hypertension and renal vasoconstriction in the cystic rats, but did not affect the progression of proteinuria or cystic expansion. Cystic rats demonstrated marked increases in renal ET-1 and eNOS levels. L-NAME reduced eNOS expression in the membrane compartment, but increased eNOS in the cytosol. Localization studies indicated that renal eNOS was abundant in nonvascular compartments, but not in renal vascular and glomerular structures, whereas renal nNOS was diffusely diminished. CONCLUSION: These alterations of endothelial-derived mediators (up-regulation of ET-1, and dysfunction of the NO system) contribute to vasoconstriction, and thereby are likely to contribute to the progressive loss of renal function in polycystic kidney disease (PKD).

Effects of cyclooxygenase-2 (COX-2) inhibition on plasma and renal renin in diabetes.

COX-2-derived prostaglandins (PG) have been suggested to be important modulators of renin release and expression. However, the role of COX-2 in various high-renin states is still being debated. In the present studies we explored the role of COX-2-derived PG on basal and angiotensin converting enzyme inhibitor (ACEI)-stimulated plasma and renal renin concentrations (PRC and RRC, RIA), and mRNA expression (RmRNA, RNAse protection assay) in experimental diabetes (DM). Groups of moderately hyperglycemic (n = 5, approximately 350 mg/dl), streptozotocin-diabetic rats (D) after 3 weeks of DM were treated with a selective COX-2 inhibitor, MF-tricyclic (MF, 5 mg/kg/day for 10 days in food), the combination of MF and the ACEI enalapril (3 mg/kg/day), enalapril alone, or vehicle (MF-free chow), for 10 days. Non-diabetic control rats, fed MF-free chow, were also studied. All groups of diabetic rats demonstrated similar glycemic control. Treatment with ACEI resulted in significant elevations in PRC, RRC and RmRNA as compared to non-ACEI treated groups of diabetic and control rats. A similar rise in these parameters was observed in the rats treated with the combination of ACEI and MF. Furthermore, in diabetic rats treated with MF alone, PRC and RRC were similar to vehicle-treated animals. Diabetic rats demonstrated higher urinary PG as compared to controls. MF-treated rats demonstrated a significant reduction in urinary PG excretion. In summary, selective COX-2 inhibition influenced neither basal renin status nor ACEI-induced renin release and expression in diabetic rats. These findings do not support a significant role for COX-2 in mediating renin status in diabetes.