Abstract-The (pro)renin receptor ([P]RR) is a transmembrane protein that binds both renin and prorenin with high affinity, increasing the catalytic cleavage of angiotensinogen and signaling intracellularly through mitogen-activated protein kinase activation. Although initially reported as having no homology with any known membrane protein, other studies have suggested that the (P)RR is an accessory protein, named ATP6ap2, that associates with the vacuolar H ϩ -ATPase, a key mediator of final urinary acidification. Using in situ hybridization, immunohistochemistry, and electron microscopy, together with serial sections stained with nephron segment-specific markers, we found that (P)RR mRNA and protein were predominantly expressed in collecting ducts and in the distal nephron. Within collecting ducts, the (P)RR was most abundant in microvilli at the apical surface of A-type intercalated cells. Dual-staining immunofluorescence demonstrated colocalization of the (P)RR with the B1/2 subunit of the vacuolar H ϩ -ATPase, the ion exchanger that secretes H ϩ ions into the urinary space and that associates with an accessory subunit homologous to the (P)RR. In collecting duct/distal tubule lineage Madin-Darby canine kidney cells, extracellular signal-regulated kinase 1/2 phosphorylation, induced by either renin or prorenin, was attenuated by the selective vacuolar H ϩ -ATPase inhibitor bafilomycin. The predominant expression of the (P)RR at the apex of acid-secreting cells in the collecting duct, along with its colocalization and homology with an accessory protein of the vacuolar H ϩ -ATPase, suggests that the (P)RR may function primarily in distal nephron H ϩ transport, recently noted to be, at least in part, an angiotensin II-dependent phenomenon. Key Words: (pro)renin receptor Ⅲ intercalated cell Ⅲ vacuolar H ϩ -ATPase Ⅲ ATP6ap2 Ⅲ prorenin Ⅲ renin-angiotensin system Ⅲ bafilomycin A little more than a decade ago, the binding characteristics and activity of a specific renin receptor in cultured mesangial cells were reported. 1 This was followed in 2002 by the identification of an apparently novel, 350 amino acid, single-transmembrane protein that binds both renin and prorenin with high affinity. 2 Ligand binding to this (pro)renin receptor ([P]RR) induced a 4-fold increase in the catalytic cleavage of angiotensinogen, as well as stimulating intracellular signaling, with activation of mitogen-activated protein kinases extracellular signal-regulated kinase (ERK) 1/2 2 and induction of transforming growth factor-␤ expression. 3 The existence of a (P)RR not only expanded our understanding of the physiology of the renin-angiotensin system (RAS) but also provided insight into the potential pathogenetic role of prorenin, the enzymatically inactive zymogen that is elevated in disease states, eg, diabetes mellitus, where it predicts the subsequent development of nephropathy and retinopathy. 4 Given its localization to the mesangium, its actions in augmenting local angiotensin II production, and its ability to increase mesangial tr...

Experimental type I diabetes mellitus is characterized by an early increase in kidney weight and glomerular volume, but changes in gene expression accompanying diabetic renal growth have not been fully elucidated. In the current study, total RNA was extracted from renal cortex and isolated glomeruli of streptozotocin-induced diabetic rats 24 hours, 48 hours, 96 hours, one and two weeks after the onset of hyperglycemia (blood glucose > 15 mmol/liter), insulin-treated diabetic rats (blood glucose < 6.0 mmol/liter), and normal rats. RNA samples were reverse transcribed (RT) and subjected to polymerase chain reaction (PCR) amplication with specific 5' and 3' primers for rat transforming growth factor (TGF-beta 1) and beta-actin. RT-PCR analysis revealed that glomerular TGF-beta 1 mRNA levels increased relative to beta-actin as early as 24 hours after the onset of hyperglycemia, reaching a plateau after 96 hours that was sustained at one and two weeks. In cortical samples, TGF-beta 1 mRNA levels increased less abruptly, reaching a peak one week after the onset of hyperglycemia. Intensive insulin treatment to normalize blood glucose levels attenuated the rise in glomerular and renal cortical TGF-beta 1 mRNA. Cryostat sections of rat kidneys were immunostained for TGF-beta 1 utilizing a polyclonal anti-porcine TGF-beta 1 antibody and semiquantitative scoring of TGF-beta 1 immunostaining revealed a twofold increase in diabetic glomeruli after two weeks compared to normal glomeruli. Increased segmental immunostaining for TGF-beta 1 was also evident in cortical tubules of diabetic rats. These studies establish that TGF-beta 1 expression in the kidney increases during the phase of rapid renal hypertrophy in diabetic rats. Normalization of blood glucose levels with insulin treatment attenuates the increase in TGF-beta 1 expression.

Inhibiting the actions of VEGF is a new therapeutic paradigm in cancer management with antiangiogenic therapy also under intensive investigation in a range of nonmalignant diseases characterized by pathological angiogenesis. However, the effects of VEGF inhibition on organs that constitutively express it in adulthood, such as the kidney, are mostly unknown. Accordingly, we examined the effect of VEGF inhibition on renal structure and function under physiological conditions and in the setting of the common renal stressors: hypertension and activation of the reninangiotensin system. When compared with normotensive SpragueDawley (SD) rats, glomerular VEGF mRNA was increased 2-fold in transgenic (mRen-2)27 rats that overexpress renin with spontaneously hypertensive rat (SHR) kidneys showing VEGF expression levels that were intermediate between them. Administration of either an orally active inhibitor of the type 2 VEGF receptor (VEGFR-2) tyrosine kinase or a VEGF neutralizing antibody to TGR(mRen-2)27 rats resulted in loss of glomerular endothelial cells and transformation to a malignant hypertensive phenotype with severe glomerulosclerosis. VEGFR-2 kinase inhibition treatment was well tolerated in SDs and SHRs; although even in these animals there was detectable endothelial cell loss and rise in albuminuria. Mild mesangial expansion was also noted in hypertensive SHR, but not in SD rats. These studies illustrate: (i) VEGF has a role in the maintenance of glomerular endothelial integrity under physiological circumstances, (ii) glomerular VEGF is increased in response to hypertension and activation of the renin-angiotensin system, and (iii) VEGF signaling plays a protective role in the setting of these renal stressors.albuminuria ͉ endothelium ͉ hypertension ͉ Ren-2 ͉ renin-angiotensin system

Endothelial nitric oxide synthase (eNOS) deficiency may contribute to the pathogenesis of diabetic nephropathy in both experimental models and humans, but the underlying mechanism is not fully understood. Here, we studied two common sequelae of endothelial dysfunction in diabetes: glomerular capillary growth and effects on neighboring podocytes. Streptozotocin-induced diabetes increased glomerular capillary volume in both C57BL/6 and eNOS 2/2 mice. Inhibiting the vascular endothelial growth factor receptor attenuated albuminuria in diabetic C57BL/6 mice but not in diabetic eNOS 2/2 mice, even though it inhibited glomerular capillary enlargement in both. In eNOS 2/2 mice, an acute podocytopathy and heavy albuminuria occurred as early as 2 weeks after inducing diabetes, but treatment with either captopril or losartan prevented these effects. In vitro, serum derived from diabetic eNOS 2/2 mice augmented actin filament rearrangement in cultured podocytes. Furthermore, conditioned medium derived from eNOS 2/2 glomerular endothelial cells exposed to both high glucose and angiotensin II activated podocyte RhoA. Taken together, these results suggest that the combined effects of eNOS deficiency and hyperglycemia contribute to podocyte injury, highlighting the importance of communication between endothelial cells and podocytes in diabetes. Identifying mediators of this communication may lead to the future development of therapies targeting endothelial dysfunction in albuminuric individuals with diabetes.

Epigenetic changes in gene expression play a role in the development of diabetic complications, including nephropathy. Histone deacetylases (HDACs) are a group of enzymes that exert epigenetic effects by altering the acetylation status of histone and nonhistone proteins. In the current study, we investigated the action of the clinically available HDAC inhibitor vorinostat in a mouse model of diabetic nephropathy, with the following aims: to define its effect on the progression of renal injury and to explore its mechanism of action by focusing on its role in regulating the expression of endothelial nitric oxide synthase (eNOS). Control and streptozotocin-diabetic wild-type and eNOS(-/-) mice were treated with vorinostat by daily oral dosing for 18 weeks. Without affecting either blood glucose concentration or blood pressure, vorinostat decreased albuminuria, mesangial collagen IV deposition, and oxidative-nitrosative stress in streptozotocin-wild-type mice. These attenuating effects were associated with a >50% reduction in eNOS expression in mouse kidneys and in cultured human umbilical vein endothelial cells. Vorinostat treatment had no effect on albuminuria, glomerular collagen IV concentration, or mesangiolysis in diabetic mice genetically deficient in eNOS. These observations illustrate the therapeutic efficacy of long-term HDAC inhibition in diabetic nephropathy and emphasize the importance of the interplay between eNOS activity and oxidative stress in mediating these effects.

The hexosamine pathway may mediate some of the toxic effects of glucose. We hypothesized that flux through this pathway might regulate the activity of nuclear factor B (NF-B)-dependent genes in mesangial cells (MCs). In MCs, RT-PCR revealed that high glucose (30 mmol/l) and glucosamine (1 mmol/l) increased mRNA levels for vascular cell adhesion molecule 1 (VCAM-1) and increased the activity of an NF-B enhancer by 1.5-and 2-fold, respectively. Overexpression of glutamine: fructose-6-phosphate amidotransferase (GFAT), the rate-limiting enzyme for flux through the hexosamine pathway, led to a 2.2-fold increase in NF-B enhancer activity; the combination of GFAT overexpression and high glucose increased activity 2.8-fold, and these increases were prevented by 40 mol/l O-diazoacetyl-Lserine (azaserine) or 6-diazo-5-oxonorleucine. High glucose, glucosamine, and GFAT overexpression increased binding of MC nuclear proteins to NF-B consensus sequences. Immunoblotting revealed that the p65 subunit of NF-B was O-glycosylated in MC cultured in physiologic glucose and that significant enhancement occurred with high glucose and glucosamine. Both glucose and glucosamine dose-dependently increased human VCAM-1 promoter activity. In addition, GFAT overexpression activated the VCAM-1 promoter (2.25-fold), with further augmentation by high glucose and abrogation by inhibitors of GFAT, NF-B, and O-glycosylation. Inactivation of the two NF-B sites in the VCAM-1 promoter abolished its response to high glucose, glucosamine, and GFAT overexpression. These results suggest that increased flux through the hexosamine pathway leads to NF-B-dependent promoter activation in MCs.

Genetic variability in the renin-angiotensin system may modify renal responses to injury and disease progression. We examined whether the M235T polymorphism of the angiotensinogen (AGT) gene, the insertion/deletion polymorphism of the angiotensin-converting enzyme (ACE) gene, and the A 1166 → C polymorphism of the angiotensin II type 1 receptor gene may be associated with disease progression in 168 Caucasian patients with IgA nephropathy. All patients had serial measurements of their creatinine clearance, proteinuria, and blood pressure (mean Ϯ SD) with a follow-up of 6.1 Ϯ 4.7 yr. The genotype frequencies for each gene were consistent with Hardy-Weinberg equilibrium, and were similar to those of 100 Caucasian control subjects. We examined two primary outcomes: ( a ) the rate of deterioration of Ccr, and ( b ) the maximal level of proteinuria. We found that patients with the AGT MT ( n ϭ 79) and TT ( n ϭ 29) genotypes had a faster rate of deterioration of Ccr than those with the MM ( n ϭ 60) genotype (i.e., median values, Ϫ 6.6 and Ϫ 6.2 vs.

Excessive reactive oxygen species play a key role in the pathogenesis of diabetic nephropathy, but to what extent these result from increased generation, impaired antioxidant systems, or both is incompletely understood. Here, we report the expression, localization, and activity of the antioxidant thioredoxin and its endogenous inhibitor thioredoxin interacting protein (TxnIP) in vivo and in vitro. In normal human and rat kidneys, expression of TxnIP mRNA and protein was most abundant in the glomeruli and distal nephron (distal convoluted tubule and collecting ducts). In contrast, thioredoxin mRNA and protein localized to the renal cortex, particularly within the proximal tubules and to a lesser extent in the distal nephron. Induction of diabetes in rats increased expression of TxnIP but not thioredoxin mRNA. Kidneys from patients with diabetic nephropathy had significantly higher levels of TxnIP than control kidneys, but thioredoxin expression did not differ. In vitro, high glucose increased TxnIP expression in mesangial, NRK (proximal tubule), and MDCK (distal tubule/collecting duct) cells, and decreased the expression of thioredoxin in mesangial and MDCK cells. Knockdown of TxnIP with small interference RNA suggested that TxnIP mediates the glucose-induced impairment of thioredoxin activity. Knockdown of TxnIP also abrogated both glucose-induced 3 H-proline incorporation (a marker of collagen production) and oxidative stress. Taken together, these findings suggest that impaired thiol reductive capacity contributes to the generation of reactive oxygen species in diabetes in a site-and cell-specific manner.