Molecular and functional studies have suggested that AT1 receptors are present in most nephron segments, yet direct demonstration of AT1 at these sites is lacking. The present study was performed to determine the intrarenal localization of the AT1 receptor utilizing a monoclonal anti-peptide (amino acid residues 8-17) antibody (6313/G2) in adult male Sprague-Dawley rats. Western blot analysis of kidney protein extracts showed a predominant 41-kDa immunoreactive band corresponding to the molecular weight of the deduced cDNA sequence. To determine optimal fixation conditions, kidney tissues were immersion fixed in Bouin's solution, 10% buffered Formalin, or 4% paraformaldehyde. Specificity of immunostaining was documented by preadsorption of the antibody with the immunogenic peptide sequence. Prominent AT1 immunostaining was visualized in the proximal tubule brush-border and basolateral membranes. In addition, distal tubules, cortical and medullary collecting ducts, and the renal arterial vasculature exhibited specific immunoreactivity. Glomerular staining for AT1 was observed in mesangial cells and podocytes. Macula densa cells stained positively. Similar localization of the AT1 receptor was obtained using the three tissue fixation methods, although the intensity of vascular and glomerular staining was highest in Bouin-fixed tissues. The present study demonstrates that the AT1 receptor is more widely distributed along the nephron than previously described and includes renal vascular smooth muscle and proximal and distal epithelial sites.

We examined the role of the plasminogen activator/plasmin system in extracellular matrix (ECM) degradation by human mesangial cells cultured on thin films of 125I-labeled ECM (Matrigel). ECM degradation (release of 125I into the medium) was dependent on exogenous plasminogen, proportional to the number of mesangial cells and amount of plasminogen added, and coincident with the appearance of plasmin in the medium. ECM degradation was completely blocked (P < 0.001) by two plasmin inhibitors, alpha-2-antiplasmin (40 micrograms/ml) and aprotinin (216 KIU/ml), and partially reduced (-33 +/- 1.8%, P < 0.01) by TIMP-1 (40 micrograms/ml), a specific inhibitor of matrix metalloproteinases. Zymography of medium obtained from cells cultured in the absence of plasminogen revealed the presence of latent matrix metalloproteinase-2 (MMP-2) which was converted to a lower molecular weight, active form in the presence of mesangial cells and plasminogen. Northern analysis of poly A+RNA prepared from cultured human mesangial cells revealed mRNA for tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), and uPA receptor (uPAR). The presence of uPA protein in medium obtained from cultured human mesangial cells was demonstrated by Western blotting and ELISA which revealed a large molar excess of PAI-1 (1.2 +/- 0.1 x 10(-9) M) over uPA (1.2 +/- 0.1 x 10(-12) M) and tPA (0.19 +/- 0.04 x 10(-9) M). ECM degradation was reduced by a monoclonal antibody (MAb) against human tPA (-54 +/- 8.6%) or human uPA (-39 +/- 5.2%) compared to cells treated with identical amounts of non-specific monoclonal IgG (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

The pathogenesis of arterial hypertension often involves a rise in systemic vascular resistance (vasoconstriction and vascular remodeling) and impairment of salt excretion in the kidney (inappropriate salt retention despite elevated blood pressure). Experimental and clinical evidence implicate an imbalance between endogenous vasoconstrictor and vasodilator systems in the development and maintenance of hypertension. Kinins (bradykinin and lys-bradykinin) are endogenous vasodilators and natriuretic peptides known best for their ability to antagonize angiotensin-induced vasoconstriction and sodium retention. In humans, angiotensin-converting enzyme inhibitors, a potent class of antihypertensive agents, lower blood pressure at least partially by favoring enhanced kinin accumulation in plasma and target tissues. The beneficial actions of kinins in renal and cardiovascular disease are largely mediated by nitric oxide and prostaglandins, and extend beyond their recognized role in lowering blood pressure to include cardioprotection and nephroprotection. This article is a review of exciting, recently generated genetic, biochemical and clinical data from studies that have examined the importance of the tissue kallikrein-kinin system in protection from hypertension, vascular remodeling and renal fibrosis. Development of novel therapeutic approaches to bolster kinin activity in the vascular wall and in specific compartments in the kidney might be a highly effective strategy for the treatment of hypertension and its complications, including cardiac hypertrophy and renal failure.

Histone deacetylases (HDACs) regulate fundamental biological processes such as cellular proliferation, differentiation, and survival via genomic and nongenomic effects. This study examined the importance of HDAC activity in the regulation of gene expression and differentiation of the developing mouse kidney. Class I HDAC1-3 and class II HDAC4, -7, and -9 genes are developmentally regulated. Moreover, HDAC1-3 are highly expressed in nephron precursors. Short term treatment of cultured mouse embryonic kidneys with HDAC inhibitors (HDACi) induced global histone H3 and H4 hyperacetylation and H3K4 hypermethylation. However, genome-wide profiling revealed that the HDAC-regulated transcriptome is restricted and encompasses regulators of the cell cycle, Wnt/␤-catenin, TGF-␤/Smad, and PI3K-AKT pathways. Further analysis demonstrated that base-line expression of key developmental renal regulators, including Osr1, Eya1, Pax2/8, WT1, Gdnf, Wnt9b, Sfrp1/2, and Emx2, is dependent on intact HDAC activity. Treatment of cultured embryonic kidney cells with HDACi recapitulated these gene expression changes, and chromatin immunoprecipitation assays revealed that HDACi is associated with histone hyperacetylation of Pax2/Pax8, Gdnf, Sfrp1, and p21. Gene knockdown studies demonstrated that HDAC1 and HDAC2 play a redundant role in regulation of Pax2/8 and Sfrp1 but not Gdnf. Long term treatment of embryonic kidneys with HDACi impairs the ureteric bud branching morphogenesis program and provokes growth arrest and apoptosis. We conclude that HDAC activity is critical for normal embryonic kidney homeostasis, and we implicate class I HDACs in the regulation of early nephron gene expression, differentiation, and survival.Kidney development depends on reciprocal inductive interactions between the metanephric mesenchyme (MM), 4 a specified region in the caudal intermediate mesoderm, and the ureteric bud (UB), an epithelial outgrowth from the Wolffian (nephric) duct (1-3). Recent years have witnessed significant progress in our understanding of the gene regulatory networks of early kidney development (3-6). For example, the Osr1/ Eya1/Pax2/Six/Sall/WT1/Hoxd11 gene regulatory network specifies the MM and is absolutely required for expression of glia-derived neurotrophic factor (Gdnf) (7,8). Gdnf, in turn, is essential for UB outgrowth and subsequent branching (9 -11).Gdnf acts via activation of a c-Ret/PI3K/ERK-dependent gene network (Wnt11, Spry1, Etv4, Etv5, Cxcr4, Myb, Met, and Mmp14) in UB tip cells to control the branching morphogenesis program (12-14). Various growth factor/receptor signaling pathways, including FGFs, bone morphogenic proteins, VEGF, semaphorins, hepatocyte growth factor, EGF, among others, share signaling components with the c-Ret pathway and are required for optimal metanephric growth and patterning (15)(16)(17)(18)(19)(20)(21)(22). Following induction of the MM, activation of the Wnt/␤-catenin signaling pathway plays a key role in nephrogenesis. Release of Wnt9b from the UB branches triggers a ␤-catenindependent mo...

The objective of this study was to investigate the singular role of elevated angiotensin II (ANG II) levels in the development of two-kidney, one-clip (2K1C) Goldblatt hypertension in the rat and specifically in the altered intrarenal ANG II levels that occur in the nonclipped kidney. As a substitute for the clipped kidney, chronic delivery of ANG II (40 ng/min) via an osmotic minipump implanted subcutaneously was used to mimic plasma ANG II levels observed in 2K1C rats during the developmental phase of hypertension. Arterial pressure increased gradually over a period of 14 days, and a pressure profile similar in magnitude and temporal pattern to that of the 2K1C rats was observed. Systemic ANG II was elevated to similar levels in the 2K1C (60 +/- 13 fmol/ml) and ANG II-infused rats (72 +/- 15 fmol/ml) compared with intact two-kidney control animals (31 +/- 6 fmol/ml; P < 0.05) or uninephrectomized rats (13 +/- 1 fmol/ml; P < 0.05). Although renin content was markedly suppressed (80%), intrarenal ANG II content of the contralateral kidneys of the 2K1C groups (86 +/- 12 fmol/g) and the ANG II-infused group (150 +/- 17 fmol/g) was greater than that of the two-kidney control (53 +/- 7 fmol/g; P < 0.05) and uninephrectomized control animals (42 +/- 5 fmol/g; P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)