p66Shc regulates renal vascular tone in hypertension-induced nephropathy.

Renal preglomerular arterioles regulate vascular tone to ensure a large pressure gradient over short distances, a function that is extremely important for maintaining renal microcirculation. Regulation of renal microvascular tone is impaired in salt-sensitive (SS) hypertension-induced nephropathy, but the molecular mechanisms contributing to this impairment remain elusive. Here, we assessed the contribution of the SH2 adaptor protein p66Shc (encoded by Shc1) in regulating renal vascular tone and the development of renal vascular dysfunction associated with hypertension-induced nephropathy. We generated a panel of mutant rat strains in which specific modifications of Shc1 were introduced into the Dahl SS rats. In SS rats, overexpression of p66Shc was linked to increased renal damage. Conversely, deletion of p66Shc from these rats restored the myogenic responsiveness of renal preglomerular arterioles ex vivo and promoted cellular contraction in primary vascular smooth muscle cells (SMCs) that were isolated from renal vessels. In primary SMCs, p66Shc restricted the activation of transient receptor potential cation channels to attenuate cytosolic Ca2+ influx, implicating a mechanism by which overexpression of p66Shc impairs renal vascular reactivity. These results establish the adaptor protein p66Shc as a regulator of renal vascular tone and a driver of impaired renal vascular function in hypertension-induced nephropathy.

Endothelin signaling via guanine exchange factor C3G in renal glomerular mesangial cells.

The guanine nucleotide exchange factor C3G is one of the mediators of endothelin-1 (ET-1) intracellular signaling cascades and is vital for kidney development and homeostasis. The aim of the current study was to analyze the specificity of ET-1-induced signaling via C3G in rat glomerular mesangial cells (GMC) and to investigate the biological significance of C3G during mesangioproliferative glomerulonephritis. In GMC, C3G expression was increased (1) in vivo after induction of the anti-Thy1 model of glomerulonephritis and (2) in cell culture experiments after fetal bovine serum incubation. To examine the consequences of C3G up-regulation, adenovirus-mediated gene transfer of C3G into cultured glomerular cells was done, and the GTP loading of the small G proteins Rap1 and R-Ras was analyzed. Overexpression of C3G in mesangial cells resulted in enhanced activation of Rap1, but failed to affect the GTP-bound status of R-Ras in ET-1-stimulated cells. C3G overexpression led to significant changes in GMC spreading and migration patterns in response to ET-1 stimulation and increased stress fiber formation, which was mimicked by Rap1A overexpression. Together, these findings suggest (1) the existence of regulatory mechanisms resulting in disease-related up-regulation of C3G in GMC and (2) that an increase in the C3G protein level may contribute to the resolution stage of mesangioproliferative glomerulonephritis by reducing GMC sensitivity to ET-1, modulating cellular motility, and actin dynamics.

Down-regulation of 20-HETE synthesis and signaling inhibits renal adenocarcinoma cell proliferation and tumor growth.

BACKGROUND: We examined the ability of inhibitors of the synthesis or actions of 20-HETE, metabolite of arachidonic acid, to inhibit proliferation of human renal carcinoma cell lines. MATERIALS AND METHODS: 786-O and 769-P cells were exposed to either 10 microM HET0016 (selective inhibitor of 20-HETE synthesis), 10 microM WIT002 (20-HETE antagonist), or vehicle. Subsequently, we assessed the effect of WIT002 on tumor growth in vivo using an ectopic mouse model of clear-cell renal carcinoma. RESULTS: Addition of HET0016 and WIT002 inhibited the proliferation of 786-O and 769-P human renal cell carcinoma lines. HET0016 and WIT002 had little effect on the proliferation of primary cultures of normal human proximal tubule epithelial cells. WIT002 (10 mg/kg, s.c.) administered daily to athymic nude mice implanted subcutaneously with 786-O cells reduced the growth of the tumors by 84 % compared to vehicle (p<0.001). CONCLUSION: 20-HETE is required for proliferation of human renal epithelial cancer.

Endothelin-converting enzyme inhibition in the rat model of acute heart failure: heart function and neurohormonal activation.

Endothelin-1 (ET-1) has been implicated in many cardiovascular diseases, including acute heart failure (AHF) due to myocardial ischemia. Previously we described the oral endothelin-converting enzyme (ECE) inhibitor, PP36, and in this study, we investigated its cardioprotective effect in more detail, and examined the role of PP36 in the neurohormonal activation in rats that had been subjected to acute myocardial ischemia due to the microsphere embolization of coronary microcirculation. PP36 treatment (3.5 x 10(-5) M/kg/day) led to a significant fourfold decrease in hypertensive response when big-ET-1 was administered to healthy, conscious rats. ECE inhibition did not affect mortality during the first 48 hours after ischemia initiation. Systemic hemodynamic, heart function, and neurohormonal activation were analyzed in the healthy control group, the AHF group, and the AHF+PP36 group two days after AHF induction. In conscious rats in the AHF+PP36 group, mean arterial pressure (MAP) was restored and became similar to that of the MAP of the control group. In anesthetized rats, in the AHF+PP36 group, MAP was not restored and was 22% lower than the MAP of the control group. Myocardial contractility was partially restored and cardiac relaxation significantly improved after PP36 application. Further analysis of cardiac output and peripheral resistance in anesthetized rats revealed no differences between the AHF group and the AHF+PP36 group. There were no differences in plasma ET-1 concentration, serum angiotensin converting enzyme activity, and in the adrenal glands' catecholamine content between the AHF group and the AHF+PP36 group. However, rats in the AHF+PP36 group demonstrated a 60% decrease in cardiac endothelial nitric oxide synthase (eNOS) protein expression, and a 56% reduction of myocardial norepinephrine release, when compared with the AHF group's animals. These results suggest that PP36 can preserve heart function during the recovery from acute ischemic injury, and may modulate the cardiac norepinephrine release and eNOS protein level.

C3G overexpression in glomerular epithelial cells during anti-GBM-induced glomerulonephritis.

The guanine nucleotide exchange factor C3G, along with the CrkII adaptor protein, mediates GTP activation of the small GTPase proteins Rap1 and R-Ras, facilitating their activation of downstream signaling pathways, which had been found to be important in the pathogenesis of glomerulonephritis. We found that expression of C3G protein was upregulated in glomerular epithelial cells in an experimental model of accelerated anti-GBM antibody-induced glomerulonephritis expression. To determine the consequence of its increased expression, we transfected C3G (using adenoviral constructs) into cultured glomerular epithelial cells and measured the activated forms (i.e., GTP-bound) forms of Rap1 and R-Ras. Activation of Rap1 was not affected by C3G; however, the basal level of GTP-bound R-Ras was decreased. Further, C3G over-expression enhanced the activation of R-Ras in response to endothelin. Overexpression of C3G also led to a significant reduction in glomerular epithelial cell spreading and decreased the cells' E-cadherin expression and augmented their migration. We found that C3G was overexpressed in accelerated anti-GBM antibody-induced glomerulonephritis and suggest that this modulates glomerular epithelial cell morphology and behavior.

Pyk2 mediates endothelin-1 signaling via p130Cas/BCAR3 cascade and regulates human glomerular mesangial cell adhesion and spreading.

Calcium-regulated non-receptor proline-rich tyrosine kinase 2 (Pyk2) is a critical mediator of endothelin-1 (ET-1) signaling in human glomerular mesangial cells (GMC). We aimed to identify which small G-protein is acting downstream of Pyk2. Dominant interfering Pyk2 construct, termed calcium regulated non kinase (CRNK) or green fluorescent protein (control) were expressed in GMC using adenovirus-mediated gene transfer. ET-1 stimulation resulted in a significant increase of Pyk2 phosphorylation accompanied by GTP-loading of Rap1 and RhoA. CRNK expression inhibited ET-1-induced autophosphorylation of endogenous Pyk2 and diminished Rap1, but not RhoA, activation. The mechanism linking Pyk2 and Rap1 included (1) increased autophosphorylation of Pyk2 associated with p130Cas, (2) augmented p130Cas Y165 and Y249 phosphorylation, and (3) enhanced p130Cas-BCAR3 complex formation. CRNK expression prevented p130Cas phosphorylation and attenuated p130Cas association with BCAR3. Downregulation of endogenous BCAR3 protein expression using an siRNA technique led to a significant decrease in Rap1 activation in response to ET-1. We observed that endogenous Pyk2 was important for GMC adhesion and spreading. Our data suggest that ET-1 stimulated the GTPase Rap1 (but neither RhoA nor Ras) by a mechanism involving Pyk2 activation and recruitment of the p130Cas/BCAR3 complex in GMC.

CrkII associates with BCAR3 in response to endothelin-1 in human glomerular mesangial cells.

Endothelin-1 (ET-1) effects in human glomerular mesangial cells (GMC) include proliferation, contraction, and extracellular matrix synthesis. Calcium-regulated nonreceptor, proline-rich tyrosine kinase 2 (Pyk2) is a critical mediator of ET-1 signaling in human glomerulae. Working in concert with Pyk2, adaptor protein CrkII and a recently discovered guanidine exchange factor for certain small GTPases BCAR3 can be involved in ET-1 signaling in the kidney. Signaling through CrkII and BCAR3 might be critical in some proliferative kidney pathologies. The current study was designed to determine the possibility of CrkII and BCAR3 interaction in response to ET-1 in human GMC and the role of Pyk2 in the association of these proteins. Using adenovirus-mediated transfer of genes encoding either green fluorescent protein (control) or dominant interfering Pyk2 construct, we demonstrated that CrkII and BCAR3 can be coprecipitated from unstimulated and ET-1 stimulated GMC; ET-1 treatment time-dependently increased CrkII/BCAR3 complex formation; and inhibition of endogenous Pyk2 autophosphorylation led to a significant decrease in CrkII/BCAR3 association both basal and stimulated.