In renal mesangium endothelin-1 (ET-1) exerts excessive contraction, proliferation and extracellular matrix accumulation leading to glomerulosclerosis and kidney failure. The molecular mechanisms of ET-1 actions in renal mesangium are insufficiently studied. In the current grant application we aim to prove that novel ET-1 mediated signaling pathways, discovered by us in cultured glomerular mesangial cells (GMC), play principal role in glomerular diseases in vivo when ET-1 production is increased and renal mesangium is dysfunctional. To achieve these goals we have generated unique rat strains in which we precisely modified rat genome using engineered Zinc Finger Nucleases (ZFNs) in combination with innovative in vivo knock-in strategy. Until recently the precise modification of rat genome was not possible, but the generation of targeted gene changes using ZFNs in inbred rat strains has become one of the major breakthroughs in the field dramatically increasing opportunities of investigators in utilizing rats for biomedical research. In our preliminary studies we have discovered novel signaling pathway stimulated by ET-1 in GMC which involves the formation of multiunit signaling complex including adaptor protein p66 Shc. We hypothesize that ET-1 signaling via adaptor protein p66 Shc in renal mesangium in vivo is contributing to kidney pathologies associated with abnormal function of renal mesangial cells.
In specific aim 1 we will test whether ET-1-mediated signaling via p66 Shc contributes to renal injury in glomerular diseases associated with enhanced ET-1 production and abnormal glomerular function. We will induce anti-Thy-1.1 nephritis and hypertension-induced nephropathy in rats which either lack p66 Shc protein or express endogenous p66 Shc with introduced mutations. The extent of renal injury will be assessed.
In specific aim 2 we will use primary GMC derived from wild type and genetically modified rat strains to uncover the molecular mechanism of p66 Shc signaling in renal mesangium. We will test the hypothesis that p66 Shc promotes GMC proliferation via inactivation of transcription factor FOXO3a and restricts GMC contractility through regulation of calcium influx. These studies are important because abnormal GMC function is detected in the majority of patients with hypertension induced nephropathy and glomerulosclerosis. The elucidation of mechanisms of ET-1-induced renal pathologies will result in understanding of the mechanisms underlying proliferation-associated and oxidative stress related renal glomerular diseases.
Endothelin-1 (ET-1) signaling in renal mesangium plays an essential role for kidney glomerular diseases. The molecular mechanisms of ET-1 actions in renal mesangium are insufficiently studied. The goal of this grant application is to prove that novel ET-1 mediated signaling pathways, discovered by us in cultured renal glomerular mesangial cells, play principal role in renal injury which occurs in glomerular diseases when ET-1 production is increased and renal mesangium is dysfunctional. To achieve these goals we will induce glomerular diseases in unique rat strains which we have generated by precision modification of rat genome using engineered Zinc Finger Nucleases (ZFNs) in combination with innovative in vivo knock-in strategy. The elucidation of mechanisms of ET-1-induced renal pathologies will result in understanding of the mechanisms underlying proliferation-associated and oxidative stress related renal glomerular diseases.
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