Renal microvascular injury occurs in a majority of patients with diabetes and hypertension-induced nephropathy. Thus, the uncovering of the molecular mechanisms of changes in microvascular reactivity of renal blood vessels is necessary for developing new therapeutic strategies to combat these diseases, which contribute significantly to escalation of health care. Based on our preliminary data we hypothesize that overexpression of adaptor protein p66Shc is implicated in the loss of microvascular reactivity during the progression of both hypertension-induced nephropathy and diabetic nephropathy. We will use genetically modified Dahl salt sensitive (SS) rats, generated by targeted modification of Shc1 gene, and primary renal vascular smooth muscle cells (SMC) derived from these rats. p66Shc-dependent regulation of microvascular reactivity will be studied in rat afferent arterioles and human renal microvessels from deceased donors with medical history of either diabetes or hypertension-induced nephropathy.
Specific Aim 1 will test the hypothesis that loss of renal microvascular reactivity in hypertension induced nephropathy is caused by p66Shc- dependent inhibition of Ca2+ influx mediated by TRPC channels and will analyze molecular mechanisms of TRPC regulation by p66Shc. We will test the role of p66Shc interaction with guanine exchange factor beta-Pix in regulation of TRPC channels activity and channel subcellular distribution. Using 2-photon imaging we will study the role of p66Shc in regulation of spontaneous intracellular Ca2+ oscillations in SMC embedded in the vascular wall of human renal resistance vessels. We will also test compound SHetA2, known to interfere with p66Shc action, for its ability to prevent p66Shc-induced decline of renal function in hypertension nephropathy. The restoration of renal vascular function will be evaluated by studying microvascular responses to purinergic activation and perfused pressure in the control SS rats and SS rats treated with compound SHetA2. We will also define whether SHetA2 has beneficial effect on vascular function in samples from diseased donors with and without hypertension.
Specific Aim 2 will test the hypothesis that p66Shc regulates arteriolar KATP channel activity and causes hyperfiltration in diabetic nephropathy. Effect of upregulation of KATP activity, in concert with impaired Ca2+ influx responses to modulators of myogenic tone, is likely to promote vasodilation of renal afferent arterioles, causing hyperfiltration. We will study whether p66Shc regulates KATP channels in human renal microvessels. We will also employ type 1 diabetic rat model of STZ-induced diabetic nephropathy, which display markers of the disease similar to those observed in human patients. p66Shc-dependent KATP channel activity will be tested by electrophysiological recording in renal SMC. We will test whether p66Shc stimulates KATP channel activity via inducing protein-protein interactions with adaptor protein 14-3-3. The proposed experiments will provide direct evidence for the role of p66Shc signaling cascade in the regulation of renal microvascular reactivity and progression of renal injury, associated with hypertension and diabetes.
Renal microvascular reactivity is decreased in hypertension-induced nephropathy and diabetes. Based on our preliminary data we propose to test whether protein p66Shc suppresses microvascular reactivity of renal blood vessels, and whether inhibition of p66Shc action will prevent hypertension- and diabetes-related vascular dysfunction. Analysis of unique genetically modified rats and primary vascular cells, derived from these rats, will be combined with assessing vascular function of isolated rat and human renal microvessels in order to uncover signaling mechanisms causing loss of microvascular reactivity and provide novel targets for treatment of human vascular diseases.
