Role of p66Shc in Regulation of Microvascular Reactivity of Renal Blood Vessels

肾微血管损伤发生在大多数糖尿病和高血压患者中， 肾病因此，揭示微血管反应性变化的分子机制， 肾血管对于开发新的治疗策略来对抗这些疾病是必要的， 大大有助于提高医疗保健水平。根据我们的初步数据，我们假设， 衔接蛋白p66 Shc的过度表达与微血管反应性的丧失有关， 高血压性肾病和糖尿病性肾病的进展。我们将利用基因 改良的Dahl盐敏感（SS）大鼠，通过靶向修饰Shc 1基因产生， 血管平滑肌细胞（SMC）。p66 Shc依赖的微血管调节 将在大鼠传入小动脉和来自死亡供体的人肾微血管中研究反应性， 糖尿病或高血压诱发的肾病病史。具体目标1将检验假设 高血压肾病中肾微血管反应性的丧失是由p66 Shc引起的， TRPC通道介导的Ca 2+内流的依赖性抑制，并将分析 TRPC通过p66 Shc调节。我们将测试p66 Shc与鸟嘌呤交换因子β-Pix相互作用的作用。 调节TRPC通道活性和通道亚细胞分布。使用双光子成像，我们将 研究p66 Shc在调节平滑肌细胞内自发性钙振荡中的作用， 人肾阻力血管壁。我们还将测试化合物SHetA 2，已知其干扰 p66 Shc作用，因为其能够预防p66 Shc诱导的高血压肾病肾功能下降。 将通过研究微血管对嘌呤能的反应来评价肾血管功能的恢复 在对照SS大鼠和用化合物SHetA 2处理的SS大鼠中的激活和灌注压力。我们将 还定义了SHetA 2是否对来自患有以下疾病的患病供体的样品中的血管功能具有有益作用： 没有高血压。具体目标2将检验p66 Shc调节小动脉KATP通道的假设 活性并导致糖尿病肾病的高滤过。KATP活性上调的作用，与 对肌源性张力调节剂的Ca 2+内流反应受损，可能促进肾血管舒张， 传入小动脉引起超滤我们将研究p66 Shc是否调节人KATP通道 肾脏微血管。我们还将采用STZ诱导的糖尿病肾病的1型糖尿病大鼠模型， 显示出与人类患者中观察到的类似的疾病标志物。p66 Shc依赖性KATP通道 将通过肾SMC中的电生理记录来测试活性。我们将测试p66 Shc是否刺激 通过诱导与衔接蛋白14-3-3的蛋白质-蛋白质相互作用的KATP通道活性。拟议 实验将为p66 Shc信号级联在肾功能调节中的作用提供直接证据。 微血管反应性和肾损伤进展，与高血压和糖尿病相关。

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.