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

p66Shc 在肾血管微血管反应性调节中的作用

• 批准号: 10198033
• 负责人: ANDREY SOROKIN
• 金额: $ 38.41万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-22 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10198033
• 关键词: Adaptor Signaling Protein; Address; Aging; Blood Vessels; Cells; Chronic Kidney Failure; Dahl Hypertensive Rats; Data; Development; Diabetes Mellitus; Diabetic Nephropathy; Disease; Disease Marker; Electrophysiology (science); Evaluation; General Population; Genes; Guanine; Guanine Nucleotide Exchange Factors; Healthcare; Homeostasis; Human; Hypertension; Image; Impairment; Injury; Injury to Kidney; Kidney; Kidney Diseases; Knock-out; Knowledge; Lead; Location; Longevity; Mediating; Medical History; Microvascular Dysfunction; Mitochondria; Molecular; Oxidative Stress; Pathogenesis; Patients; Peptides; Phosphorylation; Play; Proteins; Rattus; Regulation; Renal Blood Flow; Renal function; Resistance; Role; SHC1 gene; SHetA2; Sampling; Serine; Signal Pathway; Signal Transduction; Site-Directed Mutagenesis; Smooth Muscle Myocytes; Streptozocin; Structure; Testing; Up-Regulation; Vascular Diseases; Vasoconstrictor Agents; Vasodilation; age related; arteriole; base; combat; diabetic; diabetic rat; dietary salt; experimental study; kidney cell; kidney cortex; kidney vascular structure; mutant; novel; novel therapeutic intervention; overexpression; p66(ShcA) protein; pressure; prevent; protein protein interaction; renal damage; response; restoration; salt intake; salt sensitive; two-photon; type I diabetic; vasoconstriction

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.

大多数糖尿病和高血压患者会发生肾微血管损害 肾病。因此，微血管反应性变化的分子机制的揭示 肾血管对于开发新的治疗策略来对抗这些疾病是必要的，这 极大地促进了医疗保健的升级。根据我们的初步数据，我们假设 接头蛋白p66Shc的过度表达与微血管反应性的丧失有关 高血压肾病和糖尿病肾病的研究进展。我们将在基因上使用 靶向修饰Shc1基因产生的改良Dahl盐敏感(SS)大鼠和原肾 来源于这些大鼠的血管平滑肌细胞(SMC)。P66shc依赖的微血管调节 将研究来自已故供者的大鼠传入小动脉和人肾微血管的反应性。 有糖尿病或高血压肾病病史。具体目标1将检验这一假设 高血压肾病肾微血管反应性丧失是由p66Shc-1引起的。 TRPC通道介导的钙离子内流的依赖性抑制，并将分析其分子机制 P66Shc对TRPC的调控。我们将测试p66Shc与鸟嘌呤交换因子β-Pix的相互作用 在调节TRPC通道的活性和通道的亚细胞分布方面。使用双光子成像，我们将 P66Shc在SMC自发性细胞内钙振荡中的作用 人肾阻力血管的血管壁。我们还将测试化合物SHetA2，已知它会干扰 P66shc的作用，可能是因为它能预防p66shc引起的高血压肾病肾功能下降。 肾血管功能的恢复将通过研究微血管对嘌呤能的反应来评估。 复方SHetA2对正常SS大鼠和SS大鼠的激活和灌流压力。我们会 还要确定SHetA2是否对患有疾病的捐赠者的血管功能有有益影响 而且没有高血压。特殊目的2将验证p66Shc调节小动脉KATP通道的假设 糖尿病肾病中的活动性和引起高滤过。联合应用上调KATP活性的作用 钙离子内流对肌张力调节剂的反应受损，可能促进肾脏血管扩张 传入小动脉，导致高滤过。我们将研究p66Shc是否调节人类的KATP通道 肾微血管。我们还将采用1型糖尿病大鼠STZ诱导的糖尿病肾病模型，该模型 这种疾病的标记物与在人类患者身上观察到的类似。依赖p66shc的KATP通道 肾SMC的活动将通过电生理记录进行检测。我们将测试p66Shc是否刺激 KATP通道活性通过诱导蛋白-蛋白与接头蛋白14-3-3的相互作用来实现。建议数 实验将为p66Shc信号通路在肾脏调节中的作用提供直接证据。 与高血压和糖尿病相关的微血管反应性和肾脏损伤的进展。