Mechanistic Underpinnings of Chronic Kidney Disease Initiated by Acute Cardiorenal Syndrome

急性心肾综合征引发的慢性肾脏病的机制基础

• 批准号: 10436781
• 负责人: Michael P Hutchens
• 金额: --
• 依托单位: PORTLAND VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10436781
• 关键词: Acute; Acute Disease; Acute Renal Failure with Renal Papillary Necrosis; Albumins; Anesthesia procedures; Animal Model; Animals; Basic Science; Cardiac; Cardiac development; Cardiopulmonary Resuscitation; Cardiovascular Diseases; Cardiovascular system; Carrier Proteins; Cell Nucleus; Cells; Chest; Chronic; Chronic Kidney Failure; Chronic Kidney Insufficiency; Cicatrix; Clinical; Clinical Research; Data; Development; Dialysis procedure; Endocytosis; Epinephrine; Epithelial; Epithelial Cells; Exposure to; Extracellular Matrix Proteins; FDA approved; Fibrosis; Filtration; Fluorescence; Genes; Genetic Transcription; Glomerular Filtration Rate; HK2 gene; Heart; Heart Arrest; Heart Diseases; Heart Injuries; Heart failure; Hospitalization; Hour; Human; Immunohistochemistry; Impairment; In Vitro; Inflammatory Response; Injections; Injury; Injury to Kidney; Interruption; Intervention; Investigation; Ischemia; Isoflurane; Kidney; Kidney Diseases; LDL-Receptor Related Protein 2; Length of Stay; Link; Mediating; Methods; Micropuncture; Modeling; Mosaicism; Mouse Strains; Mus; Myocardial Infarction; Nuclear; Organ; Patients; Pharmaceutical Preparations; Plasma; Potassium Chloride; Prevalence; Prevention strategy; Proteins; Recombinant Proteins; Recombinants; Renal function; Reperfusion Therapy; Reporter; Resuscitation; Risk; Role; Signal Transduction; Survivors; Syndrome; System; Testing; Tissues; Transplantation; Tubular formation; Urine; Veterans; Western Blotting; Work; artery occlusion; base; cardiovascular disorder risk; coronary fibrosis; fibrous protein; glomerular filtration; high risk; in vivo; innovation; kidney cell; kidney dysfunction; kidney fibrosis; lentiviral-mediated; military veteran; mouse model; nanoproteomic; novel; prevent; receptor; renal artery; therapy design; translational model; translational study; treatment strategy; uptake

Acute cardiorenal syndrome imperils the survival of half of cardiac arrest survivors and that of many patients with myocardial infarction or acute heart failure. Patients who survive have high risk of chronic kidney disease (CKD). The hypothesis in this proposal is that cardiac arrest and cardiopulmonary resuscitation in the mouse (CA/CPR) causes filtration and tubular endocytosis of cardiac LIM protein (CSRP3), a soluble inducer of renal fibrosis, which initiates chronic kidney disease via Wingless (Wnt)-effector activation in renal tubular epithelium. The objectives in this proposal are: 1) Establish whether a soluble factor explains the potent induction of CKD by CA/CPR , 2) Determine whether tubular endocytosis of CSRP3 via the endocytic receptor megalin leads to renal fibrosis, and 3) Determine the intracellular mechanism of fibrosis induction by CSRP3. Aim 1 tests the hypothesis that CA/CPR potently induces CKD via a soluble factor from the heart. CA/CPR will be performed in mice with deletion of cardiac CSRP3, and CSRP3 will be administered to mice with deletion of cardiac CSRP3 to recapitulate the wild-type injury. Renal fibrosis glomerular filtration rate and urine protein will be quantified. Aim 2 tests the hypothesis that functional endocytosis via the tubular epithelial receptor megalin is required for development of CA/CPR-induced renal fibrosis. CA/CPR will be performed in mice with conditional, proximal tubular-specific deletion of megalin (and littermate controls). 7 weeks later mice will be tested for chronic kidney disease, quantifying fibrosis, glomerular filtration rate, and urine protein. Aim 3 tests the hypothesis that CSRP3 translocates to the tubular epithelial cell nucleus and induces Wnt effector genes. Tubular epithelial cells will be exposed to CSRP3, and a Wnt-effector reporter system and immunoblotting and immunohistochemistry used to determine Wnt effector transcription, and localize CSRP3. Methods: Murine CA/CPR will be employed in aim 1 and aim 2. Briefly, under isoflurane anesthesia, cardiac arrest is induced with potassium chloride and resuscitated 8 min later with chest compressions and epinephrine, analogous to resuscitation of cardiac arrest in humans. Preliminary data indicate that 7 weeks after CA/CPR, there is robust induction of renal fibrosis and reduction in glomerular filtration rate. Aim 1 employs mice with inducible deletion of cardiac CSRP3. Aim 2 will use 2 strains of mice with deletion of proximal tubular megalin, one with mosaic and lifelong deletion, and the second with inducible, complete deletion of megalin. CA/CPR and recombinant CSRP3 administration will be employed in these two strains to test the involvement of megalin. Aim 3 will be conducted in vitro, using immortalized human tubular epithelial cells (the HK2 line). Cells will be transfected with a Wnt reporter system (TOPFlash) which yields fluorescence proportional to transcription of Wnt effector genes. TOPFlash will be used to test the effect of recombinant CSRP3 administration in cells with lentiviral-mediated megalin interference. To test whether CSRP3 undergoes nuclear localization, immunohistochemistry will be used. This study is derived from clinical observation that acute cardiorenal syndrome increases risk of chronic kidney disease. The model employed, murine CA/CPR, is uniquely translational among models of acute disease causing chronic renal insufficiency. Our study evaluates a novel mechanism by which cardiac disease causes renal disease. We test intervention in this mechanism for which an FDA-approved medication which could be applied in humans is available, should clinical study corroborate our basic science findings. Veterans suffer from greatly elevated risk of cardiovascular disease, chronic kidney injury, and cardiorenal syndrome. This study tests a novel mechanism connecting cardiovascular disease and chronic kidney disease; therapy derived from this study could therefore reduce the burden of chronic kidney disease on US military veterans.

急性心肾综合征危及一半心脏骤停幸存者和许多患者的生存 患有心肌梗塞或急性心力衰竭。存活下来的患者患慢性肾病的风险很高 （CKD）。该提案的假设是，小鼠的心脏骤停和心肺复苏 (CA/CPR) 引起心脏 LIM 蛋白 (CSRP3) 的滤过和肾小管内吞作用，CSRP3 是肾病的可溶性诱导剂 纤维化，通过肾小管中的 Wingless (Wnt) 效应器激活引发慢性肾脏疾病 上皮。该提案的目标是： 1) 确定可溶性因子是否可以解释 CA/CPR 对 CKD 的有效诱导，2) 确定是否 CSRP3 通过内吞受体巨蛋白的肾小管内吞作用导致肾纤维化，并且 3) 确定 CSRP3诱导纤维化的细胞内机制。 目标 1 检验 CA/CPR 通过心脏中的可溶性因子有效诱导 CKD 的假设。 CA/CPR 将 在心脏 CSRP3 缺失的小鼠中进行，并将 CSRP3 给予心脏 CSRP3 缺失的小鼠 心脏 CSRP3 来重现野生型损伤。肾纤维化肾小球滤过率和尿蛋白会 被量化。目标 2 检验通过肾小管上皮受体巨蛋白进行功能性内吞作用的假设 是 CA/CPR 诱导的肾纤维化发展所必需的。 CA/CPR 将在小鼠中进行 有条件的、近端肾小管特异性巨蛋白缺失（和同窝对照）。 7周后，小鼠将 测试慢性肾脏疾病，量化纤维化、肾小球滤过率和尿蛋白。目标 3 测试 CSRP3 易位至肾小管上皮细胞核并诱导 Wnt 效应基因的假设。 肾小管上皮细胞将暴露于 CSRP3 和 Wnt 效应报告系统以及免疫印迹和 免疫组织化学用于确定 Wnt 效应子转录并定位 CSRP3。 方法：将在目标 1 和目标 2 中采用小鼠 CA/CPR。简而言之，在异氟烷麻醉下，心脏 用氯化钾诱导心脏骤停，并在 8 分钟后进行胸外按压和复苏 肾上腺素，类似于人类心脏骤停的复苏。初步数据显示，7周 CA/CPR 后，肾纤维化强烈诱导，肾小球滤过率降低。目标1 使用可诱导缺失心脏 CSRP3 的小鼠。目标 2 将使用 2 个品系的小鼠，其中删除了 近端管状巨蛋白，一种具有嵌合性和终身缺失，第二种具有诱导性、完全性 巨蛋白缺失。 CA/CPR 和重组 CSRP3 给药将在这两种菌株中使用 测试巨蛋白的参与。目标 3 将在体外进行，使用永生化人肾小管上皮细胞 细胞（HK2 系）。细胞将转染 Wnt 报告系统 (TOPFlash)，产生荧光 与 Wnt 效应基因的转录成正比。将使用TOPFlash来测试重组的效果 在具有慢病毒介导的巨蛋白干扰的细胞中施用CSRP3。测试CSRP3是否经过 将使用核定位、免疫组织化学。 这项研究源自临床观察，即急性心肾综合征会增加慢性心肾综合征的风险 肾脏疾病。所采用的模型是小鼠 CA/CPR，在急性呼吸系统疾病模型中具有独特的转化能力。 导致慢性肾功能不全的疾病。我们的研究评估了心脏病的一种新机制 导致肾脏疾病。我们测试了对这一机制的干预，FDA 批准的药物 如果临床研究证实我们的基础科学发现，那么可以应用于人类。退伍军人 患心血管疾病、慢性肾损伤和心肾综合征的风险大大增加。 这项研究测试了一种连接心血管疾病和慢性肾脏疾病的新机制；治疗 因此，这项研究的成果可以减轻美国退伍军人慢性肾病的负担。