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: 9892583
• 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/9892583
• 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; Epithelium; 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; Immunoblotting; 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; Military Personnel; 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; 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; 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半数心脏骤停幸存者和许多患者的生存 有心肌梗死或急性心力衰竭。存活下来的患者患慢性肾脏疾病的风险很高 (CKD)。这项建议中的假设是，小鼠的心脏骤停和心肺复苏 (CA/CPR)导致肾脏可溶性诱导物心肌LIM蛋白(CSRP3)的滤过和小管内吞 肾小管无翼(WNT)效应分子激活引发慢性肾脏疾病的纤维化 上皮组织。这项建议的目标是： 1)确定一个可溶因素是否可以解释CA/CPR诱导CKD的能力，2)确定是否 通过内吞受体megalin的CSRP3的小管内吞导致肾纤维化，以及3)确定 CSRP3诱导肝纤维化的细胞内机制。 目的1验证CA/CPR通过心脏可溶性因子诱导CKD的假设。CA/CPR将 在心脏CSRP3缺失的小鼠身上进行，在CSRP3缺失的小鼠身上注射CSRP3 心脏CSRP3重现野生型损伤。肾纤维化、肾小球滤过率和尿蛋白 被量化。目的2验证通过肾小管上皮细胞受体Megalin实现功能性内吞作用的假说 在CA/CPR诱导的肾纤维化的发展过程中是必需的。CA/CPR将在小鼠身上进行 有条件的，近端小管特异的巨蛋白缺失(和产仔对照)。7周后，小鼠将成为 检测慢性肾脏疾病，量化纤维化、肾小球滤过率和尿蛋白。AIM 3测试 CSRP3易位到肾小管上皮细胞核并诱导Wnt效应基因的假说。 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 免疫组织化学方法检测WNT效应子转录，并定位CSRP3。 方法：将小鼠CA/CPR用于目标1和目标2。简单地说，在异氟醚麻醉下，心脏 用氯化钾诱导停搏，8分钟后用胸部按压和 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，肾纤维化的诱导和肾小球滤过率的降低非常明显。目标1 使用具有可诱导的心脏CSRP3缺失的小鼠。AIM 2将使用2个品系的小鼠 近端肾小管巨蛋白，一例嵌合终生缺失，另一例可诱导完整 删除megalin。将在这两个菌株中使用CA/CPR和重组CSRP3给药 测试megalin的参与程度。目标3将在体外进行，使用永生化的人肾小管上皮细胞 细胞(HK2线)。将用产生荧光的Wnt报告系统(TOPFlash)导入细胞 与Wnt效应基因的转录成正比。将使用TOPFlash来测试重组的效果 慢病毒介导的巨蛋白干扰细胞中的CSRP3给药。测试CSRP3是否经历 将采用核定位、免疫组织化学方法。 这项研究源于临床观察，急性心肾综合征会增加慢性肾功能衰竭的风险。 肾脏疾病。所使用的模型，小鼠CA/CPR，是急性脑脊髓炎模型中唯一的翻译模型。 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 会导致肾脏疾病。我们测试了对这种机制的干预，FDA批准的药物 如果临床研究证实我们的基本科学发现，可以将其应用于人类。退伍军人 罹患心血管疾病、慢性肾脏损伤和心肾综合征的风险大大增加。 这项研究测试了一种将心血管疾病和慢性肾脏疾病联系起来的新机制；治疗 因此，从这项研究中得出的结论可以减轻美国退伍军人的慢性肾脏疾病负担。