Mechanistic Underpinnings of Chronic Kidney Disease Initiated by Acute Cardiorenal Syndrome

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 心肌梗死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，小鼠心脏骤停和心肺复苏 (CA/CPR）引起心脏LIM蛋白（CSRP 3）的过滤和肾小管内吞，CSRP 3是肾功能衰竭的可溶性诱导物。 纤维化，通过肾小管中的无翼（Wnt）效应物激活引发慢性肾病 上皮本提案的目标是： 1)确定是否有可溶性因子可以解释CA/CPR对CKD的强效诱导，2）确定是否 通过内吞受体巨蛋白的CSRP 3的肾小管内吞导致肾纤维化，和3）确定CSRP 3的肾小管内吞作用。 CSRP 3诱导纤维化细胞内机制 目的1检验CA/CPR通过来自心脏的可溶性因子有效诱导CKD的假设。CA/CPR将 在具有心脏CSRP 3缺失的小鼠中进行，并且将CSRP 3施用至具有心脏CSRP 3缺失的小鼠。 心脏CSRP 3来概括野生型损伤。肾纤维化肾小球滤过率和尿蛋白将 要量化。目的2：验证肾小管上皮细胞巨蛋白受体介导的功能性内吞作用 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， 巨蛋白的条件性近端小管特异性缺失（和同窝对照）。7周后， 检测慢性肾脏疾病，定量纤维化，肾小球滤过率和尿蛋白。Aim 3测试 CSRP 3易位到肾小管上皮细胞核并诱导Wnt效应基因的假说。 将肾小管上皮细胞暴露于CSRP 3和Wnt效应子报告系统，并进行免疫印迹， 使用免疫组织化学测定Wnt效应子转录并定位CSRP 3。 方法：采用小鼠CA/CPR模型，分别进行目标1和目标2的实验。简单地说，在异氟烷麻醉下， 用氯化钾诱导停搏，8分钟后用胸部按压复苏， 肾上腺素，类似于人类心脏骤停的复苏。初步数据显示， 在CA/CPR后，存在肾纤维化的强烈诱导和肾小球滤过率的降低。要求1 使用具有可诱导的心脏CSRP 3缺失的小鼠。目的2将使用2种缺失 近端小管巨蛋白，一个具有镶嵌和终身缺失，第二个具有诱导性，完全缺失， 删除megalin。CA/CPR和重组CSRP 3给药将用于这两种菌株， 测试巨蛋白的参与。目的3：利用人肾小管上皮细胞进行体外培养， 细胞（HK 2线）。将用产生荧光的Wnt报告系统（TOPFlash）转染细胞 与Wnt效应基因的转录成比例。TOPFlash将用于测试重组的效果 CSRP 3在具有慢病毒介导的巨蛋白干扰的细胞中的施用。为了测试CSRP 3是否经历 核定位，将使用免疫组织化学。 本研究源自临床观察，即急性心肾综合征可增加慢性 肾病所采用的模型，小鼠CA/CPR，是唯一的急性转化模型， 引起慢性肾功能不全的疾病。我们的研究评估了一种新的机制， 导致肾脏疾病。我们测试了这种机制的干预， 如果临床研究证实了我们的基础科学发现，退伍军人 患心血管疾病、慢性肾损伤和心肾综合征风险大大增加。 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，从这项研究中得出的结论可以减轻美国退伍军人慢性肾脏疾病的负担。

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.