Mechanistic Underpinnings of Chronic Kidney Disease Initiated by Acute Cardiorenal Syndrome

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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.