PROJECT SUMMARY Chronic kidney disease (CKD) has reached epidemic proportions globally. The single most important killer of CKD patients is cardiovascular disease (CVD) consisting of cardiac hypertrophy and fibrosis, and vascular calcification. More CKD patients die from CVD than reaching the need for dialysis. The treatment of traditional cardiovascular risk factors have met with limited success and we are in dire need to treat CKD-related CVD. Uremic cardiomyopathy is a complex metabolic disease with a panoply of underlying pathophysiologic factors. Our laboratory has focused on contributing mineral factors common to CVD and CKD-Mineral Bone Disorders (MBD) - which individually contributes to uremic cardiomyopathy, but each one also exacerbates the others creating a self-amplifying unrelenting vortex. We focus on the model that Klotho deficiency and phosphotoxicity exacerbate each other and both contribute to CVD. If we disrupt these disturbances simultaneously, it will be much more effective than manipulating them alone thus furnishing a novel and efficacious regimen to prevent and treat uremic cardiomyopathy. In Aim 1, we will use a well-established rodent model of CKD and uremic cardiomyopathy to test this therapeutic approach. These are empiric but important proof-of-concept studies to secure the utility of this approach. In Aim 2, we will examine the long sort after but yet unresolved question of the underlying mechanism of Klotho deficiency in CKD. We know that uremia, Klotho deficiency exacerbates phosphotoxicity by reducing phosphaturia but how Klotho is suppressed in CKD is not known. We will test the model that phosphate loading causes systemic Klotho deficiency by at least two mechanisms: direct inhibition of Klotho transcription via methylation of its promoter; and reduced Klotho shedding from the kidney using combined in vivo and in vitro approaches. Although Klotho deficiency per se have been shown to pathologic cardiac remodeling, the molecular mechanism(s) that mediate(s) its action is still an enigma. We constructed a model and propose that one principal mechanism is the balance between autophagy and apoptosis and Klotho holds the “toggle switch” by modifying the formation of the important autophagy complex Beclin 1/Bcl-2. In Aim 3, we will use both in vivo and in vitro models to test the paradigm. Using genetically modified animals, we will manipulate autophagy levels and test whether the protective action of Klotho is mediated by enhanced autophagy. We will use in vitro models to further test the model of phosphate loading and Klotho flipping the toggle switch in opposite directions. The proposed studies will uncover some fundamental biologic mechanisms of how Klotho is suppressed in CKD and phosphate loading, elucidate how Klotho protects the cell via autophagy flux, and finally provide the critical pre-clinical data to form the foundation for translation to therapy in human CKD and uremic cardiomyopathy.