Kidney disease mechanisms associated with human genetic variation

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DESCRIPTION (provided by applicant): Chronic kidney disease (CKD), like many complex disease traits, manifests from dependent interactions between genetic susceptibilities and environmental factors. The recently described APOL1 risk alleles associated with CKD and African Ancestry is one example, where only a portion of genetically-susceptible patients develop kidney disease, suggesting a second hit or stress is required to trigger variant APOL1- dependent kidney injury. To investigate the mechanism of APOL1 variants in CKD, we have chosen to focus on HIV-associated nephropathy (HIVAN), the disease most robustly associated with APOL1 risk haplotypes and clearly dependent on an environmental factor, HIV-1 infection. Our preliminary data demonstrate APOL1 is expressed in the podocyte, and APOL1 overexpression activates autophagy. Although mTor-induced core autophagy pathways have been implicated in the glomerular injury of diabetic nephropathy -- a renal pathology not associated with APOL1 variants -- we have evidence that selective autophagy pathways involving destruction of pathogens may be uniquely regulated by APOL1. We hypothesize that APOL1 functions as an autophagocytic adaptor by binding to both a docking SNARE (VAMP8) displayed on endosomes and to the autophagosomal protein LC3-II. The autophagocytic adaptor function of APOL1 is activated by binding a triggering molecule, such as the HIV protein Nef after infection, and results in the selective degradation of VAMP8-labeled endosomal cargo including viral particles and proteins. Variant APOL1 proteins are functionally defective, permitting HIV to persist in the infected cell, and supporting the new synthesis of Nef, the HIV protein critical for development of HIVAN. Our proposed experiments will address three unanswered and novel questions regarding APOL1 function in CKD. Is APOL1 synthesized in situ in kidney and what is its subcellular home? Does circulating or renal-expressed variant APOL1 mediate kidney disease? Does dysregulation of autophagy pathways activated in response to specific environmental stresses result in kidney disease in patients with APOL1 risk genotypes? These questions will be addressed with the following Specific Aims: 1. Determine APOL1 expression and intracellular location in normal kidney, and determine if APOL1 localization varies with risk genotype and/or disease diagnosis; 2. Generate in vivo models for the study of APOL1 function; 3. Characterize protein interactions between APOL1 with VAMP8 and the HIV protein Nef. Examine the effect of normal and variant APOL1 expression on autophagy in cell lines and cultured podocytes. Focusing on the unique pathways though which APOL1 regulates autophagy will most likely result in novel, mechanism-based therapies. Determining how APOL1 variation is associated with human kidney diseases will provide a unique opportunity to understand pathways causing common chronic kidney disease and use of that knowledge can be exploited to develop new strategies for therapy, prevention and risk stratification.