Chronic kidney diseases (CKDs) are a major public health problem affecting more than 20 million people in the United States. African Americans are disproportionately affected by progressive CKDs, especially focal segmental glomerulosclerosis (FSGS). Genetic variants in the carboxy-terminal domain of APOL1 are associated with FSGS and other non-diabetic CKDs in populations with African ancestry. These variants change the amino acid sequence of APOL1 and explain much of this increased risk for CKDs in these populations. The pathogenetic mechanisms that lead from these APOL1 variants to CKD are not known. The carboxy-terminus of APOL1 interacted with VAMP8, an endosomal/lysosomal SNARE protein involved in vesicular trafficking and APOL1 kidney disease-associated variants attenuated the interaction with VAMP8 secondary to protein conformational changes induced by the variants. Like SNARE proteins, APOL1 localized to vesicular structures in the podocytes of the healthy human kidney. We hypothesize that APOL1:SNARE interaction is necessary to mitigate/attenuate podocyte stress response, and APOL1 variants disrupt functional SNARE interaction and mediate cytotoxicity. To test this hypothesis, we propose the following aims: First, we will use stable cell lines expressing APOL1 and healthy human kidneys to identify additional interacting SNARE proteins. We will use live-cell confocal microscopy to track APOL1:SNARE localization in various subcellular compartments in the presence and absence of immune stimuli. Second, we will investigate the cytotoxicity of reference and variant APOL1 proteins in the absence and presence of immune stimuli and reference APOL1 in the absence and presence of SNARE knockdown in cell culture models. Through these aims, I will obtain biochemical, imaging and proteomics data necessary to address our long term goal of identifying agents for the treatment of APOL1-associated kidney diseases. Understanding the molecular mechanisms by which APOL1 variants cause proteinuric CKD is essential for the development of new therapeutic strategies. I propose to use imaging and proteomics tools to study the pathological effects of APOL1 variants on its function and to learn bioinformatics tools to allow me to develop models to investigate CKD mechanisms. As a young physician-scientist, this project will help me to acquire new skills and knowledge while studying an important public health problem that afflicts my patients.
Approximately 20 million Americans have evidence for advanced chronic kidney disease (CKD), and more than 570,000 patients have kidney failure (end-stage kidney disease [ESKD]), the latter accounting for 7% of Medicare expenditures, approximately $35 billion. African American CKD patients in the United States are four times more likely than white patients to progress to ESRD, and variants in a single gene (APOL1) explain much of this increased risk. We propose to use cell biology, live-cell imaging, and proteomic data analysis tools to determine how the variants change APOL1?s function to cause kidney diseases.
