Focal segmental glomerulosclerosis (FSGS) is a clinical-pathologic syndromes characterized by the accumulation of fibrotic proteins in glomeruli, initially involving only some glomeruli (focal) and involving portions (segments) of the affected glomeruli. FSGS can be classified as follows: idiopathic FSGS, genetic FSGS and post-adaptive FSGS (associated with glomerular hypertrophy and hyperfiltration, and due to reduced renal mass, renal toxins, obesity, and sickle cell disease). A related syndrome is collapsing glomerulopathy, associated with podocyte hyperplasia whereas FSGS is associated with podocyte depletion. Collapsing glomerulopathy can be classified as HIV-associated or idiopathic. In order to define the molecular mechanisms responsible for HIV-associated collapsing glomerulopathy, we have established mice in which transgene expression can be regulated in the glomerular podocyte using an tetracycline-regulated system. We have used this system to express the HIV-1 accessory protein Vpr in the podocyte. These mice develop proteinuria beginning 4 weeks after treatment with tetracycline. Collapsing glomerulopathy appears at 8 weeks, progressing to global glomerulosclerosis and end-stage kidney disease. Podocyte phenotype is abnormal, with reduced expression of the differentiation marker synaptopodin and de novo expression of the injury marker desmin. Increased cell proliferation is present in the glomerular tuft, parietal epithelum, and tubular epithelium. These results demonstrate that Vpr is sufficient to induce HIV-associated collapsing glomerulopathy in transgenic mice. 1) Using these transgenic mice, we have established double transgenic podocyte cell lines, which bear the podocin/rtTA, and temperature-sensitive SV40 Tag transgenes to confer conditional immortalization. These cells were found to express characteristic podocyte markers, including podocin, nephrin, and WT1. We have introduced the Vpr gene into these cells to understand Vpr-induced cell injury. Our preliminary results indicate increased apoptosis with 12 hours of Vpr expression induced by doxycycline. The mechanisms appear to involve both caspase dependent and caspase independent pathways. Furthermore, our collaborator Dr. Craig Beeson has studied cellular energetics in these cells, using a Seahorse extracellular flux anayzler. Vpr expression is associated with impaired oxygen consumption (including mitochondrial dysfunction) and reducing medium acidification (indicating impaired glycolysis). Current efforts are focusing on clarifying the mechanisms for these findings. 2) Five cross-sectional studies of HIV-infected patients suggest that the prevalence of microalbuminuria is 10-20%. A recent study from South Africa suggested that 6/7 of seven microalbuminuria patients have HIV-associated nephropathy;another recent US study suggested that microalbuminuria is associated with features of the metabolic syndrome and CD4 and viral load. We have initiated a study to determine the prevalence of persistent microalbuminuria and to detetermine the renal histology in these patients. We plan to enroll 250 patients in a prospective observational study. Exclusions will include diabetes (as this condition is known to be associated with microalbuminura), cancer, IL2 therapy,and acute inflammatory processes. Blood and urine samples will be collected at baseline, 3 months, and 6 months later. All patients with microalbuminuria will be followed for an additional 24 months, with blood and urine collections every 6 months. Analysis will include 1) comparison for relevant clinical and laboratory parameters between patients with and without microalbuminuria, 2) similar comparisons by quartile of albuminuria, and 3) linear regression between microalbuminuria and quantitative veriables. We have enrolled 135 patients at NIH and 45 patients at Washington Hospital Center. In the first 91 patients who have completed the study, 12 (13%) have microalbuminuria. We have amended the protocol to collect DNA from subjects, in order to examine whether kidney risk MYH9 alleles increase the propensity to microalbuminuria. 3) HIV infected patients, as well as other immunosuppressed patients, are at risk for BK virus nephropathy. We are working with a rhesus model, which uses cyclophosphamide immunosuppression to maintain infection, and are testing tranilast as a novel anti-viral agent.
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