Effect of Ubiquitin D variants upon APOL1-mediated kidney injury - PROJECT SUMMARY Despite the success of antiretroviral therapy in improving mortality, persons with HIV (PWH) have shortened lifespan and increased prevalence of non-infectious diseases including chronic kidney disease (CKD). HIV- associated nephropathy (HIVAN) occurs almost exclusively in persons of African ancestry and variants of the APOL1 gene, which confer resistance to trypanosomal disease, markedly increase risk of progressive CKD. 14% of African Americans have APOL1 high risk (HR) genotypes, which confer a 29-89-fold increased risk of HIVAN among PWH, likely by predisposing glomerular podocytes to HIV-induced injury. Since most persons with APOL1 HR genotypes never develop kidney disease, additional “hits”, including viral infection (HIV, SARS-coV-2), exposure to high levels of interferon, and/or additional genetic factors, are necessary to initiate progressive CKD. Our group was the first to identify a role for the ubiquitin-like protein Ubiquitin D (UBD) in the pathogenesis of kidney disease (HIVAN). We reported roles for UBD in promoting HIV-induced kidney epithelial cell injury and innate immune activation. A role for UBD in modulating kidney injury in APOL1- mediated kidney disease is supported by recently published studies demonstrating increased UBD expression in glomeruli in humans and mice with APOL1 HR genotypes and kidney disease and a recent report that UBD increases APOL1 degradation in vitro. Our analysis of the UBD gene locus identified a haplotype comprised of four missense mutations with an allele frequency of 0.43 in African Americans but only 0.02 in Europeans. Our preliminary data demonstrate that this haplotype (UBDb) is associated with a 5.8-fold increased risk of HIVAN in persons with APOL1 HR genotypes. Further, data from our laboratory indicate that there are important functional differences in the UBDb protein compared to UBDa (reference allele). However, the mechanism by which the UBDb variant synergizes with APOL1 risk alleles to promote kidney injury is unknown. Since UBD can covalently and non-covalently interact with cellular proteins and increase/decrease degradation or alter protein function, we will test our hypothesis that the UBDb variant increases the risk of HIVAN via changes in interactions with APOL1 and other proteins, leading to podocyte injury. We will test our hypothesis and address critically important questions in two Specific Aims. In Aim 1, we will determine the effects of the UBDa and UBDb alleles upon HIV-induced injury and inflammatory responses in novel human podocyte lines with APOL1 HR and non-risk genotypes. In Aim 2, we will use high throughput proteomics and targeted in vitro assays to delineate differences in covalent and non-covalent protein-protein interactions of UBD variants and effects of UBD variants on APOL1 protein ubiquitination and degradation in human podocytes with APOL1 HR and non- risk genotypes. These innovative and important studies will delineate novel mechanisms underlying the increased risk of CKD in PWH, allowing us to devise new strategies to prevent and treat CKD this population.
