New Drosophila, mouse, and human podocyte models to study HIV-APOL1-G1 associated chronic kidney diseases

University Of Virginia, Charlottesville VA

Investigators

Abstract

PROJECT SUMMARY/ABSTRACT People living with HIV (PLWH) carrying any combination of two APOL1 risk alleles (RA), named G1 and G2, are at high risk of developing HIV-chronic kidney diseases (HIV-CKD), even when treated with antiretroviral therapy (ART). A critical gap in knowledge that remains to be understood, is how APOL1-G1/G1GM, the most common RA in people with HIV-nephropathy (HIVAN) living in the US, including children and adolescents, interact with HIV-genes to precipitate CKD. We have developed Tg flies carrying APOL1-G1 derived from a child with HIVAN, specifically in nephrocytes (the equivalent of mammalian podocytes) and found that APOL1-G1 affects the endocytic activity and size of these cells precipitating their death. In addition, we developed dual APOL1-G1- HIV-Nef Tg flies and showed that both act in synergy to increase the workload of nephrocytes, affecting several trafficking and degradation pathways that accelerate their death. We also developed APOL1-G1-Tg mice driven by the APOL1-G1 promoter/enhancer sequences cloned from a child with HIVAN and showed that interferon-γ increased the expression of APOL1-G1 in podocytes and induced proteinuria. Furthermore, we found that (a) HIV-1 enters podocytes cultured from the urine of children with HIV-CKD via endocytosis and induces the expression of APOL1-G1; (b) APOL1-G1 is localized mainly in the perinuclear region and endoplasmic reticulum (ER) in these cells; (c) APOL1-G1’s ER localization in cis and trans orientation can induce cytotoxicity in HIVAN podocytes; (d) SNARE proteins and the autophagy pathway play a key role regulating the toxicity of APOL1-G1 in fly nephrocytes. Based on these data, we hypothesize that HIV gene products and cytokines precipitate HIV- CKD by inducing the expression of APOL1-G1 in podocytes affecting its interactions with SNARE proteins and cell trafficking molecules. In turn, we propose that APOL1-G1 acts in synergy with HIV to impair the balance between key autophagy and ER stress pathways, thus disrupting the homeostasis of podocytes and causing proteinuria and CKD. This hypothesis will be tested in two aims. In aim 1 we will define new mechanisms through which APOL1-G1 interacts in vivo with HIV-genes to affect the structure, function, and survival of nephrocytes in dual Tg flies carrying APOL1-G1 plus HIV-env and all other HIV-accessory/regulatory genes. In aim 2, we will determine how APOL1-G1 and HIV interact in new dual APOL1-G1 Tg mice driven by the APOL1-G1 promoter cloned from a child with HIVAN and in cultured HIVAN podocytes. Here we will expand and validate the most relevant findings and cell trafficking, autophagy, ER, and inflammatory pathways identified in the HIV-G1Tg-flies. Renal samples, primary mouse/human podocytes, and HIVAN podocyte cell lines modified by a CRISPER/Cas9 genome editing fluorescent tagging approach will be used to assess the endogenous localization and toxicity of APOL1-G1 and HIV-genes, and to validate all key findings generated in the fly and mouse models. This study will generate new experimental model systems to study the pathogenesis of HIV-CKD, perform high throughput genetic screenings, identify new mechanisms or therapeutic targets, and test new drugs to treat HIV-CKD.