An HIV-Tat inducible mouse model system of childhood HIV-associated nephropathy

Abstract

statement. We developed a new inducible mouse model system of childhood HIV-associated nephropathy, and demonstrated that HIV-Tat plays a critical role in this renal disease acting in synergy with other HIV-1 genes and heparin binding cytokines. AbstractBackground: Modern antiretroviral therapies (ART) have decreased the prevalence of HIVassociated nephropathy (HIVAN). Nonetheless, we continue to see children and adolescents with HIVAN all over the world. Furthermore, once HIVAN is established in children, it is difficult to revert its long-term progression, and we need better animal models of childhood HIVAN to test new treatments. Objectives. To define whether the HIV-1 trans-activator (Tat) gene precipitates HIVAN in young mice, and to develop an inducible mouse model of childhood HIVAN. Design/Methods: An HIV-Tat gene cloned from a child with HIVAN was used to generate recombinant adenoviral vectors (rAd-Tat). rAd-Tat and LacZ control vectors (2 x 10 9 ) were expressed in the kidney of newborn wild type and HIV-transgenic (Tg 26 ) FVB/N mice without significant proteinuria (n = 5 -8 per group). Mice were sacrificed 7 and 35 days later to assess their renal outcome, the expression of HIV-genes and growth factors, and markers of cell growth and differentiation by RT-qPCR, immunohistochemistry, and/or Western blots. Results: HIV-Tat induced the expression of HIV-1 genes (env) and heparin binding growth factors in the kidney of HIV-Tg 26 mice, and precipitated HIVAN in the first month of life. No significant renal changes were detected in wild type mice infected with rAd-Tat vectors, suggesting that HIV-Tat alone does not induce renal disease. Conclusion. This new mouse model of childhood HIVAN highlights the critical role that HIV-Tat plays in the pathogenesis of HIVAN, and could be used to study the pathogenesis and treatment of HIVAN in children and adolescents.

Association of circulating Fibroblast Growth Factor-2 with progression of HIV-chronic kidney diseases in children

“…The FGF-2 and PCNA staining was done as described before. 20, 21 Briefly, renal sections were incubated for 1 h at room temperature with affinity purified IgG fractions (2.5 µg/ml) of a rabbit polyclonal antibody directed against a unique peptide sequence of FGF-2 (provided by Dr. Baird, PRIZM Pharmaceuticals, San Diego CA, USA), or a monoclonal antibody (clone PC10) against the proliferative cell nuclear antigen (PCNA) from Zymed Laboratories (South San Francisco, CA, USA). These antibodies were validated in previous rodent and human studies.…”

“…Then all cells were fixed, stained with Diff Quick (Baxter Healthcare, Leesburg, VA, USA), and counted under the microscope with a 20X magnification lens as reported before. 21 For the syncytia formation assays, HIV-infected PBMCs were seeded on fibrin gels derived from plasma taken from children with a high viral load. 26 The HIV in situ hybridization studies were done using an HIV-probe from Lofstrand Labs Ltc.…”

“…25 The survival assays were done in cells cultured under similar tissue culture conditions and exposed to hr-FGF-2 for 78 hours as described before. 21…”

“…Total RNA was purified from frozen cell pellets with TRIzol Reagent from Invitrogen (Waltham, Massachusetts, USA) as described before. 21 cDNAs were synthesized from 2µg RNA with Quantabio’s qScript™ cDNA SuperMix (VWR, Radnor Pennsylvania, USA) following the manufacturer’s instructions. Quantitative PCR was performed with GoTaq® qPCR Master Mix (Promega, Madison, Wisconsin, USA) using 1/40 of the reverse transcription reaction.…”