HIV-1 Nef Induces Proliferation and Anchorage-Independent Growth in Podocytes

Abstract

Abstract. HIV-associated nephropathy (HIVAN) is now the third leading cause of end-stage renal disease in the African American population. HIV-1 infects renal tubular and glomerular epithelial cells or podocytes, cells that are a critical part of the filtration barrier. HIV-1 infection induces the loss of podocyte differentiation markers and increases podocyte proliferation. It has been previously shown that HIV-infection induces loss of contact inhibition. Here, the HIV-1 gene responsible for proliferative changes is identified by using cultured podocytes in vitro. The HIV-1 proviral construct, pNL4-3 was rendered noninfectious by replacing the HIV-1 gag/pol sequences with an EGFP reporter gene (pNL4-3: ⌬G/P-EGFP). This construct was then pseudotyped with VSV.G envelope to infect podocytes that were conditionally immortalized with SV-40 T antigen. In addition, mutated constructs were engineered with premature stop codons in the HIV-1 env, vif, vpr, vpu, nef, or rev genes. The parental construct and all the other mutated constructs, with the exception of nef, induced proliferation under nonpermissive conditions and anchorage-independent growth (colony formation in soft agar) under permissive conditions. In contrast, deletion of nef markedly reduced proliferation and colony formation. Although tat alone, or tat plus rev induced marginal levels of anchorage-independent growth, coexpression with nef significantly increased colony formation. Finally, stable expression of Nef in a retroviral vector, pBabepuro, was sufficient to induce increased proliferation and colony formation. Moreover, nef induced saturation density and loss of contact inhibition. These data indicate that Nef induces multiple proliferative effects in podocytes in culture and that nef may therefore be an important gene in the pathogenesis of HIVAN in vivo.

Retinoic Acid Utilizes CREB and USF1 in a Transcriptional Feed-Forward Loop in Order To Stimulate MKP1 Expression in Human Immunodeficiency Virus-Infected Podocytes

“…These cells proliferate under permissive conditions (gamma interferon at 33°C) but differentiate under nonpermissive conditions (37°C). The HIV-1 constructs have been described previously (17). Expression of HIV-1 genes was confirmed by immunoblot analysis.…”

“…Unlike most other glomerulopathies, HIVAN is characterized by proliferation and dedifferentiation of podocytes, which are glomerular visceral epithelial cells (3,4,17). Recently, we showed that the HIV-1 nef gene is the primary determinant of this unusual podocyte phenotype (17,36) and that Nef stimulates podocyte proliferation and dedifferentiation through the Src-dependent mitogenactivated protein kinase 1,2 (MAPK1,2) pathways (15). Increased MAPK1,2 phosphorylation is also observed in mouse and human kidney sections of HIVAN (15).…”

HIV-1 Upregulates VEGF in Podocytes

“…28 The vpr, tat, and nef constructs as well as a control green fluorescence protein-containing construct and a Stat3 dominant negative mutant (a gift from Dr. C.M. Horvath, The Rockefeller University, New York, NY) were individually introduced into wild-type conditionally immortalized podocytes maintained at 33°C via nucleofection (Amaxa Biosystems, Cologne, Germany), and the cells were transferred to 37°C for 3 d. 29 The transfection efficiency was evaluated by fluorescence microscopy confirming 70 to 80% efficiency.…”

Tubular Cell HIV-1 gp120 Expression Induces Caspase 8 Activation and Apoptosis

“…The gag/pol and VSV.G envelope genes were provided in trans using pCMV R8.91 and pMD.G plasmids, respectively. [3,6] Infectious viral supernatants were produced by transient transfection of 293T cells using effectene transfection reagent (QIAGEN Inc.) according to the manufacturer's instructions. Viral supernatants were collected at 48 and 72 hrs post-transfection.…”

“…[1][2][3][4][5] The latter is characterized by focal glomerulosclerosis and tubulointerstitial lesions. [6] Acute renal failure has frequently been reported as a result of tubular cell injury (apoptosis and necrosis) in HIV-1-infected patients.…”