HIV-1 Nef Induces Proliferation and Anchorage-Independent Growth in Podocytes MOHAMMAD HUSAIN,* G. LUCA GUSELLA,* MARY E. KLOTMAN, † IRWIN H. GELMAN, † MICHAEL D. ROSS,* ELISSA J. SCHWARTZ,* ANDREA CARA, ‡ and PAUL E. KLOTMAN* *Division of Nephrology and † Division of Infectious Diseases, Department of Medicine, Mount Sinai School of Medicine, New York, New York; ‡ Laboratory of Virology, Istituto Superiore di Sanita `, Rome, Italy. 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 glomer- ular epithelial cells or podocytes, cells that are a critical part of the filtration barrier. HIV-1 infection induces the loss of podo- cyte differentiation markers and increases podocyte prolifera- tion. 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 podo- cytes that were conditionally immortalized with SV-40 T an- tigen. 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 con- ditions. In contrast, deletion of nef markedly reduced prolifer- ation and colony formation. Although tat alone, or tat plus rev induced marginal levels of anchorage-independent growth, co- expression with nef significantly increased colony formation. Finally, stable expression of Nef in a retroviral vector, pBabe- puro, was sufficient to induce increased proliferation and col- ony 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. Patients with HIV type-1 (HIV-1) infection are at risk for developing clinically and morphologically diverse renal com- plications, including a chronic renal disease known as HIV- associated nephropathy (HIVAN) (1,2). The prevalence of HIVAN in the end-stage renal disease (ESRD) program has increased dramatically and is now reported to be the third leading cause of end-stage renal failure in African Americans between the ages of 20 and 64 yr (3). HIVAN patients present with heavy proteinuria, enlarged kidneys, and rapid progres- sion to renal failure. The pathologic features of HIVAN in- clude collapsing focal segmental glomerulosclerosis and pro- liferation of renal tubular, parietal, and visceral epithelial cells (podocytes). The other prominent features of HIVAN are tu- bulointerstitial infiltration with mononuclear cells, edema, fi- brosis, and microcystic tubule dilation (4,5). Expression of HIV-1 mRNA in tubular and glomerular epithelial cells in biopsies from HIVAN patients, as well as in these cell types in transgenic (Tg) mouse model of HIVAN (6,7), strongly sug- gests that HIV-1 mRNA expression in these sites contributes to the disease process. In both the murine model and human renal biopsy material, one of the prominent pathologic characteris- tics of HIVAN is the hyperproliferation of podocytes mani- fested by the expression of the proliferation marker Ki-67 and the loss of differentiation markers, synaptopodin, WT-1, GLEPP-1, and CALLA (8). Although the renal epithelial cells appear to be the main targets for HIV-1 pathogenesis, the HIV gene products responsible for tissue-specific renal pathology are not known. Our hypothesis is that the expression of one or more specific HIV-1 proteins in podocytes induces the in- creased proliferation of these cells. HIV-1 encodes three structural genes (gag, pol, env), two essential regulatory genes (tat and rev), and four accessory genes (vif, vpr, vpu, and nef) (9). An HIV-1 plasmid construct deleted for gag and pol (pNL4 –3: d1443) had earlier been used to generate a Tg mouse model of HIVAN (10,11). These animals present with renal disease that is clinically and patho- logically identical to that observed in patients with HIVAN. Thus, this construct expressing Env and the accessory proteins (Vif, Vpr, Vpu, Nef, Tat, and Rev) without Gag/Pol can induce HIVAN. Previously, the lack of an in vitro podocyte culture system prevented a detailed analysis of the effects of HIV-1 gene expression on renal podocytes. With, the establishment of Received December 12, 2001. Accepted April 6, 2002. Correspondence to Dr. Mohammad Husain, Box 1243, Division of Nephrol- ogy, Mount Sinai School of Medicine, One Gustave L Levy Place, New York, NY 10029. Phone: 212-241-8041; Fax: 212-987-0389; E-mail: mohammad. husain@mssm.edu 1046-6673/1307-1806 Journal of the American Society of Nephrology Copyright © 2002 by the American Society of Nephrology DOI: 10.1097/01.ASN.0000019642.55998.69 J Am Soc Nephrol 13: 1806–1815, 2002