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Background: Human immunodeficiency virus (HIV)-associated nephropathy is characterized by focal segmental glomerulosclerosis and microcystic tubular dilation. We have previously described a mouse transgenic for a Deltagag-pol HIV-1 genome, which develops glomerulosclerosis, cutaneous papillomas, and cataracts., Methods: We developed mice transgenic for a Deltagag-pol-nef HIV genome in order to investigate the role of the nef gene in these phenotypes., Results: One transgenic line, X5, expressed HIV mRNA in kidney and consistently manifested focal segmental glomerulosclerosis and tubular dilation by six weeks of age. Northern analysis indicated that renal transgene expression was higher in the Deltagag-pol-nef mice compared with the Deltagag-pol mice. In situ hybridization and immunostaining demonstrated HIV RNA and protein expression within the glomerular epithelial cells and tubular epithelial cells. These cell types showed histologic evidence of toxicity, including vacuolation and detachment from basement membrane, and exhibited increased rates of apoptosis. These data suggest that the renal disease seen in the Deltagag-pol-nef transgenic mouse may be caused by the expression of HIV genes within renal epithelial cells, that this expression may induce cellular toxicity, including apoptosis, and that nef is not required for the induction of renal disease. We have previously described mice bearing the nef gene, which do not manifest renal disease. In further experiments, Deltagag-pol-nef mice were bred with nef mice; these dual-transgenic mice developed renal disease that generally resembled that seen in Deltagag-pol-nef mice, but with somewhat more severe glomerulosclerosis and less severe tubulointerstitial injury., Results: The results of these transgenic studies suggest that the role of nef is complex and may act both to reduce transgene expression and to potentiate glomerular injury induced by other HIV-1 gene products.

The pathogenesis of human immunodeficiency virus (HIV)-associated focal segmental glomerulosclerosis (FSGS) has remained obscure. It has been proposed that renal parenchymal cells may be infected with HIV-1. If such infection occurs, the target cells would be expected to express viral proteins and thus could be targets for cytotoxic T lymphocytes. We previously described mice transgenic for a gag-pol-deleted HIV-1 genome that developed FSGS. In the present study, we tested the requirement for functional T cells in the evolution of renal disease in this model. We bred the HIV-transgenic mice (T26) with athymic nude mice to produce athymic T26 mice. We confirmed by flow cytometry of peripheral blood, thymus, lymph node, and spleen that the athymic T26 mice lacked mature T cells. The athymic T26 mice developed renal disease characterized by FSGS, tubular atrophy and dilatation, and interstitial infiltrate that was qualitatively identical to that seen in the parental T26 mice. Quantitative assessment of the athymic T26 mouse kidneys showed that glomerulosclerosis, tubular injury, and interstitial infiltrate were less severe compared with the parental T26 mouse kidneys. Although T26 mouse kidneys had a mixed cellular infiltrate composed of CD4 cells, CD8 cells, and macrophages, interstitial infiltrates within the athymic T26 mouse kidneys included macrophages but lacked both CD4 and CD8 cells. The renal expression of the HIV transgene was 1. 7-fold greater in T26 mice compared with athymic T26 mice. We conclude that mature T cells are not absolutely required for the development of HIV-associated nephropathy in transgenic mice but that, in their absence, renal disease is significantly milder. These data suggest that T-cell-mediated cytotoxicity directed against renal cells expressing virally encoded proteins is not an essential feature of renal pathogenesis in this model.

The HIV-1 virion-associated accessory protein Vpr affects both viral replication and cellular transcription, proliferation, and differentiation. We report that Vpr enhances the activity of glucocorticoids in lymphoid and muscle-derived cell lines by interacting directly with the glucocorticoid receptor and general transcription factors, acting as a coactivator. Vpr contains the signature motif LXXLL also present in cellular nuclear receptor coactivators, such as steroid receptor coactivator 1 and p300/CREB-binding protein, which mediates their interaction with the glucocorticoid and other nuclear hormone receptors. A mutant Vpr molecule with disruption of this coactivator signature motif lost its ability to influence transcription of glucocorticoid-responsive genes and became a dominant-negative inhibitor of Vpr, possibly by retaining its general transcription factor-binding activities. The glucocorticoid coactivator activity of Vpr may contribute to increased tissue glucocorticoid sensitivity in the absence of hypercortisolism and to the pathogenesis of AIDS.

Transgenic mice (T26) bearing the envelope, regulatory, and accessory genes of HIV- I develop renal disease resembling human HIV-associated nephropathy (HIVAN). Effects of vehicle (VEH) and the angiotensin-converting enzyme inhibitor captopril (CAP) were examined in wild-type (WT) or T26 mice treated from 7 to 100 d of age. Mortality was lower in CAP T26 mice (30 mg/kg: 8%; 100 mg/kg: 12%) than VEH T26 mice (52%). The urinary protein/creatinine ratio was increased in VEH T26 mice (19.5+/-7.60) versus WT mice (6.1+/-0.83), but not in low-dose (7.3+/-0.94) or high-dose (8.2+/-1.02) CAP T26 mice. Blood urea nitrogen was higher in VEH T26 mice (52+/-16.2 mg/dl) than VEH WT mice (24+/-0.8). Blood urea nitrogen was also elevated in CAP WT (high dose: 43+/-2.1 mg/dl) and T26 mice (high dose: 42+/-2.4 mg/dl). Glomerular injury was higher in VEH T26 mice (6.8+/-0.58) than VEH WT mice (0.2+/-0.08) or CAP T26 mice (low dose: 1.1+/-0.17; high dose: 0.7+/-0.13). Tubulointerstitial injury was also greater in VEH T26 mice (1.1+/-0.10) than VEH WT mice (0.2+/-0.08) or CAP T26 mice (low dose: 0.4+/-0.10; high dose: 0.3+/-0.10). These data validate recent nonrandomized studies of captopril in HIV-infected patients, and suggest that an angiotensin-converting enzyme substrate is an important mediator in HIVAN. A randomized placebo-controlled trial of captopril in HIVAN may be warranted.

To investigate the dynamic interplay between human immunodeficiency virus type I (HIV-1) replication and the extent of immune destruction in HIV-1-infected children, virus burden in lymphoid tissues (LN) and peripheral blood was compared with changes in LN architecture and cytokine levels constitutively expressed in LN. In agreement with results of a preliminary study, the plasma HIV-1 RNA level correlated with the amount of provirus in LN. However, the level was also associated with a degree of destruction of lymphoid follicular architecture and an alteration of immune cytokine expression. Expression of interleukin (IL)-4 was higher in LN with higher virus replication. Reduction of plasma viremia was associated with an increase in IL-2 mRNA levels in LN. These findings suggest that measurable virus burden in the peripheral blood is not a simple reflection of viral replication in LN but is also influenced by the extent of progressive immune destruction.

Skin from mice transgenic (Tg) for part of the human immunodeficiency virus type 1 (HIV-1) genome was transplanted onto normal mice of the same strain. All Tg grafts were rejected within 29 days. In contrast, skin from normal mice that was transplanted to HIV-1-Tg recipients remained viable for > 67 days. Histologic examination of Tg grafts on normal mice showed evidence of monocytic infiltrates. Monocytic infiltrates were not observed, however, when either normal or Tg skin was transplanted onto Tg mice. Immunohistologic staining verified the presence of gp120 protein expression in the Tg-transplanted skin but not in adjacent normal skin. It is concluded that the Tg mice are immunologically tolerant to the HIV-1 gene products they express.

To explore tissue-specific factors that may be important in HIV-1 transcriptional and post-transcriptional regulation, we examined a transgenic mouse model containing a mutant provirus deleted in the gag and pol region. The level of transgene expression was tissue-dependent. Skin, muscle, and tail consistently expressed the transgene abundantly; intestine, kidney, and thymus exhibited variable but generally low levels of expression; while liver expression was undetectable by Northern analysis. Individual mRNAs within the family of singly and multiply spliced messages were determined by reverse transcription (rt) of RNA samples from mouse tissues, polymerase chain reaction (PCR) amplification, and Southern hybridization with exon-specific probes. The exact percentage of Tat-coding mRNA that was multiply spliced was also determined by competitive rtPCR. When 2-, 4-, or 7-kb (full-length) mRNA species were calculated as a percentage of the total mRNA, two phenotypes of distribution were detected. Lymphoid tissue (thymus and spleen) and kidney had significantly greater amounts of unspliced message (P < 0.001) regardless of the level of expression. All other tissues expressed the multiply spliced messages encoding Tat, Rev, and Nef predominantly. Furthermore, utilization of the three major second exon splice acceptor sites for tat, rev, and nef was the same in transgenic mice as has been demonstrated in human cells but the splice acceptor site for the vpu/env was different in murine tissue. The marked tissue-dependent patterns of HIV mRNA expression suggest a potential mechanism for the organ-specific manifestations of AIDS.

The amount of human immunodeficiency virus type 1 (HIV-1) in various tissues was investigated by polymerase chain reaction (PCR) in 16 patients with end-stage HIV-1 infection and 7 patients with symptomatic but less advanced disease. During postmortem study of the 16 end-stage patients, HIV-1 DNA was found most often in lymph nodes and the spleen (both 100%), lung (93.8%), and colon (87.5%). Biopsied lymph nodes from the 7 symptomatic patients contained substantially higher copy numbers of HIV-1 RNA and DNA than did peripheral blood mononuclear cells (PBMC). Plasma viral RNA levels correlated significantly with the amount of HIV-1 RNA in PBMC (r2 = .86, P = .0025) but not with the level of viral RNA in lymph nodes in patients with symptomatic HIV-1 infection. These data suggest that although lymph nodes represent the main site for HIV-1 infection and replication, the level of circulating viral burden may not be solely determined by the magnitude of active HIV-1 replication in lymph nodes.

The mechanisms which predispose to growth failure in infants and children infected with immunodeficiency virus type-1 (HIV-1) are not fully understood. The contributions of viral replication and CD4+ T cell depletion to growth failure in an HIV-1 transgenic mouse model were investigated. Mice homozygous for the transgene, a gag-pol deletion mutant of the HIV-1 provirus pNL4-3, exhibited marked cachexia, growth retardation, lymphoproliferation with a reduction in the percentage of CD4+ T cells but an increase in the absolute number of splenic CD4+ and CD8+ T cells, thymic hypoplasia, and early death. Despite the absence of T cells, athymic nude mice, homozygous for the HIV transgene, displayed comparable growth failure. The results indicate that AIDS-like cachexia may be produced by expression of viral envelope or accessory genes, need not be accompanied by absolute depletion of CD4+ T cells, and may occur independent of T cell function.

Patients infected with HIV-1 experience several hyperproliferative skin disorders, including seborrheic dermatitis, ichthyosis, and psoriasis. Transgenic mice carrying a subgenomic HIV-1 proviral construct lacking the gag and pol genes were found to develop proliferative epidermal lesions, manifested as diffuse epidermal hyperplasia in homozygous transgenic mice and benign papillomas in heterozygous transgenic mice. Nonpapillomatous skin from both homozygotes and heterozygotes expressed viral RNA, and the viral envelope protein gp120 was localized to the suprabasal keratinocyte. Papillomas contained increased amounts of both viral mRNA and envelope glycoprotein. Exposure of transgenic mice to doses of ultraviolet B (UV-B) irradiation that induced cutaneous injury increased viral gene expression and resulted in the development of papillomas within 14-21 days. Cutaneous injury induced by phenol and liquid nitrogen had similar effects. These data support a role for HIV-1 gene products in the pathogenesis of proliferative epidermal disorders associated with HIV-1 infection. Further, they suggest that the process of wound repair increases HIV-1 gene expression in this transgenic mouse model.

Patients infected with human immunodeficiency virus type 1 (HIV-1) develop a renal syndrome characterized by proteinuria, renal failure, and focal segmental glomerulosclerosis. By using a noninfectious HIV-1 DNA construct lacking the gag and pol genes, three transgenic mouse lines have been generated that develop a syndrome remarkably similar to the human disease. In the present study, we have characterized in detail one of these lines, Tg26. In Tg26 mice, proteinuria was detectable at approximately 24 days of age, followed by severe nephrotic syndrome and rapid progression to end-stage renal failure. Renal histology showed focal segmental glomerulosclerosis and microcystic tubular dilatation. Indirect immunofluorescence studies demonstrated increased accumulation of the basement membrane components laminin, collagen type IV, and heparan sulfate proteoglycan. The viral protein Rev was present in sclerotic glomeruli. Northern blot analysis of total renal RNA showed expression of viral genes prior to the appearance of histologic renal disease, with greatly diminished viral gene expression late in the disease course. Kidneys from transgenic mice expressed increased steady-state levels of collagen alpha 1(IV) mRNA when glomerulosclerosis was present. We conclude that the presence of HIV-1 genes is associated with progressive renal dysfunction and glomerulosclerosis in transgenic mice.