HIV-associated nephropathy is a clinicopathologic entity that includes proteinuria, focal segmental glomerulosclerosis often of the collapsing variant, and microcystic tubulointerstitial disease. Increasing evidence supports a role for HIV-1 infection of renal epithelium in the pathogenesis of HIV-associated nephropathy. Using in situ hybridization, we previously demonstrated HIV-1 gag and nef mRNA in renal epithelial cells of patients with HIV-associated nephropathy. Here, to investigate whether renal epithelial cells were productively infected by HIV-1, we examined renal tissue for the presence of HIV-1 DNA and mRNA by in situ hybridization and PCR, and we molecularly characterized the HIV-1 quasispecies in the renal compartment. Infected renal epithelial cells were removed by laser-capture microdissection from biopsies of two patients, DNA was extracted, and HIV-1 V3-loop or gp120-envelope sequences were amplified from individually dissected cells by nested PCR. Phylogenetic analysis of kidney-derived sequences as well as corresponding sequences from peripheral blood mononuclear cells of the same patients revealed evidence of tissue-specific viral evolution. In phylogenetic trees constructed from V3 and gp120 sequences, kidney-derived sequences formed tissue-specific subclusters within the radiation of blood mononuclear cell-derived viral sequences from both patients. These data, along with the detection of HIV-1-specific proviral DNA and mRNA in tubular epithelium cells, argue strongly for localized replication of HIV-1 in the kidney and the existence of a renal viral reservoir.

NK-T-B antigen (NTBA) is a CD2 family member that functions as a coreceptor in human NK cell activation. Several receptor/ligand interactions occur between different members of this molecular family. In this study, in order to identify the natural ligand of NTBA, we produced a chimeric protein formed by the NTBA extracellular region fused with the Fc portion of human IgG1 (termed NTBA-Fc*). NTBA-Fc* specifically binds to NTBA cell transfectants but not to cells transfected with other CD2 family members including CD2, CD48, CD84, CD150, CD229, and CD244. Moreover, NTBA-Fc* also binds to NTBA + but not to NTBA -T cell lines. Enzyme-linked immunosorbent assays, plasmon resonance analysis, as well as NTBA-Fc*-mediated down-regulation of NTBA surface expression further confirmed the occurrence of NTBA/NTBA homophilic interaction. Functionally, in NK cells, NTBA-Fc* promoted a strong production of IFN-+ and TNF- § . Moreover, NTBA-transfected targets displayed increased susceptibility to NK-mediated killing as compared to untransfected cells and this effect was specifically inhibited by anti-NTBA mAb. Altogether our data indicate that NTBA is characterized by self recognition.

NK cells express receptors characterized by opposite functions that finely regulate their activities. Among inhibitory receptors, some are specific for different groups of MHC class I alleles, while others are still orphan receptors. On the contrary, various activating receptors are involved in the triggering of NK-mediated natural cytotoxicity. In general, their engagement induces human NK cells to kill target cells that are either HLA class I-negative or -deficient. Thus, the process of NK cell triggering mediated by Natural Cytotoxicity Receptors can be mainly considered as a non MHC-restricted mechanism. Here, a brief description of the molecular nature of these receptors, as well as, of their 3D-structures and of the implications for ligand recognition, is given.

Growing evidence suggests that human immunodeficiency virus (HIV)-1 infection of podocytes plays a central role in the glomerular disease of HIV-associated nephropathy (HIVAN).As an approach to identify host genes involved in the pathogenesis of the sclerotic glomerular lesion in HIVAN, representational difference analysis of cDNA was used to identify differentially expressed genes in HIV-1 transgenic and nontransgenic podocytes. We isolated a novel member of the small leucine-rich repeat (SLR) protein family, podocan, that is expressed at high levels in the HIV-1 transgenic podocytes. In normal embryonic kidney, a 3.2-kb podocan transcript was detected at low levels, and expression increased dramatically within 24 h following birth. Expression of a 2.3-kb transcript became evident after birth and gradually increased to 50% of the total podocan RNA in the mature kidney. Phylogenetically, podocan represents a new class in the SLR protein gene family, an expanding protein family sharing homology with the small leucine-rich repeat proteoglycans. The 3.2-kb transcript encodes a predicted 611-amino acid secretory protein with 20 leucine-rich repeats, a unique N-terminal cysteine-rich cluster pattern and a highly acidic C-terminal domain. In situ hybridization of normal kidney revealed podocan mRNA expression in podocytes and likely vascular endothelial cells within the glomerulus. The immunohistochemical staining pattern of podocan protein in normal kidney glomeruli was consistent with that of the glomerular basement membrane, and staining was markedly increased in sclerotic glomerular lesions in the transgenic HIVAN model. Thus, podocan defines a new class within the SLR protein family and is a previously unrecognized component of the sclerotic glomerular lesion that develops in the course of experimental HIVAN.

Gene transfer into kidney holds great potential as a novel therapeutic approach. We have studied the transduction of kidney in vivo after delivery of lentiviral vectors by various routes of administration. A lentiviral vector expressing the bacterial lacZ gene from the cytomegalovirus early promoter was used. The lentiviral vector was delivered into the kidneys of BALB/c mice by retrograde infusion into the ureter, by injection into the renal vein or artery, or by direct injection into the renal parenchyma. Expression of the reporter gene was achieved independently of the route of administration, although it appeared more efficient after parenchymal or ureteral administration. After parenchymal or ureteral infusion, expression of the transgene was localized to the outer medulla and corticomedullary junction. In the case of parenchymal injection, expression of the reporter gene extended to the cortex. Detection of the transgene in the renal proximal tubules was confirmed by in situ polymerase chain reaction after parenchymal or ureteral infusion. On delivery of the lentiviral vector through the renal artery or vein, expression of the reporter gene was markedly lower than was observed with parenchymal or ureteral infusion and was limited to the inner medullary collecting ducts. No apparent histological abnormality was observed after virus administration and transgene expression was stable for at least 3 months. These results provide the first evidence that lentiviral vectors can stably transduce renal cells in vivo and may be effective vehicles for gene delivery to the kidney.

These findings indicate that intrathecal injections of lentivectors can provide significant levels of transgene expression in the meninges. Unlike intracerebral injections of lentivectors, intrathecal gene delivery through the spinal canal appears to produce a wider diffusion of the transgene. This approach is less invasive and may be useful to address those neurological diseases that benefit from the ectopic expression of soluble factors impermeable to the blood-brain barrier.

Lentiviral vectors can effectively deliver exogenous genes to the kidney in vivo, resulting in persistent expression of the introduced gene.

The phenotype of HIV-1 gp120 envelope derived from renal epithelium and peripheral blood mononuclear cells (PBMC) of patients with HIV-associated nephropathy was investigated in vitro. Chimeric viruses were derived from kidney or blood and used to infect primary CD4+T cells, cell lines expressing single co-receptors and a renal epithelial cell line HPT-1. HIV-1 variants derived from renal epithelium were dual tropic whereas simultaneously derived viruses from PBMC were R5-tropic. Utilization of alternative co-receptors CCR3, BONZO and BOB, also differed.