Detection and localization of HIV-1 DNA in renal tissues by in situ polymerase chain reaction.

The localization of HIV-1 DNA in renal tissues is critically important for understanding pathogenesis of HIV-associated nephropathy (HIVAN), but the clarification has been technically challenging. We applied in situ polymerase chain reaction (IS-PCR) to human renal tissues to demonstrate viral entry into the renal epithelial cells in vivo. To test the specificity of this method and to determine the cell types infected, we used IS-PCR followed by in situ hybridization (ISH) and IS-PCR followed by immunohistochemistry and histochemical counterstains. Brief 2 hour fixation in 4% paraformaldehyde had 92.9% sensitivity and 100% specificity for detection of viral DNA in renal biopsies of HIVAN patients, compared to 70.8% sensitivity and 66.7% specificity in renal biopsies fixed overnight in 10% formalin. Under optimized conditions, the only signals detectable in HIV-1 seronegative cases were false positives attributable to renal tubular apoptosis. In HIVAN cases, positive signal was observed in podocytes, parietal cells, renal tubular cells, and interstitial leukocytes. Immunohistochemical co-labeling for pan-T cell and macrophage markers revealed that the interstitial leukocytes with positivity for HIV-1 DNA included both T cells and macrophages. Application of ISH after IS-PCR showed the same distribution of signal as observed using IS-PCR alone, confirming the specificity of the technique. IS-PCR is a powerful technique to detect viral DNA in human tissue sections, but requires proper use of negative controls to set optimal fixation, protein digestion, and amplification conditions.

HIV-associated nephropathy: an urban epidemic.

Human immunodeficiency virus-associated nephropathy (HIVAN) is the most common form of chronic renal disease in HIV-1-seropositive patients. Over 85% of cases of HIVAN occur in African-American patients and it is the third leading cause of ESRD in blacks age 20 to 64. Changes in incidence rates of HIVAN have coincided with changes in AIDS incidence rates. The demographics of the AIDS/HIV-1 epidemic indicate that the risk pool for HIVAN will continue to grow and that urban Nephrology centers will continue to see high rates of HIVAN. In addition, improvements in survival rates of HIV-1-seropositive patients on hemodialysis and improved treatment of HIVAN with highly active antiretroviral therapy (HAART) and angiotensin-converting enzyme (ACE)-inhibitors will result in an increased prevalence of HIVAN in the end-stage renal disease (ESRD) and pre-ESRD patient populations.

Nuclear factor-kappa B binding to the HIV-1 LTR in kidney: implications for HIV-associated nephropathy.

BACKGROUND: We have recently shown that renal epithelium is infected by HIV-1 and supports HIV-1 transcription in seropositive patients with renal disease. To investigate the regulation of HIV-1 gene expression in kidney, an HIV-1 transgenic mouse model was used to analyze the host transcriptional proteins that bind the 5' long-terminal repeat (LTR). METHODS: Viral gene expression was assessed in transgenic mouse tissue using Northern blotting and mRNA in situ hybridization. The transcription factors involved in LTR binding were determined using electrophoretic mobility shift assays. Cytoplasmic and nuclear extracts were prepared from tissues with varied levels of transgene expression. The binding of transcription factors to specific LTR fragments was determined using DNA competition experiments and supershifts with transcription factor-specific antibodies. RESULTS: Tissue-specific expression of the transgene was variable, with viral gene expression in the kidney at an intermediate level as compared with other tissues. Overall, the level of transgene expression directly correlated with abundance of nuclear factor-kappa B (NF-kappa B) in the nuclear extracts. High expressing tissue, however, had a constitutively active form of NF-kappa B. In contrast, the kidney contained an inducible NF-kappa B, which bound the LTR in combination with Sp1, suggesting a requirement for an activating event in renal HIV-1 expression of the LTR. CONCLUSIONS: These studies indicate that the regulation of the HIV-1 LTR in the kidney is similar to lymphoid tissues, and may explain, in part, why the HIV-1 life cycle is supported in kidney.

Effect of tissue processing on the ability to recover nucleic acid from specific renal tissue compartments by laser capture microdissection.

The anatomic heterogeneity of the nephron poses obstacles to microdissection of individual renal compartments for analysis of gene expression. We have systematically analyzed the effects of fixation time and nuclear staining on the ability to recover nucleic acid from individual renal compartments by laser capture microdissection (LCM). Formalin-fixed kidney sections from Wistar rats and archival human renal biopsies were used for DNA analysis. From 1 to 10 individual glomeruli and from 1 to 10 individual proximal tubules were captured sequentially onto polymer films. DNA for beta-globin could be amplified by PCR from even a single glomerulus or tubule. Optimal conditions for DNA amplification were brief (1- or 2-day) formalin fixation. Use of nuclear counterstains, including Weigert's hematoxylin, Harris's hematoxylin, Mayer's hematoxylin, or methyl green, did not adversely affect the ability to extract and amplify DNA. For RNA extraction, glomeruli and tubules were microdissected from renal cryostat sections stored for up to 6 months. By RT-PCR, mRNA expression of the glomerulus-specific gene, Wilms' tumor-1, was identified in as few as 5 microdissected glomeruli and of the tubule-specific gene, aminopeptidase N, in as few as 5 microdissected tubules, with no cross-contamination between renal compartments. Our findings indicate that the LCM method can successfully microdissect pure glomerular and tubular tissue compartments and that the optimal fixation and staining conditions are those employed routinely for renal biopsies, namely overnight formalin fixation and hematoxylin counterstain for DNA extraction, and cryostat sectioning with hematoxylin counterstain for RNA extraction. The specificity of LCM coupled with the sensitivity of RT-PCR should prove a powerful tool for the analysis of gene expression in specific renal compartments from archival human renal biopsies.

HIV-associated nephropathy: case study and review of the literature.

Human immunodeficiency virus type 1 (HIV-1)-seropositive patients are at risk for the development of a variety of acute and chronic renal diseases. The most common cause of chronic renal failure in HIV-1-seropositive patients is HIV-associated nephropathy (HIVAN). HIVAN occurs almost exclusively in black patients and the majority of published cases are of patients who present with acquired immunodeficiency syndrome (AIDS). This disease is currently the third leading cause of end-stage renal disease in blacks aged 20-64. Because HIV-1-seropositive patients may develop a wide variety of acute and chronic renal diseases, definitive diagnosis requires renal biopsy. Emerging data suggest a direct role of HIV-1 infection of kidney cells in the pathogenesis of HIVAN. There have been no well-controlled clinical trials in the treatment of HIVAN. The therapeutic agents with the most promise are angiotensin-converting enzyme inhibitors and antiretroviral medications. Long-term renal prognosis may be changing in the setting of improved aggressive antiretroviral therapy. Patient survival is determined primarily by the stage of HIV-1 infection. In this article, we present the case history of a patient who developed HIVAN. We then review the current literature concerning the epidemiology, differential diagnosis, etiology, and treatment of HIVAN.

Glomerulosclerosis and viral gene expression in HIV-transgenic mice: role of nef.

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.

HIV-1 induces renal epithelial dedifferentiation in a transgenic model of HIV-associated nephropathy.

BACKGROUND: Human immunodeficiency virus-associated nephropathy (HIVAN) is the most common cause of renal failure in HIV-1-seropositive patients. Recent studies using an HIV-1 transgenic mouse model have demonstrated that expression of HIV-1 in the kidney is required for the development of HIVAN. What has remained unclear, however, is the renal cell type responsible for pathogenesis and the essential pathological process. METHODS: To address these issues, we used a transgenic murine model of HIVAN. We identified the cell types in kidney in which HIV transgene expression occurs using in situ hybridization. We evaluated evidence of proliferation by immunocytochemical analysis using an antibody to Ki-67 and cell type-specific markers, including WT-1, synaptopodin, Na+,K+-ATPase, adducin, and desmin. TUNEL assay was used to evaluate apoptosis. RESULTS: We found that glomerular and tubular epithelial cells express the HIV-1 transgene early in the disease process when renal architecture is well preserved. Transgene expression is lost, however, in tubular epithelial cells when they lose their differentiated cuboidal phenotype. In glomerular epithelial cells, dedifferentiation occurs with reduced expression of WT-1 and synaptopodin, in association with activation of desmin expression. Tubular microcysts also form with mislocalization of Na+,K+-ATPase expression to the lateral and apical cellular membranes. CONCLUSIONS: These studies support the hypothesis that the glomerular and renal epithelial cells are the primary targets of HIV-1 pathogenesis in the kidney. The essential pathologic process is dysregulation of the epithelial cell cycle with increased proliferation, apoptosis, cellular dedifferentiation, and altered cellular polarity.

Treatment of HIV-associated nephropathy.

HIV-Associated Nephropathy (HIVAN) is the most common cause of chronic renal disease in HIV-1 infected patients. The disease occurs predominantly in blacks between the ages of 20 and 64. In this population it is currently the third leading cause of end-stage renal disease. The majority of patients with HIVAN have an AIDS-defining condition when the kidney disease is diagnosed. Without treatment they progress to end-stage renal disease within weeks to months. Patients with HIVAN should be treated with highly active antiretroviral therapy (HAART). Treatment should prolong survival and may improve or stabilize kidney function. Steroids have short-term benefits but long-term benefits have not been shown. Converting enzyme inhibitors (CEI) seem to stabilize kidney function and appear to be most effective when administered early in the course of HIVAN. A randomized controlled trial comparing HAART therapy to HAART and CEI should be performed.

Transduction of renal cells in vitro and in vivo by adeno-associated virus gene therapy vectors.

There has been an increasing interest recently in the possibility of treating renal diseases using gene therapy. The ability to pursue gene therapy for renal diseases has been limited by the availability of an adequate system for gene delivery to the kidney. Adeno-associated virus (AAV) is a defective virus of the parvovirus family that has a number of properties attractive for renal gene delivery: recombinant AAV contains no viral genes; expression of genes delivered by these vectors does not activate cell-mediated immunity; the virus is able to transduce nondividing as well as dividing cells; and both wild-type and recombinant AAV integrate into the host chromosome resulting in long-term gene expression. Studies were performed to determine whether AAV can deliver reporter genes to kidney cells in vitro and in vivo. These studies show that AAV can deliver reporter genes with approximately equal efficiency to human mesangial, proximal tubule, thick ascending limb, collecting tubule, and renal cell carcinoma cells in primary culture. Immortalized mouse mesangial cells are transduced at a much greater efficiency. Transduction can be enhanced by pharmaceutical agents up to sevenfold in primary cells (transducing up to 20% of primary cells per well) and as much as 400-fold in immortalized mesangial cells. AAV delivered in vivo by intraparenchymal injection results in at least 3 mo of reporter gene expression in tubular epithelial, but not glomerular or vascular, cells at the injection site. These data indicate that AAV can deliver genes to renal cells both in vitro and in vivo resulting in prolonged gene expression, and thus AAV can be a useful tool for renal gene delivery.

Chronic rejection of mouse kidney allografts.

BACKGROUND: Chronic renal allograft rejection is the leading cause of late graft failure. However, its pathogenesis has not been defined. METHODS: To explore the pathogenesis of chronic rejection, we studied a mouse model of kidney transplantation and examined the effects of altering the expression of donor major histocompatibility complex (MHC) antigens on the development of chronic rejection. RESULTS: We found that long-surviving mouse kidney allografts develop pathological abnormalities that resemble chronic rejection in humans. Furthermore, the absence of MHC class I or class II antigens did not prevent the loss of graft function nor alter the pathological characteristics of chronic rejection. Expression of transforming growth factor-beta (TGF-beta), a pleiotropic cytokine suggested to play a role in chronic rejection, was markedly enhanced in control allografts compared with isografts. However, TGF-beta up-regulation was significantly blunted in MHC-deficient grafts. Nonetheless, these differences in TGF-beta expression did not affect the character of chronic rejection, including intrarenal accumulation of collagens. CONCLUSIONS: Reduced expression of either class I or II direct allorecognition pathways is insufficient to prevent the development of chronic rejection, despite a reduction in the levels of TGF-beta expressed in the allograft. This suggests that the severity of chronic rejection is independent of the level of MHC disparity between donor and recipient and the level of TGF-beta expression within the allograft.

HIV-associated nephropathy is a late, not early, manifestation of HIV-1 infection.

BACKGROUND: Human immunodeficiency virus-associated nephropathy (HIVAN) can be the initial presentation of HIV-1 infection. As a result, many have assumed that HIVAN can occur at any point in the infection. This issue has important implications for appropriate therapy and, perhaps, for pathogenesis. Since the development of new case definitions for acquired immunodeficiency syndrome (AIDS) and better tools to assess infection, the relationship of HIVAN to the time of AIDS infection has not been addressed. In this study, we reassessed the stage of infection at the time of HIVAN diagnosis in 10 patients, and we reviewed all previously published cases applying the new case definitions to assess stage of infection. METHODS: HIVAN was confirmed by kidney biopsy in HIV seropositive patients with azotemia and/or proteinuria. CD4+ cell count and plasma HIV-1 RNA copy number were measured. We also reviewed all published cases of HIVAN to determine if AIDS-defining conditions, by current Centers for Disease Control definitions, were present in patients with biopsy-proven HIVAN. RESULTS: Twenty HIV-1 seropositive patients with proteinuria and an elevated creatinine concentration were biopsied. HIVAN was the single most common cause of renal disease. CD4+ cell count was below 200/mm3 in all patients with HIVAN, fulfilling Centers for Disease Control criteria for an AIDS-defining condition. HIV-1 plasma RNA was detectable in all patients with HIVAN. In reviewing previous reports, an AIDS-defining condition was present in virtually all patients with HIVAN. CONCLUSION: HIVAN develops late, not early, in the course of HIV-1 infection following the development of AIDS. This likely accounts for the poor prognosis noted in previous publications and has implications for pathogenesis. In addition, given the detectable viral RNA levels, highly active antiretroviral therapy is indicated in HIVAN. Highly active antiretroviral therapy may improve survival as well as alter the natural history of HIVAN.

Involvement of the protein kinase C substrate, SSeCKS, in the actin-based stellate morphology of mesangial cells.

Activation of protein kinase C is a key signal transduction event in mesangial cell dedifferentiation and proliferation, yet little is known about downstream substrates or their roles in normal or diseased glomeruli. SSeCKS, a novel protein kinase C substrate originally isolated as a src-suppressed negative mitogenic regulator in fibroblasts, controls actin-based cytoskeletal architecture and scaffolds key signaling kinases such as protein kinase C and protein kinase A. Based on the morphologic similarity between SSeCKS-overexpressing fibroblasts and stellate mesangial cells, we hypothesized that SSeCKS might play a role in mesangial cell morphology in a protein kinase C-dependent manner. Immunoblotting, in situ staining and northern blotting detected abundant expression of SSeCKS in human and rodent mesangial cells and glomerular parietal cells but not in renal tubular epithelia. Immunofluorescence analysis showed enrichment of SSeCKS in mesangial cell podosomes and along a cytoskeletal network distinct from F-actin. Activation of protein kinase C by phorbol ester resulted in a rapid serine phosphorylation of SSeCKS and its subsequent translocation to perinuclear sites, coincident with the retraction of stellate processes. These effects were blocked by concentrations of bis-indolylmaleimide that selectively inhibit protein kinase C. Finally, ablation of SSeCKS expression using retroviral anti-sense vectors induced (1) an elongated, fibroblastic cell morphology, (2) production of thick, longitudinal stress fibers and (3) repositioning of vinculin-associated focal complexes away from the cell edges. These data suggest a role for SSeCKS as a downstream mediator of protein kinase C-controlled, actin-based mesangial cell cytoskeletal architecture.

Enhanced proliferation, apoptosis, and matrix accumulation by mesangial cells derived from HIV-1 transgenic mice.

BACKGROUND: Mice, transgenic for HIV-1 genes, have been demonstrated to develop renal lesions mimicking HIV-associated nephropathy. Focal glomerulosclerosis (FGS) has been reported to be the predominant glomerular lesion in these animals. In the other models of FGS, the accumulation of mesangial matrix and mesangial cell proliferation have been shown to be the preceding abnormalities. We evaluated the proliferation, apoptosis, and matrix accumulation by mesangial cells derived from mice transgenic for HIV-1 genes as well as from nontransgenic mice. METHODS: Mesangial cells were cultured from mice transgenic for HIV-1 genes (HTrMC) and nontransgenic mice (NTrMC) of the same age and sex. The growth rate of HTrMC and NTrMC was determined under identical conditions. Morphologic evaluation of apoptosis was performed by staining cells with Hoechst (H)-33342 and propidium iodide. Accumulation of mesangial cell collagen type IV, laminin, and fibronectin was measured by the dot blot assay. Total RNA was extracted from HTrMC and NTrMC and Northern blots were generated. These blots were probed with specific probes for TGF-beta, proteoglycan (P16), and GAPDH. RESULTS: Mesangial cells (HTrMC) derived from transgenic mice had greater (P < 0.004) proliferation when compared to mesangial cells (NTrMCs) from nontransgenic mice (HTrMCs, 4.2 +/- 0.3 vs NTrMCs, 3.0 +/- 0.2 x 10(4) cells/well). HTrMCs also showed enhanced (P < 0.0001) apoptosis compared to NTrMCs (HTrMCs, 13.2 +/- 1.5% vs NTrMCs, 3.1 +/- 0.5% apoptotic cells/field). HTrMCs accumulated an increased (P < 0.02) amount of collagen type IV (HTrMCs, 5659.7 +/- 472.8 vs NTrMCs, 3882.2 +/- 339.7 ng/well); whereas NTrMCs accumulated a greater amount of laminin when compared to HTrMCs (HTrMCs, 12.8 vs NTrMCs, 29.6 +/- 2.9 ng/well). HTrMCs also showed an enhanced mRNA expression of TGF-beta and an attenuated expression of proteoglycan (P16). CONCLUSIONS: These results suggest that mesangial cells derived from mice transgenic for HIV-1 genes have enhanced proliferation and collagen accumulation. The enhanced expression of TGF-beta may have contributed to enhanced HTrMC proliferation and the accumulation of collagen. The present study provides the basis for a hypothesis that mesangial cells may be contributing to the development of focal glomerulosclerosis in mice transgenic for HIV-1 genes.

The human immunodeficiency virus (HIV) epidemic and HIV-associated nephropathy.

Human immunodeficiency virus (HIV)-associated nephropathy (HIVAN), the single most common cause of end-stage renal failure in seropositive patients, has increased in incidence by 30% each year since 1991. Occurring almost exclusively in blacks, HIVAN became the third leading cause of ESRD in blacks, ages 20 to 64, in 1995. During that year, the absolute number of new acquired immune deficiency syndrome (AIDS) cases declined for the first time since the epidemic began. The decrease occurred predominantly in white males, whereas in blacks with heterosexual exposures for risk factors, the incidence actually increased. Also in 1995, the number of AIDS-related deaths declined for the first time. If these trends continue, we can expect a continued increase in the number of blacks living with AIDS. We estimate that 1% to 4% will develop renal failure from HIVAN. The incidence of HIVAN can be expected to increase unless new approaches are successful in preventing the spread of HIV-1 in all segments of the population or in treating the renal complications of HIV-1 infection.

Pathogenesis of human immunodeficiency virus (HIV)-associated nephropathy.

Human immunodeficiency virus-associated nephropathy (HIVAN) is the third leading cause of end-stage renal failure in Blacks between the ages of 20 and 64. Because the incidence of HIV infection has continued to increase in Blacks as survival has improved, the pool of patients alive and at risk for developing HIVAN has vastly expanded. This suggests that HIVAN will continue to increase in importance to the end-stage renal disease program. The racial predilection for the disease in Blacks implies that genetic or environmental cofactors are involved. Evidence in human and animal models has shown that proliferation of renal epithelial cells is the predominant feature of the disease and that apoptosis occurs. The prospect that renal infection is necessary to stimulate cells to proliferate remains a possibility but is not yet proven. Cytokine dysregulation may also be involved in disease progression, but evidence is lacking that altered cytokine production is the proximate cause of HIVAN. Many issues remain to be resolved including the potential for renal infection in vivo, the mechanisms responsible for proliferation and apoptosis, and factors that provide racial susceptibility to HIVAN. Advances in our understanding of pathogenesis will be required to control the growth of HIV-related renal diseases in the ESRD population.

Adeno-associated virus gene transfer into renal cells: potential for in vivo gene delivery.

The human parvovirus adeno-associated virus (AAV), type 2, has a number of features that make it an attractive choice as a vector for gene delivery to the kidney. AAV vectors permit long-term gene expression in vivo by integration into the host genome, have potential for site-specific integration on chromosome 19, do not express viral genes or generate a cellular immune response, and demonstrate enhancement of gene expression by chemotherapeutic agents that are approved for use in vivo. These properties confer advantages to AAV over other viral and nonviral methods for gene transfer. Preliminary experiments in our laboratory suggest that AAV is able to transfer genes to both renal cells in culture and the kidney in vivo. Thus, AAV has the potential to be an important gene transfer vector for the kidney in vivo.

Optimizing ribozymes for somatic cell gene therapy.

Therapeutic ribozymes are created through a multistep process that requires trial and error. There are few established rules governing ribozyme design, but guidelines are emerging. It is not yet known whether hammerheads and hairpins, the two ribozymes most widely studied as potential gene therapy agents, have the inherent capability to ablate single genes. Their capacity for specificity and selectivity remains to be explored through rigorous experimentation. These experiments require a battery of control molecules, the characteristics of which are outlined here. Methods for completing the steps in the ribozyme development process, from the selection of a target gene to the quantitation of RNA levels, are also presented and discussed.