Infection of human primary renal epithelial cells with HIV-1 from children with HIV-associated nephropathy
1998; Elsevier BV; Volume: 53; Issue: 5 Linguagem: Inglês
10.1046/j.1523-1755.1998.00900.x
ISSN1523-1755
AutoresPatricio E. Ray, Xuehui Liu, D. Alan Henry, Louis Dye, Lian Xu, J M Orenstein, Ted E. Schuztbank,
Tópico(s)HIV-related health complications and treatments
ResumoInfection of human primary renal epithelial cells with HIV-1 from children with HIV-associated nephropathy. Children affected with human immunodefficiency virus (HIV)-associated nephropathy (HIVAN) usually develop significant renal glomerular and tubular epithelial cell injury. The pathogenesis of these changes is not clearly understood. Human renal tubular epithelial cells (RTEc) do not express CD4 surface receptors, and it is not clear whether these cells can be infected by HIV-1. Certain strains of HIV-1, however, have been shown capable of infecting CD4-negative epithelial cell lines. We hypothesized that the inability of laboratory strains of HIV-1 to infect renal epithelial cells may be due to a limited tropism, as opposed to wild-type viruses derived from children with HIVAN, and that viruses derived from these children are capable of infecting RTEc from the same patient. Here, we have demonstrated that HIV-1 isolates from children with HIVAN can productively infect RTEc through a CD4 independent pathway, and that infected mononuclear cells can transfer the virus to human RTEc. Human RTEc sustained low levels of viral replication and HIV-1 inhibited the growth and survival of cultured human RTEc. Thus, HIV-1 may directly induce degenerative changes in RTEc of children with HIVAN. Infected macrophages may play a relevant role in this process by transferring viruses to RTEc. Infection of human primary renal epithelial cells with HIV-1 from children with HIV-associated nephropathy. Children affected with human immunodefficiency virus (HIV)-associated nephropathy (HIVAN) usually develop significant renal glomerular and tubular epithelial cell injury. The pathogenesis of these changes is not clearly understood. Human renal tubular epithelial cells (RTEc) do not express CD4 surface receptors, and it is not clear whether these cells can be infected by HIV-1. Certain strains of HIV-1, however, have been shown capable of infecting CD4-negative epithelial cell lines. We hypothesized that the inability of laboratory strains of HIV-1 to infect renal epithelial cells may be due to a limited tropism, as opposed to wild-type viruses derived from children with HIVAN, and that viruses derived from these children are capable of infecting RTEc from the same patient. Here, we have demonstrated that HIV-1 isolates from children with HIVAN can productively infect RTEc through a CD4 independent pathway, and that infected mononuclear cells can transfer the virus to human RTEc. Human RTEc sustained low levels of viral replication and HIV-1 inhibited the growth and survival of cultured human RTEc. Thus, HIV-1 may directly induce degenerative changes in RTEc of children with HIVAN. Infected macrophages may play a relevant role in this process by transferring viruses to RTEc. 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Thus, it is possible that HIV-1 may adapt to host tissues by using cell surface receptors or by acquiring adhesion molecules from host cells29.Lee M.M. Hildreth J.E.K. HIV acquires functional adhesion receptors from host cells.AIDS Res Hum Retroviruses. 1995; 11: 1007-1013Crossref Scopus (8) Google Scholar and gaining a selective tropism to infect these cells. We hypothesized that the inability of HIV-1 to infect renal epithelial cells may be due to a limited tropism of cloned laboratory strains, as opposed to wild-type virus, and that HIV-1 isolates derived from children with HIVAN are capable of infecting renal tubular epithelial cells derived from the same patient. This study demonstrates that HIV-1 can infect renal epithelial cells and supports the hypothesis that HIV-1 may cause direct cytotoxicity of renal epithelial cells from children with HIVAN. Primary HIV-1 isolates were initially derived from peripheral blood mononuclear cells (PBMCs), from three patients and from the peritoneal fluid of one additional patient after informed consent was obtained (Table 1). HIV-1 (SP-PF1) and (BS-3) were isolated from two children with HIVAN also affected with hemolytic uremic syndrome (HIVAN-HUS). HIV-1 (RW-2) was isolated from a child with HIV-HUS, and DC-4 from a child with HIVAN. All children with HUS were in renal failure. This study was approved by the Children's Hospital Institutional Review Board. PBMCs were separated on Ficoll-Hypaque gradients and propagated in 10 ml RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum, 5% interleukin-2 and 5 μg/ml PHA from Cellular Products (Buffalo, NY, USA). PBMCs (1 × 107) and peritoneal cells (5 × 106) were co-cultured with 1 × 107 mitogen-stimulated normal human PBMCs from healthy HIV-1 negative donors. The culture media was changed every three days. All cultures were fed with fresh HIV-1 negative PBMC (5 × 106) once a week. The HIV-1 IIIB isolate from Dr. Robert Gallo37.Popovic M. Sarngadharan M.G. Read E. Gallo R.C. Detection, isolation and continuous production of cytopathic retrovirus (HTLV-III) from patients with AIDS and pre-AIDS.Science. 1984; 224: 497-500Crossref PubMed Scopus (2526) Google Scholar was obtained through the AIDS Research and Reference Reagent Program (Division of AIDS, NIAID, NIH, Bethesda, MD, USA). Samples from infected PBMCs and cell-free supernatants were frozen and saved. Virus supernatants were harvested from infected PBMCs and titrated simultaneously by limiting dilution on HIV-1 negative PBMCs. The viabilities of these cultures were monitored by vital dye exclusion as described previously by Pise, Newburger and Holland38.Pise C.A. Newburger P.E. Holland C.A. Human immunodeficiency virus type 1-infected HL-60 cells are capable of both monocytic and granulocytic differentiation.J Gen Virol. 1992; 73: 3257-3261Crossref PubMed Scopus (7) Google Scholar. Only, cultures yielding infectious titers of > 106 TCID50/ml were used.Table 1Primary viral isolates used in this study Open table in a new tab Primary renal tubular epithelial cells (RTEc) were isolated from renal biopsies, autopsies, and urine of five children with HIV-associated renal disease. RTEc cultures were established as described previously34.Wilson P.D. Monolayer of microdissected renal tubule epithelial segments.J Tiss Cult Meth. 1991; 13: 137-141Crossref Scopus (16) Google Scholar, by cloning homogeneous micro-colonies of RTEc on collagen I-coated dishes. RTEc exhibit a characteristic epithelial morphology, growing in cobblestone-like monolayers Figure 1. They lack Factor VIII-related antigen, characteristic of endothelial cells, and express cytokeratin, γ-GTP, or other markers of cell differentiation depending on each specific RTEc type34.Wilson P.D. Monolayer of microdissected renal tubule epithelial segments.J Tiss Cult Meth. 1991; 13: 137-141Crossref Scopus (16) Google Scholar. To preferentially enhance the growth of proximal tubular cells, which are capable of endocytosis, a potential mechanism of HIV-1 viral entry into the cells, all primary colonies were plated on collagen-coated plastic dishes and grown in the following selective renal proximal tubule culture medium: Click RPMI (Quality Biological Inc. Gaithersburg, MD, USA), supplemented with 1% fetal bovine serum (Lot #A9234J; Gemini Bioproducts, Calabasas, CA, USA), 5 ml/500 ml (100 xpen/strep), 1 mm Hepes, 2 mm L-glutamine, insulin (5 μg/ml), transferrin (5 μg/ml), and dexamethasone 5 × 10-8m, obtained from Sigma (St. Louis, MO, USA). Insulin was omitted in collecting tubule culture media. All cells were used between passages 1 and 3 while retaining epithelial characteristics. RTEc were also isolated from HIV-negative children (Table 2). The diagnosis of HIV infection was excluded based on the clinical history and HIV-1 serology. HIV-1 serology, however, was not available in two patients. All RTEc derived from children not infected with HIV-1 were screened by HIV-1 PCR and p24 antigen measurements in culture supernatants. RTEc derived from proximal, thick ascending limb of Henle, and distal convoluted tubules were generously provided by Dr. Patricia Wilson (Mount Sinai Medical Center, New York, NY, USA). Mouse primary RTEc were isolated and grown as previously described11.Ray P.E. Bruggeman L.A. Weeks B.S. Kopp J.B. Bryant J.L. Owens J.W. Notkins A.L. Klotman P.E. Role of bFGF and its low affinity receptors in the pathogenesis of HIV-associated nephropathy in transgenic mice.Kidney Int. 1994; 46: 759-772Abstract Full Text PDF PubMed Scopus (88) Google Scholar. The non permissive renal canine epithelial cell line MDCK was obtained from ATCC (Rockville, MD, USA). The HeLa-CD4+ cell clone 1022 from Dr. Bruce Chesebro35.Chesebro B. Wehrly K. Metacalf J. Griffin D.E. Use of a new CD4-positive HeLa cell clone for direct quantification of infectious human immunodeficiency virus from blood cells of AIDS patients.J Infect Dis. 1991; 163: 64-70Crossref PubMed Scopus (62) Google Scholar was obtained through the AIDS Research and Reference Reagent Program (Division of AIDS, NIAID, NIH, Bethesda, MD, USA). This adherent cell line was used as a positive control since HIV-1 IIIB and many primary HIV isolates derived from patients PBMC are capable of infecting these cells36.Chesebro B. Wehrly K. Development of a sensitive quantitative focal assay for human immunodeficiency virus infectivity.J Virol. 1988; 62: 3779-3788Crossref PubMed Google Scholar.Table 2Summary of all primary epithelial cell cultures used in this studyView Large Image Figure ViewerDownload (PPT) Open table in a new tab To determine whether RTEc derived from HIV-infected children were not previously infected at the outset of the experiments, the following studies were performed: (1) polymerase chain reaction (PCR) for HIV-1 proviral DNA; (2) p24 antigen measurements in culture supernatants (p24 antigen values > 2.0 sd above the mean from all control uninfected RTEc cells were considered positive); (3) determined whether HIV-1 could be rescued by co-culturing a representative sample of cells from each clone with stimulated human HIV-1 negative PBMCs; (4) studied selected RTEc clones by electron microscopy to exclude the presence of viral particles. RTEc derived from HIV-infected children were not used in other experiments if at least one of these results was positive. Each RTEc clone or cell type was divided in two groups. One group of cells was used as control and the other group was exposed to HIV-1. In cell-associated infection experiments, RTEc were exposed to HIV-1 infected PBMCs. In cell-free infection experiments, RTEc were exposed to HIV-1 infected supernatants derived from infected PBMCs. Supernatants were filtered through a 0.4 μm pore size membrane. Control cells were exposed to stimulated HIV-1 negative PBMCs or uninfected supernatants. All experiments were done in triplicate. In typical experiments, human RTEc were plated on 24-well plates, six-well plates, or T25 flasks, at a density of approximately 2,500 cell/cm2. RTEc were grown until they were confluent. In cell-associated infection experiments, RTEc were exposed to HIV-1 infected PBMCs (approximately 2.4 × 103 cells/cm2), while in cell-free infection experiments, RTEc were exposed to HIV-1 infected supernatants [> 1 × 107 infection units (IU)/ml]. Since we did not use dextran or polybrene to facilitate the infection process, high viral titers were used, ranging from 1 to 10 MOIs, in all experiments unless indicated otherwise. RTEc were exposed to HIV-1 for 24 to 48 hours. Cells were then washed three times to remove the input virus and refed with fresh culture media. Cultures were followed for a minimum period of three weeks. Two additional washes and culture media changes were done during this period. At the end of each experiment, all cells were washed and harvested for HIV-1 studies (see infection criteria below), or co-cultured with HIV-1 negative PBMCs to determine whether HIV-1 could be rescued. Supernatants from control and HIV-exposed RTEc were used to infect normal PBMCs. To eliminate the possibility that adherent viruses were being detected, non-permissive epithelial cells (MDCK and mouse renal tubular epithelial cells) were exposed to a similar number of infectious units in both cell free and cell-associated infection experiments. In addition, the HIV-1 IIIB isolate, which does not infect human epithelial cells, was used as another control to investigate whether adherent viruses could be rescued from the cell surface of human RTEc by PBMCs. The following criteria were used to define RTEc infection: (1) at least two consecutive samples with absorbance values > 2.0 sd above the p24 antigen control values, and one cutoff p24 antigen value of > 12.5 pg/ml after removal of input virus; (2) two consecutive measurements detecting an increase in p24 antigen values in the culture media after each wash; (3) positive HIV proviral DNA by PCR in RTEc cultured for more than two weeks. Positive cultures were defined by a cutoff value of > 800 ECL units/1 × 106 cells (> 2.0 sd above control values). (4) Positive HIV RNA by nucleic acid sequence based amplification (NASBA) in mRNA extracted from RTEc cultured for more than two weeks. Positive cultures were defined by a cutoff value of > 700 copies/1 × 106 (>2.0 sd above control values). (5) Infection of HIV-1 negative PBMCs co-cultivated with RTEc previously exposed to HIV-1 and grown in culture for more than twenty days. All five criteria were required in both cell-free infection and cell-associated infection experiments, and in all experiments using RTEc isolated from HIV-infected or HIV negative children. To confirm the presence of positive cytoplasmic HIV-1 staining in RTEc infected in cell-associated experiments, we performed immunohistochemistry and in situ hybridization studies. Co-localization experiments were done using HIV-1 probes and immunohistochemical markers for mononuclear and epithelial cells by combining in situ hybridization and immunohistochemical techniques. Finally, we performed transmission electron microscopy (TEM) studies to confirm the presence of viral particles inside RTEc. For blocking experiments, human renal proximal tubular epithelial cells (RPTEc) were seeded at a density of 1 × 106 cells per well. After 24 hours RPTEc were preincubated with anti-CD4 monoclonal antibodies, OKT4 from Ortho Diagnostics Systems (anti-V3/V4 domains; 10 μg/ml), or Leu 3a (Becton Dickinson, Mountain View, CA, USA; 10 μg/ml) for one hour. HIV-1 infected PBMCs were preincubated with these antibodies for one hour. RPTEc and PBMCs were co-cultured in medium containing 10 μg/ml of OKT4 or anti-Leu 3a antibodies for 12 to 24 hours. Infection was assessed as previously described. Controls included human RPTEc exposed to infected PBMCs in the absence of anti-CD4 monoclonal antibodies. The presence of CD4 protein in human RTEc was examined in cells fixed with 2% paraformaldehyde by fluorescein-activated cell scanning analysis, following standard procedures and using anti-Leu 3a monoclonal antibody (Becton Dickinson) and an isotype-matched irrelevant mouse monoclonal antibody as a control for non-specific staining. To determine whether macrophages were able to infect RPTEc in the absence of cell fusion, RPTEc were seeded on permeable Nucleopore filters (3 μm) which were placed in Transwell cell culture chambers (Costar, Cambridge, MA, USA), above the macrophages. Macrophages, isolated from primary PBMCs by their ability to attach to the culture dishes, were infected with cell-free HIV-1 (DC-4). RPTEc seeded on the upper surface of five filters were placed in the Transwell cell culture chambers above the infected macrophages. Controls included human RPTEc derived from the same clone, seeded on two filters and exposed to uninfected macrophages, as well as non permissive MDCK cells seeded on three filters and exposed to HIV-1 infected macrophages. After 48 hours of exposure, the filters with the attached RPTEc were transferred to new culture dishes in the absence of macrophages, the culture media was changed, and the cells were followed for 10 to 14 days. P24 antigen values were measured by an antigen capture enzyme immunoassay from Abbott Laboratory (North Chicago, IL, USA), following the manufacturer's directions. The upper detection limit of the assay was 200 pg/ml. Electrochemiluminescent detection of the HIV-1 specific amplification products were measured as described by Schutzbank and Smith39.Schutzbank T.E. Smith J. Detection of Human immunodeficiency virus type 1 proviral DNA by PCR using and electrochemiluminescence-tagged probe.J Clin Microbiol. 1995; 33: 2036-2041PubMed Google Scholar. Briefly, cells were removed and suspended in 250 μl of lysis buffer (50 mm KCl, 10 mm Tris-HCl, pH 8.3, 2.5 mm MgCl, 0.45% Nonidet P-40, 0.45% Tween 20, and 10 μg/ml proteinase K). The lysates were incubated at 60°C for one hour, followed by 100°C for 10 minutes to inactivate the proteinase K. A 50 μl sample of each specimen was transferred to a thin walled, 200 μl MicroAmp tube from Perkin Elmer (Norwalk, CT, USA) containing 50 μl of amplification master mix (50 mm KCl, 10 mm Tris-HCl, pH 8.3, 2.5 mm MgCl2, 500 μm each dNTP, 500 μm each SK38 and SK39-biotin primers and 2.5 units of AmpliTaq DNA polymerase (Perkin Elmer). The HIV-1 gag gene-specific PCR primers, SK38/5′ biotinylated-SK 39, and the detection probe SK 19, were synthesized by Genosys Biotechnologies Inc. (The Woodlands, TX, USA). Each PCR study was done in the presence of 3 negative controls (HIV-negative PBMCs) and two positive controls (10 and 50 copies of HIV-1 proviral DNA). In addition, control and infected human RTEc, as well as nonpermissive epithelial cells (MDCK or mouse RTEc) were run simultaneously in duplicates. RNA was extracted from control and infected renal epithelial cells as described previously11.Ray P.E. Bruggeman L.A. Weeks B.S. Kopp J.B. Bryant J.L. Owens J.W. Notkins A.L. Klotman P.E. Role of bFGF and its low affinity receptors in the pathogenesis of HIV-associated nephropathy in transgenic mice.Kidney Int. 1994; 46: 759-772Abstract Full Text PDF PubMed Scopus (88) Google Scholar. Quantification of viral genomic RNA was performed using a commercially available HIV-1 Q NASBA kit, from Organon Technica, (Research Triangle Park, NC, USA) following the manufacturer's directions. For indirect immunofluoresence staining, cells were fixed in methanol for five minutes, washed three times in PBS and incubated for one hour at room temperature with HIV-positive human serum diluted 1:100 in PBS containing 3% BSA. After three washes in PBS, the coverslips were incubated for one hour at room temperature in rabbit anti-human fluorescein isothiocyanate (FITC)/green (Sigma) diluted in 1:80 in PBS with 3% BSA. Background and nonspecific staining were determined by using mock-infected cells. For HIV-1 p24 antigen staining we used a Kal-1 monoclonal antibody to HIV-1 p24 antigen from Dako (Glostrup, Denmark) and a peroxidase-avidin-biotin complex (ABC) method from Dako11.Ray P.E. Bruggeman L.A. Weeks B.S. Kopp J.B. Bryant J.L. Owens J.W. Notkins A.L. Klotman P.E. Role of bFGF and its low affinity receptors in the pathogenesis of HIV-associated nephropathy in transgenic mice.Kidney Int. 1994; 46: 759-772Abstract Full Text PDF PubMed Scopus (88) Google Scholar. All sections were counterstained with hematoxylin. A panel of different antibodies was used to identify the cell type infected by HIV-1; mouse monoclonal EBM-11 (1:4,000 dilution) and
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