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*
Department of Pathology, University of Washington, Seattle,
Washington
Medizinische Poliklinik, Klinikum Innenstadt der LMU, Munich,
Germany
Division of Pediatric Infectious Disease, Oregon Health Sciences
University, Portland, Oregon
§
Department of Medicine and Pathology, George Washington University Medical
Center, Washington, DC
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Department of Pathology, New York University, New York, New
York
¶
National Institutes of Health, Bethesda, Maryland.
Correspondence to Dr. Charles E. Alpers, University of Washington, Department of Pathology, Box 356100, 1959 NE Pacific Street, Seattle, WA 98195. Phone: 206-548-6409; Fax: 206-548-4928; E-mail: calp{at}u.washington.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The pathogenesis of HIV-associated renal disease is poorly understood (2,3). Proposed mechanisms include: (1) direct HIV infection of renal parenchymal cells (e.g., visceral epithelial cells, tubular epithelial cells, and renovascular endothelial cells); (2) indirect injury to the kidney by renal cellular uptake of circulating virally encoded molecules; or (3) indirect injury to the kidney through release of cytokines by infected mononuclear cells in the circulation or infiltrating the kidney (2,3). Furthermore, different mechanisms might be involved in the pathogenesis of the different variants of HIV-associated renal disease. Circulating immune complex deposition and in situ mechanisms of immune-mediated renal disease may underlie the pathogenesis of glomerulonephritis in HIV-infected patients (7). In addition, there may be a role for hepatitis C coinfection in some cases of HIV-associated immune complex glomerulonephritis (8). Some renal disease in HIV-infected individuals, however, may represent chance occurrence unrelated to HIV infection itself.
New directions in AIDS research of viral entry, tropism, and pathogenesis were initiated by the recent discovery that members of the chemokine receptor family act as necessary coreceptors together with CD4 for entry of HIV-1 into susceptible cells (15). HIV-1 strains previously characterized as T lymphocyte-tropic have been shown to bind to the chemokine receptor CXCR4 as a condition of entry into mammalian cells, while macrophage-tropic strains of HIV-1 require the chemokine receptor CCR5. Expression of these molecules appears to play a key role in determining which tissues are permissive for direct HIV infection.
Expression of the HIV coreceptors CCR5 and CXCR4 in human kidneys with features of HIV-associated renal disease is completely unknown. In previous studies from our laboratories, expression of both chemokine receptors CCR5 and CXCR4 was not identified by either immunohistochemical (16) or in situ hybridization (17,18) techniques in intrinsic renal parenchymal cells in normal human kidneys, in renal allograft nephrectomies, and in human renal biopsies with a variety of different glomerular and interstitial diseases. However, in vitro studies show that the expression of certain chemokine receptors in mononuclear cells is regulated by a number of stimuli (reviewed in references (19) and (20). An upregulation of CCR5 or CXCR4 expression in intrinsic renal cells might therefore require specific cytokine stimulation present in the course of HIV infection.
In this study, we tested the hypothesis that the relevant chemokine coreceptors for HIV infection not constitutively expressed in normal kidney may be upregulated in the setting of HIV infection and thereby permit infection of renal tissue. We assessed the cellular sites of expression of chemokine receptor CCR5 by immunohistochemistry and in situ hybridization, and CXCR4 by in situ hybridization, in renal biopsies obtained from HIV-infected patients with features of HIVAN, HIV-associated immune complex glomerulonephritis, and HIV-associated thrombotic microangiopathy. Additional tissues examined within this study consisted of human renal biopsy material with features of a collapsing glomerulopathy from patients without clinical evidence of HIV infection. Furthermore, we provide corresponding data for the presence of HIV-1 protein by immunohistochemistry and for the presence of HIV-1 RNA by in situ hybridization in each of these cases.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Formalin-fixed, paraffin-embedded HIV-1-infected human peripheral blood mononuclear cell (PBMC) pellets were generated as controls for immunohistochemical detection of HIV-1 p24 antigen and for in situ hybridization of HIV-1 RNA. Human PBMC were obtained from HIV antibody-negative volunteer donors as described previously (25). Briefly, the PBMC layer was removed after centrifugation of whole blood, washed, and resuspended in RPMI 1640 containing 16% fetal calf serum, penicillin (100 U/ml), streptomycin (100 µg/ml), and glutamine (0.3 mg/ml). The mononuclear cells were stimulated with phytohemagglutinin (5 µg/ml) and 5% nonrecombinant human interleukin-2 (Pharmacia, Piscataway, NJ) as described (25). Forty-eight hours after stimulation, the human PBMC were infected with a cell-free solution of HIV-1 strain LAI. A full-length clone of HIV-1LAI [pBRU3] was obtained from Dr. Michael Emerman, Fred Hutchinson Cancer Research Center, Seattle, WA (26). Cell-free stock solutions of HIV-1LAI were generated by transfection of this plasmid into 293T cells). Cells were harvested 48 h after infection and counted in a hemocytometer. Several different preparations were generated by mixing defined concentrations of HIV-1-infected PBMC with defined concentrations of uninfected PBMC. The different HIV-1-infected/uninfected PBMC preparations contained HIV-1-infected PBMC in concentrations ranging from 5 to 50%. These mixed PBMC samples were subsequently centrifuged, and the remaining cell pellet was fixed in 10% phosphate-buffered formalin and embedded in paraffin using standard protocols for tissue preparation. Four-micrometer-thick sections were generated as controls for immunohistochemical and in situ hybridization procedures.
Antibodies
CCR5. A murine monoclonal antibody MC5 directed against human
chemokine receptor CCR5 has previously been characterized for specificity by
Western blotting and fluorescence-activated cell sorter analysis
(16), and was found suitable
for the specific detection of CCR5 in formalin-fixed, paraffin-embedded tissue
sections after heat-mediated antigen retrieval procedures
(16).
HIV-1. A murine monoclonal antibody p24, clone Kal-1, directed against an epitope of the core protein p24 of HIV-1 was purchased from DAKO (Carpinteria, CA). Specificity of this antibody for the detection of HIV-1 p24 has been demonstrated previously by immunoprecipitation, Western blotting, and immunohistochemistry (27), and it has been demonstrated previously to recognize HIV-1 p24 in formalin-fixed, paraffin-embedded tissue sections (27,28).
Immunohistochemistry
Immunohistochemistry was performed on formalin-fixed, paraffin-embedded
tissue sections according to protocols that we have used previously
(29). Four-micrometer sections
of tissue samples were deparaffinized in xylene and rehydrated in graded
ethanols. Endogenous peroxidase was blocked by incubation in 3% hydrogen
peroxide. Sections that were subsequently incubated with the anti-CCR5
antibody were pretreated by steam heating for 20 min in Antigen Unmasking
Solution (Vector Laboratories, Burlingame, CA), according to the instructions
of the manufacturer. Nonspecific binding was blocked by incubation in 10%
normal horse serum (Vector). The sections were then incubated for 1 h at room
temperature with the primary antibody diluted in phosphate-buffered saline
plus 1% bovine serum albumin (Sigma, St. Louis, MO). After washes in
phosphate-buffered saline, the sections were incubated with biotinylated horse
anti-mouse antibody (Vector). A Tyramide Signal Amplification
(TSATM-Indirect, NENTM Life Science Products, Boston,
MA) was performed according to the manufacturer's instructions. Finally,
3,3' diaminobenzidine (with nickel chloride enhancement) was used as a
chromogen. Sections were counterstained with methyl green, dehydrated, and
coverslipped. The complete biopsies were examined, and the number of CCR5
protein-expressing cells per glomerular cross-section was calculated in each
case. Negative controls for the immunohistochemical procedures consisted of
substitution of the primary antibody with isotype-matched, irrelevant murine
monoclonal antibodies (DAKO).
Molecular Probes
CCR5. A 1.1-kb sequence of DNA coding for human CCR5 was subcloned
into pcDNAI/amp (Invitrogen, San Diego, CA) (obtained through the AIDS
Research and Reference Reagent Program, Division of AIDS, NIAID, and NIH,
originally provided by Dr. Nathaniel Landau) and then linearized with
HindIII and transcribed with Sp6 for the antisense probe or
linearized with SphI and transcribed with T7 for the sense probe.
Sensitivity and specificity of the CCR5 antisense riboprobe has been
demonstrated previously by Northern analysis and by in situ
hybridization (17).
CXCR4. A 1.1-kb sequence of DNA coding for human CXCR4 was subcloned into pcDNAI/amp (Invitrogen) (obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, and NIH, originally provided by Dr. Nathaniel Landau) and then linearized with HindIII and transcribed with Sp6 for the antisense probe or linearized with XbaI and transcribed with T7 for the sense probe. Sensitivity and specificity of the CXCR4 antisense riboprobe have been demonstrated previously by Northern analysis and by in situ hybridization (18).
HIV-1. Four DNA templates that collectively represent 90% of the HIV-1 genome were purchased from Lofstrand Labs (Gaithersburg, MD): gag, SacI-BglII, 1.4 kb; gag/pol, BglII-EcoRI, 2.6 kb; pol/vif/vpr/rev/tat/vpu, KpnI-KpnI, 2.2 kb; env/vpr/rev/tat/vpu, EcoRI-BamHI, 2.7 kb. Antisense and sense riboprobes were generated from all templates using Sp6 or T7 according to the manufacturer's instructions. Radioactivity was introduced by synthesis of RNA using 35S-UTP. The generated probes were combined to yield one sense or antisense probe cocktail. Specific detection of HIV-1 RNA in formalin-fixed, paraffin-embedded tissue sections by in situ hybridization with an estimated sensitivity of 30 to 300 copies of target RNA has been described previously by numerous groups using identical RNA probes (30,31,32,33,34,35).
In Situ Hybridization
HIV-1 RNA, CCR5 mRNA, and CXCR4 mRNA were detected in tissue sections using
in situ hybridization techniques according to protocols used
previously (29). Riboprobes
for in situ hybridization were generated from cDNA using
35S-UTP. Four-micrometer sections of formalin-fixed,
paraffin-embedded tissue samples were deparaffinized and rehydrated through
xylene and graded ethanols, washed with 0.5 x SSC (Life Technologies,
Grand Island, NY), and digested with proteinase K (5 µg/ml; Sigma).
Sections that were subsequently hybridized with HIV-1-specific riboprobes were
analyzed in duplicate.
In one section, the in situ hybridization was performed according to the following protocol. The second section was additionally pretreated by steam heating for 20 min in Antigen Unmasking Solution (Vector) before the hybridization, according to the manufacturer's instructions. Prehybridization was performed for 2 h by adding 100 µl of prehybridization buffer (0.3 M NaCl, 20 mM Tris, pH 8.0, 5 mM ethyl-enediaminetetra-acetic acid, 1x Denhardt's solution, 10% dextran sulfate, and 10 mM dithiothreitol). The hybridizations were started by adding 500,000 cpm of 35S-labeled riboprobe in 50 µl of prehybridization buffer and allowed to proceed overnight at 50°C. After hybridization, sections were treated with RNase A (20 µg/ml; Sigma), followed by three high-stringency washes in 0.1x SSC/0.5% Tween 20 (Sigma) for 40 min each at 50°C, and several 2x SSC washes. After the tissue was dehydrated and air-dried, it was dipped in NTB2 nuclear emulsion (Kodak, Rochester, NY) and exposed in the dark at 4°C for 2 wk (HIV-1) or 6 wk (CCR5, CXCR4), respectively. After developing, the sections were counterstained with hematoxylin and eosin, dehydrated, and coverslipped. Positive cellular labeling was defined as five or more silver grains concentrated over a single cell on the slides hybridized with the antisense probe, and little or no signal present on the sense control slides. The complete biopsies were examined, and the numbers of CCR5 mRNA and CXCR4 mRNA expressing cells per glomerular cross-section were calculated in each case. Positive controls for the detection of CCR5 and CXCR4 mRNA consisted of several allograft nephrectomy specimens with features of severe rejection, as published previously (17,18).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All cases of the different disease groups analyzed in this study showed variable degrees of tubulointerstitial inflammation ranging from very mild, focal, perivascular infiltrates of leukocytes to severe, diffuse interstitial mononuclear cell infiltrates. CCR5-expressing cells were demonstrable by both immunohistochemistry (Figure 1, E and F) and in situ hybridization (Figure 1, G and H) in these infiltrates in the tubulointerstitial compartment of all analyzed cases. The expression of CCR5 was restricted to infiltrating mononuclear cells (Figure 1, E through H). The number of CCR5-expressing cells correlated with the degree of tubulointerstitial mononuclear cell infiltration. No difference in the CCR5 expression pattern on infiltrating leukocytes was seen between patients with HIV-associated renal disease or HIV-negative disease controls (data not shown). Tubular epithelial cells, whether located in areas with absence of interstitial inflammation, at sites of severe tubulointerstitial mononuclear cell infiltration, or at sites of microcystic tubular dilation, showed no detectable CCR5 expression (Figure 1, E through H). CCR5 expression was completely absent in endothelial cells and smooth muscle cells of the vascular compartment.
The number of leukocytes exhibiting expression of the CCR5 protein as detected by immunohistochemistry correlated with the number of leukocytes synthesizing detectable CCR5 mRNA in all individual cases (Figure 1, compare Panels F and H). However, in the glomerular compartment slightly more CCR5-positive leukocytes were identified by immunohistochemistry compared with in situ hybridization (Table 1).
CXCR4 Expression in HIV-Associated Renal Disease
By in situ hybridization, CXCR4 mRNA expression remained
undetectable in cells clearly identifiable as renal parenchymal cells of the
glomerular, tubular, and vascular compartments in all analyzed cases. At sites
of collapsing glomeruli, CXCR4 mRNA expression was typically absent
(Figure 2, A and B). However,
within all analyzed cases small numbers of individual CXCR4 mRNA-expressing
cells were identified within glomeruli
(Figure 2, C and D)
(Table 1). We were unable to
clearly determine whether these CXCR4 mRNA-expressing cells were intrinsic
glomerular cells or circulating leukocytes, although we favor the latter
interpretation. All of the biopsy tissue was consumed during the course of the
present studies, and so material was unavailable for double-labeling studies
that might confirm this interpretation.
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Within the tubulointerstitial compartment, CXCR4 mRNA expression was restricted to infiltrating mononuclear leukocytes at sites of tubulointerstitial inflammation (Figure 2, E and F). There was no difference in the number of detectable CXCR4 mRNA-expressing leukocytes between HIV-1-positive or -negative renal tissues. CXCR4 mRNA expression was undetectable in tubular epithelial cells, including those at sites of tubulointerstitial inflammation and at sites of microcystic tubular dilation (Figure 2, E and F). Renovascular endothelial cells did not show detectable CXCR4 mRNA expression.
Comparison of CCR5 and CXCR4 expression in serial sections of the analyzed specimens revealed that both chemokine receptors were expressed in a large percentage of interstitial infiltrating mononuclear leukocytes. The number of CCR5- or CXCR4-expressing cells, respectively, correlated positively with the degree of tubulointerstitial inflammation. No obvious differences in the distribution pattern of CCR5- or CXCR4-expressing infiltrating leukocytes were evident. Because of the technical limitation of serial sections, we could not properly address the question of whether CCR5 and CXCR4 were coexpressed in the same subset of infiltrating mononuclear leukocytes, or whether CCR5 and CXCR4 were expressed in different leukocyte subsets. As indicated above, exhaustion of the biopsy samples precluded double-labeling studies that would address this issue.
Specificity of the HIV-1 p24 Immunohistochemistry and of the HIV-1
RNA in Situ Hybridization
Immunohistochemical detection of HIV-1 proteins in formalin-fixed,
paraffin-embedded renal tissue sections has been very unreliable and
frequently resulted in nonspecific immunostaining in tissue obtained from
HIV-negative controls
(36,37).
We therefore performed a series of experiments to control the specificity of
our procedures. As positive controls, formalin-fixed, paraffin-embedded PBMC
pellets, containing HIV-1-infected PBMC, reproducibly demonstrated positive
immunostaining (Figure 3, A through
C). Furthermore, the number of cells with positive
immunohistochemical signal within the pellet closely reflected the number of
HIV-1-infected PBMC mixed in the pellet preparation
(Figure 3, A through D). No
immunostaining was detectable in HIV-1-infected PBMC when the primary antibody
was substituted by an irrelevant, isotype-matched mouse IgG preparation (data
not shown). PBMC pellets that contained no HIV-1-infected cells showed no
detectable immunostaining with the HIV-1 p24 antibody
(Figure 3D). Renal biopsy
sections from patients with collapsing glomerulopathy and negative HIV
serology (n = 8) consistently showed absent immunohistochemical
staining when incubated with the HIV-1 p24 antibody (data not shown).
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PBMC pellets containing HIV-1-infected PBMC consistently showed detectable in situ hybridization signal for HIV-1 RNA (Figure 3E). The number of PBMC with positive hybridization signal closely reflected the number of HIV-1-infected PBMC mixed in the pellet preparation. No difference in the number of HIV-1 RNA-positive PBMC was seen in the in situ hybridization procedures with and without heat-mediated pretreatment of the PBMC pellet specimens before hybridization (data not shown). No hybridization was seen when identical procedures were performed with substitution of a control sense probe for hybridization (Figure 3F). All renal biopsies obtained from HIV-negative control patients showed absent hybridization signal for HIV-1 RNA (data not shown).
Detection of HIV-1 in Biopsies from Patients with HIV-Associated
Renal Disease
By immunohistochemistry, HIV-1 p24 protein was not detected in glomerular
(Figure 4B), tubulointerstitial
(Figure 4E), or vascular
compartments of all cases of HIVAN (n = 13), HIV-associated
glomerulonephritis (n = 3), and HIV-associated thrombotic
microangiopathy (n = 1).
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In situ hybridization detected no HIV-1 RNA in glomerular (Figure 4, C and D), tubular (Figure 4F), or vascular cells in all analyzed cases. However, in one case of HIVAN, HIV-1 RNA was detectable within one small lymphoid follicle located in the tubulointerstitium (Figure 4, G and H). HIV-1 RNA was clearly restricted to infiltrating mononuclear leukocytes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Major insights into the mechanisms of HIV infection have been obtained from the discovery that members of the chemokine receptor family act as necessary coreceptors, together with CD4, for entry of AIDS viruses into mammalian cells (reviewed in reference (15). Chemokine receptors represent a family of structurally and functionally related seven transmembrane-spanning, G protein-coupled receptors (19, 20, 38). Recent observations indicate that certain chemokine receptors may also facilitate infection by immunodeficiency viruses in a CD4-independent manner (39, 40). By facilitating entry into cells, these receptors determine viral tropism. CXCR4 is a coreceptor for strains of HIV-1 that infect T lymphocyte cell lines (T-tropic strains), and CCR5 serves as a coreceptor for HIV-1 isolates that infect macrophages and activated T lymphocytes (M-tropic strains) (15). Additional in vivo observations support the central role of CCR5 and CXCR4 in the pathogenesis of HIV infection in humans. Individuals homozygous for a mutant allele of the CCR5 gene bearing a 32-nucleotide deletion (CCR5 delta 32), which leads to an inactive variant of CCR5, have been linked to protection from HIV-1 infection (41, 42). The consequence of the heterozygous state is not clear, but it may delay the progression to AIDS in infected individuals (41,42,43). Additionally, individuals homozygous for a mutation of the only identified CXCR4 ligand, stromal cell-derived factor-1, appear to have some degree of protection against disease progression after HIV infection (44).
The expression of chemokine receptors is not limited to leukocytes and lymphoid tissues. Tissue-specific expression of the HIV coreceptors CCR5 and CXCR4 most likely play a role in determining whether tissues are permissive for direct HIV infection in vivo. CCR5 mRNA was detectable by Northern blotting in RNA isolated from small intestine, ovary, and lung (45), and CCR5 protein was detectable by immunohistochemistry in the central nervous system (on neurons, astrocytes, and microglia) and on endothelium, vascular smooth muscle cells, and fibroblasts in several analyzed parenchymal tissues (46). Two recent studies by members of our group investigated the expression of CCR5 in normal kidneys, kidneys with various glomerular diseases, kidneys with interstitial diseases, and rejected allograft nephrectomies (16, 17). By either in situ hybridization (17) or immunohistochemistry (16), CCR5 was undetectable in intrinsic renal cell types of the glomerular, tubular, or vascular compartments. The expression of CCR5 was restricted to infiltrating mononuclear leukocytes at sites of tubulointerstitial and vascular injury. The total number of CCR5-expressing cells correlated with the number of CD3-positive T lymphocytes at sites of tubulointerstitial inflammation (16).
T lymphocytes, monocytes, and neutrophils express the chemokine receptor CXCR4 (47), however, CXCR4 is by far the most widely expressed of the functional chemokine receptors in nonhematopoietic cells (38). High transcript levels have been demonstrated in several tissues, including heart, brain, liver, and colon (48). We have recently reported that CXCR4 mRNA expression is absent in intrinsic glomerular, tubular, and renovascular cells in native normal kidneys and in rejected allograft nephrectomies (18). In the presence of renal interstitial inflammation, CXCR4 expression was localized to a large fraction of infiltrating leukocytes (18).
The present study does not provide evidence for an upregulated renal parenchymal expression of the HIV coreceptors CCR5 or CXCR4 in kidneys with features of HIV-associated renal disease. Expression of the two analyzed chemokine receptors was clearly limited to infiltrating leukocytes at sites of tubulointerstitial inflammation. Individual CCR5- and CXCR4-expressing cells were identified within glomeruli. Those cells likely represented circulating leukocytes. Because of the complete exhaustion of the biopsy material, a combination of in situ hybridization for chemokine receptor mRNA with immunohistochemical labeling of cellular phenotypes as described previously (18) was impossible. However, a previous detailed immunohistochemical analysis of the immune cell populations present in HIV-associated kidney disease (14) quantified the numbers of macrophages and T lymphocytes in glomeruli obtained from patients with HIVAN and with HIV-associated immune complex glomerulonephritis. In that study (14), the total number of leukocytes per glomerular cross-section was 2.7 ± 1.1 (mean ± SEM) in HIVAN and 5.5 ± 2.8 in HIV-associated immune complex glomerulonephritis. The numbers of chemokine receptor CCR5- and CXCR4-expressing cells in glomeruli, identified in the present study (as detailed in Table 1), correlate well with the previously published number of circulating leukocytes in HIV-associated renal disease.
Chemoattraction of CCR5- and CXCR4-expressing mononuclear leukocytes to sites of tubulointerstitial inflammation is not specific for HIV-associated renal disease. Similar numbers of CCR5- or CXCR4-expressing leukocytes were detected in HIV-negative collapsing glomerulopathy (present study) and in HIV-negative patients with a variety of different glomerular and interstitial diseases (16,17,18).
We have previously investigated whether the major HIV receptor CD4 is expressed by intrinsic renal parenchymal cells. Unpublished studies by our group of immunohistochemically detectable expression in human renal tissues and published studies of cultured human mesangial cells (49) have failed to demonstrate detectable expression of CD4 on any cells except infiltrating leukocytes in kidney tissues. A single report, published as a letter but without further corroboration in the literature, has asserted that CD4 can be expressed by human mesangial cells (50).
Previous investigations that were aimed to detect HIV-1 proteins or genomic material in human renal biopsies from HIV-infected individuals revealed conflicting data. Cohen et al. demonstrated the presence of HIV-1 protein in the cytoplasm of tubular epithelium and in a few glomerular visceral epithelial cells in human biopsies with HIVAN by immunohistochemistry and of HIV-1 genomic material within a small number of tubular epithelial cells and within both glomerular parietal epithelial and visceral epithelial cells by in situ hybridization (51). Kimmel et al. detected HIV-1 protein by immunofluorescence in acid-treated kidney sections obtained from patients with HIV-associated immune complex glomerulonephritis (7) and HIV-associated IgA nephropathy (9). Others, however, have not been able to replicate these results (52, 53). Furthermore, Nadasdy et al. and a recent study by Yamamoto et al. testing several different HIV-1 antibody preparations detected positive immunostaining not only in renal biopsies with features of HIVAN but also in uninfected control tissues, raising serious concerns regarding the specificity of immunohistochemical staining with some HIV-1 antibodies (36, 37). In a previous study, two of the authors of the present study were able to demonstrate HIV-1 DNA in microdissected glomeruli, tubules, interstitial cells, and inflammatory infiltrating cells from renal biopsies of HIV-infected patients using PCR amplification techniques (22). Identification of this genomic material did not correspond to manifestations of HIV-associated renal disease, as HIV genome was equally detectable in renal tissues of HIV-infected patients with and without clinical or pathologic evidence of renal disease (22). Although PCR amplification is likely to be the most sensitive technique for the detection of HIV genomic material, the difficult microdissection procedures entail a risk of contamination by circulating or infiltrating leukocytes, and therefore there remains some uncertainty about whether infection of renal parenchymal cells has been conclusively demonstrated in this study. Our study, using less sensitive techniques, cannot exclude the possibility of latent infection of tissues or the possibility of low-level replication with virus copy numbers below the threshold for detection by either immunohistochemistry or in situ hybridization.
A second part of this present study therefore readdressed the still conflicting question of whether productive HIV-1 infection (detection of HIV-1 protein or RNA) occurs in renal parenchymal cells in HIV-associated renal disease in vivo. We used highly sensitive immunohistochemistry and in situ hybridization techniques that identified productive HIV-1 infection in lymphoid and neuronal tissues (30,31,32,33,34,35, 54) and that have not reportedly been used for studies in kidney tissues. Immunohistochemical detection of HIV-1 was performed with a well-characterized, commercially available murine monoclonal antibody (DAKO, anti-p24, clone Kal-1) (27, 28) according to the recently published protocol of Strappe et al. (54). In situ hybridization utilized radiolabeled RNA probes generated from commercially available cDNA templates of the HIV-1 genome (30) according to the initial protocol of Fox et al. with some modifications to achieve a higher sensitivity. These modifications included pretreatment of the formalin-fixed, paraffin-embedded tissue sections with proteinase K and heat-mediated procedures as described (34).
Both immunohistochemistry and in situ hybridization demonstrated excellent specificity in PBMC control pellets and uninfected control tissues and were sensitive enough to easily detect productively infected leukocytes in the PBMC pellet preparations. However, both HIV-1 p24 antigen and HIV-1 RNA remained completely undetectable in renal parenchymal cells in all analyzed cases of HIV-associated renal disease. In one single renal biopsy with HIVAN, HIV-1 RNA was localized to mononuclear cells at a site of extensive tubulointerstitial inflammation.
Our data do not provide evidence for productive HIV-1 infection of renal parenchymal cells in the pathogenesis of HIV-associated renal disease. We recognize that our methods of HIV-1 detection, although successfully established in lymphoid organs, have some limits to their sensitivity. Both immunohistochemistry and in situ hybridization are likely suitable to identify productive infection, i.e., infection associated with viral replication and antigen expression. These techniques do not detect a true latent infection of cells, which might be detectable only by such sensitive techniques as PCR. The inability to detect HIV-1 RNA in more than one case with HIV-associated renal disease might further be explained by the limited tissue material that was available in some cases.
Renal parenchymal expression of the HIV cofactors CCR5 and CXCR4 is not upregulated in HIV-associated renal disease, whereas large numbers of infiltrating mononuclear leukocytes at sites of tubulointerstitial inflammation express CCR5 or CXCR4. Although productive HIV-1 infection of kidney parenchyma is undetectable, small numbers of HIV-1-infected mononuclear cells can be detected at sites of tubulointerstitial inflammation in kidneys with features of HIV-associated renal disease. Whether this finding is related to the pathogenesis of the renal injury and whether the presence of HIV in the kidney is capable of inducing cytopathic effects in susceptible individuals independent of direct renal infection, or whether it just represents nonspecific trapping of some HIV-infected leukocytes as "innocent bystanders" has not yet been ascertained (14). We believe that infected leukocytes circulating within the kidney, the altered cytokine milieu within the host occurring as a consequence of HIV infection and the development of AIDS, and certain host susceptibility factors such as racial background all contribute to the development of HIVAN. It has been difficult to directly test the importance of each of these variables in isolation, and hence the pathogenesis of HIV-associated renal disease remains far from being understood.
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Acknowledgments |
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Footnotes |
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F. Eitner, T. Ostendorf, C. Van Roeyen, M. Kitahara, X. Li, K. Aase, H.-J. Grone, U. Eriksson, and J. Floege Expression of a Novel PDGF Isoform, PDGF-C, in Normal and Diseased Rat Kidney J. Am. Soc. Nephrol., April 1, 2002; 13(4): 910 - 917. [Abstract] [Full Text] [PDF]
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