HIV-persistent infection and cytokine induction in mesangial cells: a potential mechanism for HIV-associated glomerulosclerosis

Abstract

HIV-associated nephropathy (HIVAN), a disease affecting up to 10% of HIV-seropositive individuals, is a clinical-pathological entity characterized by heavy proteinuria, enlargement of the kidney and rapid progression to renal failure [1,2]. The incidence of HIVAN has increased by 30% each year since 1991 and is becoming a prominent cause of end-stage renal disease [1]. Clinical and experimental findings suggest a direct role of HIV-1 in renal pathogenesis, but this hypothesis has not yet been proved [1,3]. We recently reported that tubular epithelial cells are susceptible to HIV-1 and undergo death by apoptosis after infection [4], a potential mechanism of HIV-associated severe tubulopathy [2]. Virus susceptibility of glomerular cells remains controversial [5,6]. Because glomerulopathy (mainly characterized by focal segmental glomerulosclerosis) plays a prominent role in the clinical manifestations and progression of HIVAN [1,2], we analysed the effects of HIV-1 interaction with glomerular cells. Primary and immortalized cultures of glomerular mesangial cells (MC) and glomerular epithelial cells (GEC or podocytes) were established and infected with T-tropic HIV-1 strains, as described [4,7]. MC primary cultures resulted in sustained HIV replication; in contrast, GEC were not susceptible to virus infection (Fig. 1a, upper panel). The distinct susceptibility of the glomerular cells to HIV-1 could be related to the different expression of virus receptors. By flow-cytometric analysis we found that MC bear on the membrane CD4 and chemokine coreceptors, whereas podocytes did not express HIV receptors (Fig. 1b). The expression of CXCR-4, the major coreceptor of T-tropic strains, can explain the susceptibility of MC to the HIV variants used in our study. However, the detection of surface CCR5 and CCR3 indicates that MC might also be susceptible to M-tropic strains. The role of these viral phenotypes in HIVAN pathogenesis has recently been enlightened by finding similar glomerulopathy in rhesus macaques infected with an M-tropic strain of SIV [8].Fig. 1.: (a) Upper panel: Production of infectious HIV-1 progeny by primary cultures of glomerular cells. Glomerular mesangial cells (MC) and glomerular epithelial cells (GEC) were infected with T-tropic HIV strains (m.o.i. 0.1 SFU/cell) or mock-infected with control supernatants, as described [4]. The IIIB strain was obtained from chronically infected H9 cells, whereas the P1 strain was derived from a syncytium-inducing isolate adapted to T lymphoblastoid C8166 cells [4]. The kinetics of virus release in the culture supernatant was measured as syncytium-forming units (SFU) in the C1866 T cell line. Consistent results were obtained with primary MC and GEC derived from five different donors. Lower side: Establishment of HIV-1-persistent infection in an immortalized line of MC: time course of virus production (filled boxes) and cell viability (filled circles) of MC cultures infected with HIV-1P1. (b) Surface expression of HIV receptors in mesangial cells. Upper side: Cytofluorimetric profile of immortalized MC stained with FITC-conjugated anti-CD4 cell monoclonal antibodies (mAb) (1 μg/106 cells: shaded histogram). Negative control was obtained by analysing MC challenged with FITC-conjugated isotype-matched irrelevant mAb (empty histogram). Lower side: Surface expression of CXCR-4 and CCR5 evaluated by incubating MC with specific mAb. Cytofluorimetric analysis of immortalized MC stained with isotype-matched irrelevant mAb and FITC-labelled goat anti-mouse IgG was used as a control. (c) Reverse transcriptase–polymerase chain reaction analysis of cytokine expression in uninfected (−) and HIV-infected (+) MC. RNA samples (1 μg) were taken from MC cultures infected for 14 days and from uninfected control. The figure shows amplicons obtained with 20 polymerase chain reaction cycles in order to compare the amounts of specific transcripts under conditions of linear amplification. PDGF, Platelet-derived growth factor; TGF-β, transforming growth factor beta.Because the production of viral progeny lasted for several days after acute infection of the MC, we investigated the possibility that HIV-1 causes persistent infection in these cells by using an established line of differentiated MC. As shown in Fig. 1a (lower panel), continuous production of HIV-1P1 by the infected MC cultures was detected over the period of observation. Both proviral DNA and genomic viral RNA were found in persistently infected MC; immunofluorescence assays demonstrated the presence of HIV-1 p24 in 10–15% of the cultured MC (not shown). This suggests that in our model HIV persistence is maintained by a mechanism of ‘carrier culture’ in which only a small fraction of MC exposed to HIV-1 are infected at any given time, possibly related to the limited number of MC expressing HIV receptors. A variety of glomerular disorders, included HIVAN, are characterized by mesangial hypercellularity and mesangial matrix increase. Cytokines and growth factors play a relevant role in the development of these pathological features [9]. Because HIV-1 replication did not cause overt cytopathic effects in MC, we evaluated its influence on the cellular synthesis of cytokines. In HIV-infected MC cultures we revealed a stimulation of cytokine production as from day 3 post-infection by measuring the cytokines released in the culture medium (not shown). MC challenged with heat-inactivated HIV-1 or mock-infected with control supernatants did not present an alteration of cytokine synthesis. Reverse transcriptase–polymerase chain reaction analysis performed under conditions of linear amplification demonstrated an overexpression of cytokine-specific messenger RNA in HIV-infected MC (Fig. 1c). This evidence suggests that the increased production of these mediators can be related to HIV-induced upregulation of cytokine gene transcription. In particular, we found that HIV-1 persistent infection stimulated MC to express platelet-derived growth factor and transforming growth factor beta (TGF-β) (Fig. 1c), crucial mediators of the pathological mechanisms of glomerulopathy [10]. Platelet-derived growth factor induces MC proliferation, whereas TGF-β stimulates the synthesis and inhibits the degradation of mesangial matrix proteins [9]. Consistent with our findings, increased expression of TGF-β was detected in renal biopsies from HIV-1 patients [11]. In our study, we also found that HIV-1 infection upregulated MC synthesis of IL-6, TNF-α and IL-8 (Fig. 1c). The role of these pro-inflammatory cytokines in glomerulonephritis is known, especially for the recruitment of inflammatory cells and the potentiation of neutrophil- and monocyte-mediated glomerular injury [9]. In summary, our results demonstrate that podocytes are not susceptible to HIV-1, but the virus can establish persistent infection of MC stimulating the production of cytokines known to be involved in the development of glomerulosclerosis in vivo. Preliminary observations indicate that highly active anti-retroviral therapy can reduce the manifestations of nephropathy in AIDS patients [12]. Insights gained from our model in vitro will probably thus improve not only the understanding of HIVAN pathogenesis, but also the design of effective therapeutic strategies. Pier Giulio Conaldia Antonella Bottellia Alison Wade-Evansb Luigi Bianconec Andreina Baja Vincenzo Cantaluppic Caterina Serrad Antonina Doleid Antonio Tonioloa Giovanni Camussic