Background. In experimental extracapillary glomerulonephritis (EG) podocytes migrate, proliferate and change phenotype, and play a pivotal role in crescent formation. Hepatocyte Growth Factor (HGF) is an injury-induced effector of tissue repair that causes cell migration, growth and transdifferentiation via its receptor Met.Methods. In 11 patients with EG we measured serum levels of HGF and investigated whether serum induces the release of HGF by Peripheral Blood Mononuclear Cells (PBMC). In renal biopsies we studied the expression of Met. In cultured podocytes we studied Met expression, migration, growth and morphological changes induced by recombinant (r) HGF.Results. In patients with EG average serum levels of HGF (0.73 ng/ml) were higher than in normal volunteers (N, 0.10 ng/ml, p<0.01) and in patients with non-crescentic glomerular disease (GD, 0.18 ng/ml, p<0.01). Serum of EG induced a significant HGF release by PBMC (mean 0.58 ng/ml) in comparison with serum of N and GD (0.07 and 0.06 ng/ml, respectively, both p<0.001). Met was strongly expressed in crescents. Cultured podocytes expressed Met, and rHGF induced in podocytes a time- and dose-dependent migration, growth and epithelial to mesenchymal transdifferentiation.Conclusions. These results suggest that HGF/Met system participates in the process of crescent formation by inducing podocyte migration, growth and mesenchymal transformation.

Aims/hypothesis: Studies on the biology of the microvascular endothelial cells (MECs) that surround and penetrate the pancreatic islets are hampered by difficulties in isolating and culturing large numbers of pure cells. We aimed to morphologically and functionally characterise primary MECs purified and cultured from human islets, and to establish a simian virus 40 (SV40)-immortalised cell line from these primary cultures. Materials and methods: Human islet MECs were extracted and purified using anti-CD105 coated immunomagnetic beads, and endothelial markers and surface molecules analysed by flow cytometric analysis. An immortalised cell line was then established by using a chimeric adeno5/SV40 virus. Results: Islet MECs expressed classic and specific endothelial markers, a high basal level of intercellular adhesion molecule-1, and low levels of E-selectin and TNF (previously known as TNF-alpha) inducible vascular cell adhesion molecule-1. IFNG (previously known as IFN-gamma) induced expression of HLA class II molecules. The immortalised islet MECs expanded rapidly, exhibited increased DNA synthesis, and were passaged approximately 30 times, without signs of senescence. They retained the endothelial characteristics of the parental cells, and behaved as the primary cells in terms of TNF stimulation of expression of adhesion molecules and support of leucocyte adhesion and transmigration. Conclusions/interpretation: The immortalised islet MECs that we have established could effectively represent a substitute for primary counterparts for in vitro studies on the role of the microvasculature in pathophysiological processes involved in type 1 and type 2 diabetes.

Numerous studies indicate that enteroviruses, such as the Coxsackievirus (CV) group, are linked to autoimmune diseases. Virus tropism and tissue access are modulated by vascular endothelial cells (ECs), mainly at the level of the microvasculature. Data on the permissiveness of ECs to CV are, however, scanty and derived from studies on large vessel ECs. To examine the susceptibility of microvascular ECs to infection of group B CV (CVB), human dermal microvascular ECs (HMEC-1) were infected with three CVB strains, and the immunological phenotype of the infected cells was analyzed. All CVB persistently infected the EC cultures without producing overt cytopathic effects. Infected ECs retained endothelial characteristics. Release of infectious particles in cell supernatants persisted for up to 3 mo of culture. Infection up-regulated expression of the adhesion molecules ICAM-1 and VCAM-1, with the highest values detected during the first 30 days of infection (p < 0.05 vs uninfected HMEC-1). CVB infection increased production of the proinflammatory cytokines, IL-6, IL-8, and TNF-α, which may account for the enhanced expression of adhesion molecules. Parallel infection of macrovascular HUVEC had less evident effects on induction of ICAM-1 and did not significantly increase expression of VCAM-1. Moreover, mononuclear cell adhesion to CVB-infected HMEC-1 monolayers was increased, compared with uninfected monolayers. These results provide evidence that small vessel ECs can harbor a persistent viral infection, resulting in quantitative modification of adhesion molecule expression, which may contribute to the selective recruitment of subsets of leukocytes during inflammatory immune responses. Furthermore, our data confirm that the behavior against a viral challenge of ECs in large vessels and microvessels may differ.

Human immunodeficiency virus-associated nephropathy (HIVAN) is etiologically related to the viral infection, but the mechanisms of virus-induced renal injury remain undetermined. Peculiar histopathological features of HIVAN are the enhanced proliferation and the loss of differentiation markers of glomerular epithelial cells (podocytes). We found that podocytes were not permissive to HIV-1 replication. In this study we investigated the effects of the HIV-1 regulatory protein Tat on primary cultures and on a continuous line of podocytes. Our results demonstrated that Tat induced hyperproliferation of these cells in a dose-dependent manner. This activity was primarily mediated by the basic domain of the viral protein. Proteoglycans were required for this phenomenon because Tat-induced increase of podocyte growth was significantly impaired by inhibition of proteoglycan synthesis with beta-D-xyloside. In podocyte cultures Tat promoted both the transcription and the release of basic fibroblast growth factor, which contributed to the enhanced cell proliferation. Moreover, Tat deregulated the podocyte phenotype causing down-regulation of maturity markers such as WT-1 and synaptopodin, alteration of cytoarchitecture, and impairment of permselectivity. Together, these results demonstrate that the interaction of extracellular Tat with podocytes can induce alterations that mimic the pathological changes of podocytes detected in HIVAN.

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

HIV-associated nephropathy (HIVAN) is characterized by a combination of glomerular and tubulointerstitial lesions, but its pathogenesis is unclear. Here, we analyzed the effects of HIV-I on cultured renal glomerular cells. Mesangial cells resulted to express CD4 and several chemokine receptors. and were found to sustain persistent virus replication. HIV-l-infected mesangial cells did not show overt cytopathic effects, but an increased synthesis of cytokines known to be involved in glomerulosclerosis pathogenesis. Podocytes were negative for HIV-1 receptors and non-permissive to virus replication, but HIV-1 Tat induced hyperproliferation of podocytes and cytoskeletal reorganization. These results indicate that the interaction of HIV with glomerular cells produces distinct pathogenic effects on different target cells and that these effects may play pathogenetic roles in the development of HIVAN.

HIV-infected patients suffer several renal syndromes, which can progress rapidly from renal insufficiency to end-stage renal disease. Histologically, HIV-induced nephropathy is characterized by prominent tubulopathy with apoptosis of tubular cells. Clinical and experimental evidence suggests that renal injury may be directly related to virus infection. Although HIV-1 is a polytropic and not solely lymphotropic pathogen, the susceptibility of renal cells to HIV-1 remains to be determined. This paper demonstrates in vitro the permissiveness of proximal tubular epithelial cells (PTEC) to HIV-1 and describes the effects of PTEC infection to explain the pathogenesis of tubular damage in vivo. The results indicate that PTEC express HIV-specific receptor and coreceptors and sustain virus replication. We observed that HIV-1 infection causes the death of tubular cells by triggering an apoptotic pathway involving caspase activation. Fas upregulation but not Fas ligand expression was found in the infected PTEC. However, after HIV-1 infection, tubular cells became susceptible to apoptosis induced through Fas stimulation. Caspase inhibition prevented the death of the infected PTEC in spite of persistent viral replication. These findings may explain the prominent histopathology of HIV-associated nephropathy and demonstrate that the apoptosis of nonlymphoid cells can be directly induced by HIV-1.

The six group B coxsackieviruses (CVBs) are highly prevalent human pathogens that cause viremia followed by involvement of different organs. Clinical and experimental evidence suggests that CVBs can induce kidney injury, but the susceptibility of human renal cells to these viruses is unknown. By using pure cultures of human glomerular and tubular cells, we demonstrated that all CVBs are capable of productively infecting renal cells of three different histotypes. Distinct pathogenic effects were observed. Proximal tubular epithelial cells and, to a lesser extent, glomerular podocytes were highly susceptible to CVBs; in both cases, infection led to cytolysis. In contrast, glomerular mesangial cells supported the replication of the six CVBs but failed to develop overt cytopathologic changes. Mesangial cells continued to produce infectious progeny for numerous serial subcultures (i.e., more than 50 days), especially with type 1, 3, 4, and 5 viruses. In the above cells, persistent infection induced the de novo synthesis of platelet-derived growth factor A/B and enhanced the release of transforming growth factor beta1/2. These two factors are important mediators of progression from glomerular inflammation to glomerulosclerosis. CVB replication appeared also to impair the phagocytic and contractile activity of mesangial cells. Loss of these properties--which are important in glomerular physiopathology--may contribute to the development of progressive nephropathy. The results show that CVBs induce distinct effects in different types of cultured renal cells and suggest that CVB infections may be associated with both acute and progressive renal injury.