Renal manifestations in children co-infected with HIV and disseminated tuberculosis.

Many children in Cape Town are co-infected with human immunodeficiency virus (HIV) and tuberculosis (TB). Granulomatous TB interstitial nephritis is a recognized entity. Our objective was to establish if TB plays a role in renal disease in HIV-infected children. We identified children co-infected with TB and HIV from our database and reviewed their biopsies and clinical notes. Since 2002, 12 renal biopsies or postmortem examinations were performed on HIV-infected children at our institution. The clinical scenario and renal biopsies in four cases (median age 73 months, range 24-108 months) were consistent with TB involvement. The mean CD4 count and percentage of these four patients were 508 cells/microl and 23%, respectively. All four patients presented with culture-proven disseminated TB (not yet on treatment) and had nephrotic range proteinuria and hypoalbuminemia. Three of these patients had renal impairment. The prominent features of the renal biopsies were a severe interstitial inflammatory infiltrate and mild to moderate mesangial proliferation. An interstitial granuloma was seen in one patient. With treatment for the TB, the proteinuria resolved and renal function improved in all four patients. Based on these results, we conclude that TB contributes to proteinuric renal disease in HIV-infected children and that the renal disease improves following TB treatment.

HBV X gene transfection upregulates IL-1beta and IL-6 gene expression and induces rat glomerular mesangial cell proliferation.

The X gene of HBV encodes a 17-kD protein, termed HBx, which has been shown to function as a transcriptional trans-activator of a variety of viral and cellular promoter/enhancer elements. The aim of this study was to investigate the effect of HBx on gene expression of interleukin (IL)-1 and IL-6, and proliferation of rat mesangial cells in vitro. The X gene of HBV was amplified by PCR assay, and inserted into the eukaryotic expression vector pCI-neo. The structure of recombinant pCI-neo-X plasmid was proved by restrict endonuclease digestion and sequencing analysis. pCI-neo-X was transfected into cultured rat mesangial cell line in vitro via liposome. HBx expression in transfected mesangial cells was detected by Western blot. The IL-1beta and IL-6 mRNA expression in those cells was assayed by semiquantitative RT-PCR. Mesangial cell proliferation was tested by MTT. The results showed that HBx was obviously expressed in cultured mesangial cell line at 36th and 48th h after transfection. The expression of IL-1beta and IL-6 mRNA was simultaneously increased. The cell proliferation was also obvious at the same time. It was concluded that HBx gene transfection could induce IL-1beta and IL-6 gene expression and mesangial cell proliferation. HBx may play a critical role in mesangial cell proliferation through upregulation of the IL-1beta and IL-6 gene expression.

[Microbial nucleic acids in the pathogenesis of glomerulonephritis].

Recent advances in the understanding of innate pathogen recognition revealed that nucleic acids have immunomodulatory functions in inflammation. A set of Toll-like pattern-recognition receptors recognize various types of microbial nucleic acids, i.e. double-stranded viral RNA (TLR3), single-stranded viral RNA (TLR7 and TLR8), and viral and bacterial CpG-DNA (TLR9). All of these TLRs are differentially expressed in the healthy and diseased kidney and TLR ligation was shown to initiate and modulate experimental glomerulonephritis. In this review we summarize the arising evidence in this field and discuss new hypotheses for the pathogenesis of kidney diseases that are triggered by infectious organisms.

Cytomegalovirus-related congenital nephrotic syndrome with diffuse mesangial sclerosis.

This case report describes congenital nephrotic syndrome in a 2-month-old girl associated with cytomegalovirus infection. Histological examination on renal biopsy showed diffuse mesangial sclerosis and cytomegalic inclusion bodies in the tubular cells and in some glomeruli. Cytomegalovirus (CMV) polymerase chain reaction (PCR) titer in serum was high. Remission of pulmonary and renal symptoms was achieved with ganciclovir in 3 weeks. No recurrence of proteinuria was observed during the follow-up period of 14 months. These finding suggested a causal relationship between congenital nephrotic syndrome and cytomegalovirus infection.

A novel paramyxovirus?

In public databases, we identified sequences reported as human genes expressed in kidney mesangial cells. The similarity of these genes to paramyxovirus matrix, fusion, and phosphoprotein genes suggests that they are derived from a novel paramyxovirus. These genes are sufficiently unique to suggest the existence of a novel paramyxovirus genus.

Differential effects of Sendai virus infection on mediator synthesis by mesangial cells from two mouse strains.

BACKGROUND: Recently, we observed that the severity of glomerulonephritis in an experimental model of immunoglobulin A nephropathy (IgAN) induced by Sendai virus differs between C57BL/6 and BALB/c mouse strains. The determinants of differing renal insufficiency are not understood. In the present study, we examine the capacity for mesangial cells to support Sendai viral replication and assess the direct effects of Sendai virus on the production of selected cytokines, chemokines, and eicosanoids by mesangial cells, comparing C57BL/6 to BALB/c mouse strains. METHODS: Sendai virus replication was measured by viral plaque assay using LLCMK2 cells. Production of cytokines [interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha)], chemokines (JE and KC), and eicosanoids [prostaglandin E2 (PGE2) and thromboxane B2 (TxB2)] in culture medium was evaluated by sandwich enzyme-linked immunosorbent assay (ELISA) or competitive enzyme immunoassay (EIA) after 48 hours' incubation with infectious or inactivated Sendai virus. RESULTS: Sendai virus replicates equally well in mesangial cells from both strains, and infection evokes increased IL-6, JE, KC, and PGE2 production in relation to viral dose. BALB/c mesangial cells produce significantly more IL-6 and JE than those from C57BL/6, and the dose response for KC is steeper in BALB/c mesangial cells than those from C57BL/6. Synthesis of PGE2 in BALB/c mesangial cells is higher than that of C57BL/6 mesangial cells, both under basal conditions and in response to infectious Sendai virus, again in a dose-dependent manner. There is no TNF-alpha or thromboxane response to viral stimulation. CONCLUSION: We conclude that different mesangial cell responses to this common mucosal viral pathogen might influence the severity of IgAN in our model system.

Classical swine fever: pathogenesis of glomerular damage and immunocharacterization of immunocomplex deposits.

Twenty-six pigs were inoculated with a virulent isolate (Quillota strain) of classical swine fever (hog cholera) virus to determine the chronological development of lesions in the renal glomeruli and the pathogenesis of glomerular damage and immunocomplex deposition. The study included the use of histopathological, ultrastructural and immunohistochemical (detection of viral antigen gp55, myeloid-histiocyte antigen, IgM, IgG and C1q) techniques. The main changes in glomerular structure were observed from 7 days post-inoculation (dpi) onwards, at which time the glomeruli showed macrophage infiltrations in the mesangium, and viral infection in circulating cells, glomerular endothelial cells and podocytes. Moreover, significant subcellular changes were detected in podocytes, which appeared swollen, with fusion of foot processes. Immunocomplex deposits immunoreactive for IgM, IgG and C1q were detected in mesangial, subepithelial and subendothelial areas from 10 dpi, but viral antigen was not detected as a component of these deposits; fusion of foot processes had increased in severity, especially near immunocomplex deposits. All these changes had increased still further in the final phase of the experiment (14 dpi), with neutrophil infiltrations in the mesangium.

Efficient gene transfer to rat renal glomeruli with recombinant adenoviral vectors.

Recombinant adenoviruses are attractive vectors for renal gene transfer since they can efficiently transduce nondividing cells. However, despite the fact that renal glomeruli are easily accessible via the renal circulation, attempts to deliver foreign genes specifically into renal glomeruli, using adenoviral vectors, have had limited success in rodents. A simple intraarterial injection of adenoviral vectors into the renal circulation or incubation of the virus with the kidney for an extended period of time was found to be insufficient for this purpose. In this study, we have established an efficient gene transfer protocol to express foreign genes in rat renal glomerular cells, using adenoviral vectors. We demonstrated, for the first time, that rat glomerular endothelial cells could be efficiently transduced by slowly infusing a recombinant adenovirus (Ad.CBlacZ) into the right renal artery for a period of 15 min. High levels of lacZ expression were achieved in renal glomeruli without causing significant damage to renal glomeruli or other kidney structures. The virus-mediated expression lasted for at least 21 days. These data demonstrate the feasibility of using recombinant adenoviral vectors as a tool with which to study the effect of foreign gene expression on the structure and function of rat renal glomeruli in vivo.

Productive infection of primary human endothelial cells by pig endogenous retrovirus (PERV).

The potential risk of viral transmission in the setting of xenotransplantation has gained major attention. Different porcine cell types have been shown to release retroviral particles, which are infectious for human cell lines in vitro. However, there are only a few data on whether PERV (pig endogenous retrovirus) is able to infect primary human cells. In this study we have analyzed endothelial cells, vascular fibroblasts, mesangial cells, mononuclear cells, hematopoetic stem cells and bone marrow stromal cells for PERV transmission. We now provide evidence for primary human endothelial cells, vascular fibroblasts, and mesangial cells to be susceptible to PERV transmission. PERV infection was productive in endothelial cells and mesangial cells. Our data confirm and extend former reports concerning the PERV infection of human cells. The PERV infection of different primary human cells represents further significant evidence for a viral risk during xenotransplantation. In this context, special attention should be directed towards productive infection of human endothelial cells: in the setting of xenotransplantation this cell type will have close contact with porcine cells and PERV particles.

Infection of mesangial cells with HIV and SIV: identification of GPR1 as a coreceptor.

BACKGROUND: Mesangial cells are an important component of the glomerulus. Dysfunction of mesangial cells is thought to be involved in the development of human immunodeficiency virus type 1 (HIV-1)-associated nephropathy (HIVAN). HIVAN is a structural renal failure frequently observed in patients with acquired immune deficiency syndrome. However, the susceptibility of mesangial cells to HIV-1 is disputable. More than ten G protein-coupled receptors, including chemokine receptors, have been shown to act as HIV-1 coreceptors that determine the susceptibilities of cells to HIV-1 strains with specific cell tropisms. METHODS: We examined the susceptibility of mesangial cells to various HIV-1, HIV type 2 (HIV-2) and simian immunodeficiency virus (SIV) strains. Expression of CD4 and HIV/SIV coreceptors was examined by Western blotting and polymerase chain reaction. RESULTS: Mesangial cells were found to be susceptible to HIV-1 variant and mutants that infect brain-derived cells, but highly resistant to T-tropic (X4), M-tropic (R5) or dual-tropic (X4R5) HIV-1 strains. In addition, mesangial cells were also susceptible to HIV-2 and SIV strains that infect the brain-derived cells. Among HIV/SIV coreceptors we tested, the expression of GPR1 mRNA was detected in mesangial cells. Expression of CD4 mRNA and protein was also detected in them. Mesangial cells and GPR1-transduced CD4-positive cells showed similar susceptibilities to the HIV-1 variant and mutants and HIV-2 and SIV strains. CONCLUSIONS: CD4 and GPR1 mRNAs were detected in mesangial cells. Mesangial cells were susceptible to HIV/SIV strains that use GPR1 as a coreceptor. Our findings suggest that an orphan G protein-coupled receptor, GPR1, is a coreceptor expressed in mesangial cells. It remains to be investigated whether the interaction of mesangial cells with specific HIV-1 strains through GPR1 plays a role in the development of HIVAN.

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.

Distinct pathogenic effects of group B coxsackieviruses on human glomerular and tubular kidney cells.

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.

The lesional changes and pathogenesis in the kidney in African swine fever.

African swine fever is a viral haemorrhagic disease of pigs which has been used as a model for the study of viral haemorrhagic diseases in man. The acute course of the disease is characterized by acute proliferative glomerulonephritis, with viral replication in mesangial cells and occasional focal necrosis of the renal tubular system; hyperplasia of the collecting ducts is associated with evident virus replication. Haemorrhages have been attributed to endothelial dysfunction, aggravated by virus replication in endothelial cells in the final stages of the disease. The renal interstitium displays intense oedema and an infiltrate largely composed of macrophages. Virus replication has also been observed in fibroblasts and in the smooth-muscle cells of arterioles and venules. In subacute-chronic forms of the disease, various types of glomerulonephritis are observed, ranging from mesangial proliferative glomerulonephritis to focal and segmental hyalinosis associated with immune-mediated phenomena. No striking changes are reported in the renal tubular system. Interstitial haemorrhages are associated with diapedesis due to immunologically mediated events. The interstitium has also been found to contain a lymphohistiocytic infiltrate with abundant plasma cells. No evidence has been reported of viral replication in any cell population.

Highly efficient adenovirus-mediated gene transfer into renal cells in culture.

In summary, we have shown that adenovirus vector efficiently introduced foreign gene into cultured renal cells both of mesangial and tubular origin. Genes transferred were properly expressed to produce the molecules of expected function. It was possible to introduce the gene into nearly 100% of the cells treated. Expression of the gene began as early as 12 hours after the infection, increased until 48 hours and persisted at least up to eight days. Finally, the vector was non-toxic to the cells, as judged from simple toxicity tests. Successful application of adenovirus vector enables for us to study function of pertinent molecules in suitable host cells and opens a new way for examining renal cellular physiology and pathophysiology.

Ultrastructural study of the renal tubular system in acute experimental African swine fever: virus replication in glomerular mesangial cells and in the collecting ducts.

Despite the considerable attention given to kidney lesions in African swine fever (ASF), a number of questions remain to be answered. Structural and ultrastructural examination showed that a highly virulent isolate of ASF virus (Malawi 83) replicated in glomerular mesangial cells and renal collecting duct epithelial cells, with hyperplasia of the latter in infected pigs. Replication in mesangial cells may be due to their contact with the bloodstream, as well as to their phagocytic capacity and high metabolism rate. Virus replication in macrophages and endothelial cells of interstitial capillaries, and the necrosis of these infected cells gave rise to a large number of free virus in interstitial tissue. This, together with the lesser thickness of the basal membrane of collecting ducts in comparison to the rest of the tubular system, probably facilitates ASFV infection of tubular epithelial cells. Virus replication in these cells may account for the presence of virus in the urine of pigs with acute ASF where haematuria is not observed.