Renal tubulointerstitial damage caused by persistent proteinuria is attenuated in AT1-deficient mice: role of endothelin-1.

Using angiotensin II (AngII) type 1A receptor-deficient mice [AT1(-/-)], in which we induced protein overload nephropathy, we explored the potential implication of AngII and endothelin-1 (ET-1) in the tubulointerstitial damage because of persistent proteinuria. At day 7, AT1(-/-) showed marked proteinuria to a similar extent to that of wild-type mice (WT). However, at day14, AT1(-/-) had significantly less proteinuria, renal damage, transforming growth factor-beta, and matrix mRNA expression and mortality. AT1(-/-) also showed a significant diminution in the activation of the transcriptional factors nuclear factor-kappaB and AP-1. Unexpectedly, AT1(-/-) had a higher interstitial infiltration than WT. The administration of the angiotensin-converting enzyme inhibitor quinapril to WT caused a marked improvement in proteinuria and renal lesions, resembling that seen in untreated AT1(-/-). However, the interstitial infiltration persisted in AT1(-/-) when treated with quinapril. Because ET-1 may participate in the recruitment of mononuclear cells, we also studied the implication of this peptide. AT1(-/-) had a significantly higher ET-1 expression in tubular epithelial cells than WT. The administration of the dual ETA/ETB antagonist bosentan to AT1(-/-) considerably reduced the interstitial infiltrates. Bosentan also exerted a beneficial effect on proteinuria, renal lesions, and mortality in WT. These data show that in overload nephropathy, proteinuria and renal lesions are, to a large extent, AngII-dependent. The up-regulation of ET-1 in tubular epithelial cells in AT1(-/-), associated with interstitial infiltrates, suggests that the combination of drugs interfering with both vasopeptides may be of therapeutic interest in renal diseases with severe proteinuria and tubulointerstitial damage.

Administration of IgG Fc fragments prevents glomerular injury in experimental immune complex nephritis.

Most human nephritis is due to glomerular deposition and/or formation of immune complexes (IC). In cultured mesangial cells, Fc receptor stimulation induces proliferation, matrix synthesis, and release of several mediators implicated in the initiation and progression of glomerular injury. Since Ig Fc fragments in vitro modified these phenomena, we studied the effects of systemic administration of IgG Fc fragments on the evolution of experimental IC nephritis. Fc fragment injection (1 mg/day i.p.) to rats with ongoing nephritis (proteinuria 20-50 mg/24 h vs 9 +/- 0.2 mg/24 h in controls) markedly ameliorates proteinuria, renal function, and morphological renal lesions. This was accompanied by a reduction in the renal synthesis of chemokines (monocyte chemoattractant protein-1, IFN-inducible protein-10, and cytokine-induced neutrophil chemoattractant-1), matrix proteins, and growth factors (platelet-derived growth factor, and TGF-beta), and in the activity of transcription factors. The treatment did not affect the glomerular deposition of IgG IC and complement C1q. In contrast, a decrease in the renal expression and production of C3 was observed without changes in serum complement levels. In vitro, very low complement consumption and no C3b covalent interaction were observed with Fc fragments, confirming that they did not modify systemic complement activity. These results indicate that the administration of Fc fragments prevents the development of glomerular damage in an aggressive model of proliferative glomerulonephritis through mechanisms involving a reduced local generation of complement, chemokines and growth factors. Modulation of IC-mesangial cell interaction by Fc fragment administration could represent a new approach to the treatment of severe immune nephritis.

In situ non-radioactive detection of nuclear factors in paraffin sections by Southwestern histochemistry.

BACKGROUND: Transcriptional activities of genes require intermediary regulators (nuclear factors) that bind to specific segments of nuclear DNA. A method to localize in situ the distribution of these factors using nonradioactive oligonucleotides in paraffin wax-embedded tissues is described. The distribution of two nuclear factors, activated protein-1 (AP-1) and nuclear factor-kappaB (NF-kappaB), was studied in two experimental models of immune complex glomerulonephritis in rats and atherosclerosis in rabbits. METHODS: Sections were fixed with 0.2% paraformaldehyde and were digested with pepsin A. Oligonucleotides containing the consensus sequence of NF-kappaB and AP-1 were 3'-labeled with digoxigenin. The preparations were incubated with the labeled probes (4 degreesC, overnight). After washing, the sections were incubated with an antidigoxigenin antibody conjugated with alkaline phosphatase, and the color reaction was developed. In addition, this method was combined with standard immunohistochemistry to identify the cell-type-specific localization of these DNA-binding factors. RESULTS: Kidney sections from rats with immune complex nephritis showed positive nuclear staining for AP-1 in the nuclei of several glomerular and tubulointerstitial cells. Arteries from rabbits with focal atherosclerosis presented nuclear staining for NF-kappaB in the neointima and media. The nuclear staining was highly specific, as assessed by several negative controls. In addition, Southwestern histochemistry in rabbits, followed by immunohistochemistry, demonstrated that the NF-kappaB activity was present in the area occupied by macrophages and smooth muscle cells. CONCLUSION: These results show a novel method of in situ transcription factors detection using nonradioactive probes in paraffin wax-embedded tissues, which allows a simultaneous visualization of the cell-type-specific localization of these nuclear factors.

Mesangial cells possess an asialoglycoprotein receptor with affinity for human immunoglobulin A.

Asialoglycoprotein receptor (ASGP-R), a hepatic lectin involved in the clearance of galactose-terminal glycoproteins, is also present in extrahepatic tissues, but its expression in renal cells is not well established. This study examines the presence of ASGP-R in cultured mesangial cells (MC), key cells involved in the removal of macromolecules deposited in the glomerulus. The binding of asialo-orosomucoid (ASOR) to rat MC was saturable and galactose-specific. In addition, MC internalized and degraded ASOR in a Ca(2+)-dependent manner. Parallel studies were performed in a homologous system (human MC), obtaining similar binding curve and competition with unlabeled ASOR and carbohydrates. The purified receptor from rat MC consisted of two proteins (41 and 55 kD) with similar size to the hepatic receptor. Both subunits were detected by mRNA expression analysis (ratio 2:1). Because the hepatic receptor presents avidity for the carbohydrates of IgA1, a protein deposited in the glomerulus of patients with IgA nephropathy, the interaction of IgA1 with the mesangial ASGP-R was explored. As for the interaction with ASOR, catabolism of IgA1 by rat and human MC was Ca(2+)-dependent and was reduced with galactose. In addition, the interaction of ASOR with rat MC was partially inhibited by incubation with IgA1 and its desialylated form, but not by IgA2, as demonstrated in binding experiments and in receptor purification. It is concluded that MC possess ASGP-R specific for galactose residues of several glycoproteins, including IgA1. These data could be important for a better understanding of the pathogenesis of IgA nephropathy.

Interaction of IgA with Fc alpha receptors of human mesangial cells activates transcription factor nuclear factor-kappa B and induces expression and synthesis of monocyte chemoattractant protein-1, IL-8, and IFN-inducible protein 10.

The mechanisms of glomerular damage in IgA nephropathy remain undefined. Mesangial cells (MC) possess Fc receptors for IgA (Fc alpha R), and their occupancy triggers cytokine expression, cell proliferation, and extracellular matrix synthesis. In cultured human MC we examined the effects of soluble IgA aggregates (AIgA) on the activation of nuclear factor-kappa B (NF-kappa B) and the production of proinflammatory chemokines monocyte chemoattractant protein-1 (MCP-1), IL-8, and IFN-inducible protein-10 (IP-10). The exposure of MC to AIgA rapidly activated a NF-kappa B complex constituted of p50 and p65 subunits. NF-kappa B activation was dose dependent, abolished by preincubation with IgA Fc fragments (indicating that AIgA effects occur via specific Fc alpha R), and attenuated by kinase inhibitors. MC stimulation with AIgA increased the mRNA expression of MCP-1, IL-8, and IP-10 in a time- and dose-dependent manner. Maximal expression of IL-8 was observed at 3 h (4.5-fold), while IP-10 and MCP-1 peaked at 6 h (5-fold for both). AIgA also induced biosynthesis and release of the chemokines, which presented biological activity in neutrophil and monocyte chemoattractant assays, peaking at 6 and 9 h, respectively. MC pretreatment with the antioxidant pyrrolidine dithiocarbamate inhibited NF-kappa B activation and chemokine mRNA expression. This study shows that stimulation of Fc alphaR in MC induces gene expression of MCP-1, IL-8, and IP-10, a process partially mediated by NF-kappa B activation. These data may be of importance for a better understanding of the pathogenesis of glomerular damage in IgA immune complex-related diseases.

The 80-kD fibronectin fragment increases the production of fibronectin and tumour necrosis factor-alpha (TNF-alpha) in cultured mesangial cells.

The presence of fibronectin (FN) fragments has been demonstrated in several inflammatory processes, and they have been implicated in the recruitment of mononuclear cells. However, the interaction of these FN fragments with resident cells has hardly been studied. We have hypothesized that the 80-kD FN fragment, which includes the RGD cell binding domain of FN, could contribute to the pathogenesis of glomerular damage through the interaction with mesangial cells (MC) via alpha5beta1 integrin. Since an increase in the glomerular deposit of matrix components, particularly FN, is frequently observed in progressive glomerulonephritis, we studied whether its synthesis is modulated by the 80-kD FN fragment and the native FN molecule. While the 80-kD FN fragment stimulated FN in a dose-dependent manner, both at the mRNA and protein level, the whole FN molecule exerted a dual effect. High doses produced FN inhibition, while low doses elicited a certain increase. This stimulation was abrogated by the presence of Sam-1, a MoAb against the alpha-subunit of the alpha5beta1 integrin. Since cytokines play a fundamental role in glomerular injury, we studied the production of TNF-alpha, one of the most powerful mediators of inflammation. TNF-alpha synthesis was induced by the 80-kD FN fragment, in a dose-dependent manner, but not by native FN. When MC were incubated with the 29- and 31-kD FN fragments, which lack the RGD cell binding domain, TNF-alpha secretion was not detected. These results strongly suggest that in cultured MC, the 80-kD FN fragment induces the synthesis of matrix proteins such as FN, and cytokines such as TNF-alpha, via alpha5beta1 integrin. This mechanism could contribute to the perpetuation of renal injury.

Receptors for immune complexes activate gene expression and synthesis of matrix proteins in cultured rat and human mesangial cells: role of TGF-beta.

Most human glomerulonephritis are induced by the deposition and/or formation of immune complexes in the glomerular region. Recently, it has been demonstrated that cultured glomerular mesangial cells (MC) express Fc receptors for IgA and IgG (Fc-alpha and Fc-gamma receptors). In this work, we studied whether the interaction of IgA and IgG complexes with MC induces accumulation of mesangial matrix, the histologic hallmark of progressive glomerular diseases. The exposure of MC to IgA and IgG complexes increased extracellular matrix components, such as fibronectin (FN) and collagens, at both the mRNA and protein levels in a time- and dose-dependent manner. Monomeric IgA or F(ab')2 fragments did not increase FN production, indicating that a constant region of IgA and cross-linking of Fc-alpha receptors are required. We also explored the role of TGF-beta, a profibrogenic cytokine, in the regulation of matrix synthesis. Both IgA and IgG complexes caused in MC an augmentation in TGF-beta1 mRNA and TGF-beta activity and the conversion of latent TGF-beta to the biologically active form. The coincubation of cells with complexes and a neutralizing Ab to TGF-beta significantly reduced the FN synthesis. These results indicate that the Fc receptor occupancy of MC increases the production of extracellular matrix proteins. The autocrine TGF-beta synthesis appears to be largely responsible for this effect. These findings could have implications for a better understanding of the glomerulosclerosis process in immune complex nephritis.

Stimulation of Fc(alpha) receptors induces tyrosine phosphorylation of phospholipase C-gamma(1), phosphatidylinositol phosphate hydrolysis, and Ca2+ mobilization in rat and human mesangial cells.

Although knowledge of IgA Fc receptor (Fc(alpha)R) structure and gene organization has progressed in the past few years, signal transduction pathways elicited by its activation have hardly been studied. Previously, we have demonstrated that mesangial cells (MC) possess Fc(alpha)R stimulation triggers several biologic responses. In this work, we studied the early biochemical signals triggered by Fc(alpha)R stimulation in MC. MC incubation with aggregated IgA (AIgA) elicited a dose-dependent increase in cytosolic Ca2+ ([Ca2+]i). The response was rapid and transient, and slowly fell to the original baseline. Ca2+ mobilization was dependent on the Fc region of the IgA, because Fc, but neither Fab fragment nor carbohydrates, inhibited the [Ca2+] rise. The initial induction of [Ca2+]i rise was due to Ca2+ mobilization from inositol trisphosphate (IP3)-sensitive intracellular stores, while sustained levels were maintained through extracellular Ca2+ influx. Stimulation of Fc(alpha)R also resulted in production of IP3, temporally correlated with Ca2+ mobilization. Protein tyrosine kinase inhibitors abolished [Ca2+]i rise, indicating that tyrosine phosphorylation of some substrates is required for Ca2+ mobilization. Stimulation through Fc(alpha)R gave rise to a marked increase in tyrosine phosphorylation of several proteins, including the 147-kDa band, similar in size to phospholipase C-gamma(1) (PLC-gamma(1)). Tyrosine phosphorylation of PLC-gamma(1) reached a maximum 30 s after stimulation, as determined by immunoprecipitation and Western blot. Collectively, these results indicate that the Fc(alpha)R signaling pathway in MC involves PLC-(gamma(1) activation, IP3 formation, and Ca2+ mobilization, and is linked to activation of tyrosine kinases.

Anti-Fas antibodies induce cytolysis and apoptosis in cultured human mesangial cells.

Death of renal cells often occurs during the acute and resolution phases of some forms of glomerulonephritis. The apoptotic Fas protein belongs to a recently described family of cytokine receptors with similarities to tumor necrosis factor (TNF) receptors, and may contribute to the necrobiology of renal cells. Fas transduces a signal for apoptosis in sensitive cells after binding by specific antibodies or following contact with natural Fas ligand. We have studied Fas in cultured human mesangial cells. Cytoflurography demonstrated Fas expression on the surface of human mesangial cells that was increased by stimulation with interferon gamma (IFN gamma). Agonistic anti-human Fas antibodies were cytotoxic to these cells. Cytotoxicity was time- and dose-dependent, and was modulated by pre-stimulation of the mesangial cells with IFN gamma and/or by co-treatment with actinomycin-D. Mesangial cell death following exposure to anti-Fas antibodies has features consistent with apoptosis, such as internucleosomal DNA fragmentation, nuclear shrinkage and condensation, and decreased DNA content. These data suggest that Fas and its ligand could play a mechanistic role in human glomerular cell injury.

Cyclosporin A (CsA) modulates the glomerular production of inflammatory mediators and proteoglycans in experimental nephrosis.

Nephrosis is characterized by glomerular epithelial cell injury and a decrease in the glomerular basement membrane (GBM) proteoglycan content. Although CsA is a useful treatment for a group of patients with this disease, its mechanism of action is unclear. We have previously shown that in experimental nephrosis there is an increase in the glomerular production of tumour necrosis factor-alpha (TNF-alpha) and platelet-activating factor (PAF). Here we have studied the effect of CsA on kidney generation of TNF-alpha and PAF in puromycin aminonucleoside (PAN) nephrosis as well as on the synthesis of proteoglycans by cultured glomerular epithelial cells. Rats receiving CsA had, on day 8 of PAN injection, a significant reduction in proteinuria, blood cholesterol levels and in interstitial mononuclear cells. A diminution in glomerular production and urinary excretion of TNF-alpha and PAF was also noted. In in vitro studies, at 24 h of incubation PAF and TNF-alpha induced in glomerular epithelial cells a significant decrease in proteoglycan synthesis. Neither PAF nor TNF-alpha had any significant effect on glomerular epithelial cell proliferation. CsA alone induced a dose-response increase in proteoglycan synthesis and a slight decrease in cell proliferation. CsA also reversed the inhibitory effect of PAF and TNF-alpha on proteoglycan synthesis. However, CsA did not alter the pattern of proteoglycan production, remaining around 50% chondroitinase ABC-, 15% heparitinase-sensitive. Our results indicate that PAF and TNF-alpha could be implicated in the pathogenesis of nephrosis through the inhibition of proteoglycan synthesis by glomerular epithelial cells. The beneficial effect of CsA in nephrosis may be due to the recovery of the GBM charge selectivity caused by the normalization of glomerular PAF and TNF-alpha synthesis and the increase in proteoglycan synthesis by glomerular epithelial cells.

Soluble IgA and IgG aggregates are catabolized by cultured rat mesangial cells and induce production of TNF-alpha and IL-6, and proliferation.

IgA nephropathy, a primary glomerulonephritis, is principally characterized by mesangial deposits of IgA immune complexes. Recently, it has been demonstrated that cultured glomerular mesangial cells (MC) express Fc alpha and Fc gamma receptors. In this work, we studied whether the interaction of soluble aggregates of IgA and IgG (AIgA and AIgG) with MC triggers a number of responses, including generation and release of inflammatory mediators, cell proliferation, and catabolism of the complexes. Aggregates bound to MC and were catabolized in a time-dependent manner. The percentage of cell-associated or -degraded proteins decreased in the presence of Fc, but not with Fab fragments. Both AIgA and AIgG elicited the synthesis and release of TNF-alpha and IL-6 in a dose-dependent manner, reaching a maximum between 6 and 12 h, respectively. Northern blot analysis showed that both aggregates induced the expression of mRNA encoding TNF-alpha and IL-6. Because MC proliferation is a morphologic feature commonly observed in patients with IgA nephropathy, we examined whether immune aggregates could be involved in this phenomenon. Both AIgA and AIgG induced an increase in MC number, assessed by [3H]thymidine and methylene blue uptake. The presence of anti-TNF-alpha or anti-IL-6 Abs in the medium decreased the proliferative effect triggered by aggregates. These results show that Fc alpha and Fc gamma receptor occupancy of MC induces the synthesis and release of inflammatory cytokines and proliferation, as well as the phagocytosis of stimulatory proteins. These findings could have implications for the understanding of inflammation and repair in IgA nephropathy.

The potential role of inflammatory and fibrogenic cytokines in the glomerular diseases.

In recent years increasing evidence has been accumulated on the role of cytokines in mediating glomerular and renal damage. Many such cytokines are released in the inflamed glomeruli by leukocytes and intrinsic glomerular cells. Cytokines not only recruit inflammatory cells into the injured glomeruli, but also induce a variety of responses on glomerular cells that range from a direct toxic effect to shape changes, proliferation, and induction of the release of inflammatory mediators and extracellular matrix, and could promote further glomerular damage. Moreover, exogenous administration of cytokines has induced glomerular injury in healthy animals and has enhanced renal damage in animals with glomerulonephritis. Anti-cytokine strategies have proved to be effective therapeutical alternatives in experimental models of glomerular diseases and may provide a more specific approach to the management of human glomerulonephritis.

Evidence for a specific IgA receptor in rat and human mesangial cells.

Previous works have demonstrated that mesangial cells (MC) possess Fc receptors for IgG. We have investigated whether serum IgA could bind to MC in culture through a specific receptor. The binding of 125I-IgA (4 degrees C, 60 min) to MC was dose dependent and saturable, and the Scatchard analysis revealed a population of 1 x 10(5) binding sites per cell with a Ka of 7.8 x 10(8) M-1. This receptor was specific for IgA because unlabeled IgA and its Fc fragment were able to inhibit the binding, whereas IgG, IgM, and the IgA F(ab) fragment were not. Parallel experiments showed that human IgA bound in a similar manner both in a homologous system (human MC) and in heterologous systems (rat MC and peritoneal macrophages). In saturation studies using neuraminidase-desialylated IgA (desIgA), which has more carbohydrate residues exposed to binding, similar results to those of untreated IgA were obtained. Incubation with several carbohydrates decreased the IgA and desIgA binding to MC, obtaining maximal inhibition with simultaneous addition of galactose and N-acetyl-galactosamine. The possible IgA receptor (IgA-R) was purified from MC by affinity chromatography on an IgA-Sepharose column and appeared on SDS-PAGE gels as a 60-kDa band. The expression of IgA-R molecules on MC was enhanced by the presence of IgA in the culture medium. Finally, a band comparable with that of U937 cells was shown by Northern blot assay hybridizing mRNA from rat and human MC with a cDNA probe for the Fc alpha R. Taken together, these results strongly suggest the existence of a 60-kDa protein on the surface of MC that is able to bind IgA with high affinity.

Involvement of lipid mediators in the pathogenesis of experimental nephrosis in rats: its pharmacological modulation.

The administration of a single-injection of Adriamycin (ADR) to rats results in marked proteinuria and glomerular morphological changes that are similar to minimal change disease in humans. We have hypothesized that Adriamycin, by itself or through the release of some mediators from resident glomerular cells, could provoke a damage to epithelial glomerular cells. Sprague-Dawley rats received a single injection of Adriamycin, 7.5 mg/kg bw, allocated randomly in several groups and treated throughout 2 weeks of follow-up. All control nontreated animals developed important nephrotic syndrome and degenerative lesions of epithelial glomerular cells. Isolated glomeruli from animals injected with adriamycin 14 days before synthesized thromboxane (TxB2) and platelet activating factor (PAF) in amounts above the rates of control glomeruli. Animals treated with three structurally different PAF receptor antagonists did not present proteinuria or only to a very low extent (p less than 0.0005). In these rats no alterations in epithelial cells were noted. Furthermore, no significant changes in the TxB2 production were noted in rats treated with BN 52021, a PAF receptor antagonist. Leukotrienes also seem to participate since treatment with a 5-lipoxygenase inhibitor partially corrected proteinuria. Moreover, glomeruli from animals with nephrosis and treated with this compound presented only a discrete reduction in the PAF synthesis. On the whole, these data suggest a key role for PAF in the pathogenesis of adriamycin nephropathy. Other lipid meditors, released in cascade simultaneously or thereafter, could perpetuate the renal damage.

Cooperation between tumor necrosis factor (TNF) and platelet-activating factor (PAF) in the inflammatory response.

In this paper we have examined the cooperation between TNF and PAF in the generation of superoxide anion (O2-) in vitro by polymorphonuclear cells (PMNs), as well as their ability to induce joint inflammation when injected into the knee of healthy rabbits. TNF and PAF directly stimulated the generation of a small amount of O2- by PMNs. TNF pretreatment of PMNs induced a certain synergy in the O2- production, when these cells were later stimulated with PAF. When PAF receptor antagonists were added, the O2- release was inhibited. The injection of either TNF or PAF into the knee joint of normal rabbits induced a dose-dependent accumulation of leukocytes in the synovial cavity 24 h after administration. When TNF was administered 1 h before PAF, a synergistic response in the accumulation of leukocytes in the joint fluid was noted. The administration of BN 52726 by the intraperitoneal route markedly inhibited the cell accumulation induced by TNF and PAF. Histological signs of inflammation were noted in the synovial lining of joints injected with TNF and PAF. These results suggest that TNF can amplify the inflammatory response induced by PAF. PAF antagonists can inhibit this effect and thus may be of therapeutic value in different pathological situations.