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Fibronectin (FN) is the main extracellular matrix component in glomerulosclerotic lesions. There are different FN isoforms that result from alternative splicing at the EDA and EDB regions of FN mRNA. Increased inclusion of EDA and EDB, which can be elicited by TGFbeta, may be conducive to the development of glomerulosclerosis (GS). TGFbeta and IL-4 have previously been shown to play a role in the development of GS. In this study, the mRNA splicing patterns for EDA+ and EDB+ fibronectin were investigated in vivo in various experimental sclerotic glomerulopathies, in vitro in rat mesangial cells (MC) that were stimulated by TGFbeta or transfected with IL-4, and in human kidney biopsies with GS from patients with various kidney diseases. Analysis of glomerular FN mRNA demonstrated inclusion of both ED regions in rats with anti-Thy1 nephritis or chronic serum sickness and in mice with anti-GBM glomerulonephritis. Inclusion of both the EDA and EDB regions was associated with glomerular TGFbeta expression. In contrast, in mice with Th2-mediated graft-versus-host disease, a model for lupus nephritis, the FN transcripts included neither the EDA nor the EDB region, and renal TGFbeta expression was absent. Compared to normal MCs in culture, MCs transfected with IL-4 produced lower amounts of FN and demonstrated less EDA inclusion, while MC that had been treated with TGFbeta showed increased production of FN and more EDA inclusion. Renal biopsies from patients with renal diseases, except those taken from patients with lupus nephritis, showed higher TGFbeta levels, higher FN levels, and more EDA inclusion than controls. TGFbeta may be a key player in the development of GS by inducing local FN production and alternative splicing of FN mRNA. In lupus glomerulonephritis, in which the involvement of TGFbeta in GS is less prominent, Th2 cytokines such as IL-4 probably account for increased intrarenal collagen synthesis and subsequent FN accumulation from the circulation. In conclusion, neither alternative FN splicing, nor a high transcription level of TGFbeta, appears to be a general prerequisite for the development of GS.

Fibronectin (FN) is an extracellular matrix component which appears in different isoforms, due to alternative mRNA splicing of the ED-A, ED-B, and IIICS regions, and subsequent post-translational modifications. The FN isoforms, some of which occur specifically during fetal development and in fibrogenic diseases, have been reported to play a role in various biological functions, such as regulation of the matrix assembly, adhesion, and proliferation. The contribution of these FN isoforms to the pathogenesis of chronic renal diseases, which are also fibrogenic disorders, is not well known. This study therefore examined the distribution of FN isoforms in renal diseases by immunohistochemistry, with a panel of isoform-specific monoclonal antibodies (MAbs), applied to 63 abnormal renal biopsies and ten normal controls. Normal kidneys contained total FN (MAb IST4) both in the mesangial and in the interstitial extracellular matrix (ECM), but only traces of ED-A-positive FN (MAb IST9), and no ED-B-positive FN (MAb BC1) or oncofetal FN (MAb FDC6) was found in normal renal tissue. All patients with renal disease demonstrated increased total FN staining of the interstitium and the mesangium. Periglomerular fibrotic lesions and fibrous crescents showed massive accumulation of total FN, whereas the amount of total FN in the ECM of obsolescent glomeruli was decreased, compared with that in normal mesangial ECM. Oncofetal (FDC6), EDB-negative (MAb IST6), ED-A-positive, and ED-B-positive FN isoforms were found in glomerular ECM accumulations and in fibrous crescents. Tubulointerstitial fibrotic lesions predominantly contained the ED-A-positive FN isoform, whereas in globally sclerotic glomeruli, predominantly ED-B-positive FN was observed. The expression of FN isoforms was similar in all renal diseases studied. These results show that in various renal diseases, oncofetal (FDC6) FN and ED-A- and ED-B-positive isoforms of FN accumulate at locations of chronic lesions, independently of the aetiology of the disease. The deposition of these isoforms in human renal tissue may play a role in the modulation of the immune response by attracting monocytes and lymphocytes to the injured kidney. Furthermore, because the ED-B-positive FN isoform is highly susceptible to proteolytic degradation, its accumulation may play a role in scar formation and tissue repair. ED-B-positive FN forms a temporary scaffold supporting the cells, which can easily be cleared by proteolytic degradation once new tissue has been produced at the site of injury.

The expression of collagen type IV chains in the renal tubulointerstitium was investigated during the development of chronic serum sickness (CSS) in rats, a model for immune complex-mediated renal disease. Immunohistochemical studies showed increased expression of alpha4(IV) collagen early during disease development, followed by an increase in alpha1(IV) through alpha3(IV) collagen subchain expression, especially in the tubular basement membrane. Dot-blot and in situ hybridization analysis showed a transient increase in steady-state mRNA levels for all collagen IV subchains during the development of CSS, which was most abundant for alpha1(IV), alpha2(IV), and alpha4(IV). Statistical correlations were found between the mRNA levels of alpha1(IV) and alpha2(IV) collagen and between alpha3(IV) and alpha4(IV), in line with the results of others which showed that these chains are co-distributed as heterotrimer collagen type IV molecules. However, additional correlations were found between the mRNA levels coding for alpha1(IV) and alpha3(IV) collagen, and between alpha1(IV) and alpha4(IV) mRNAs in the course of CSS. These abnormal correlations support the hypothesis that changes occur in the co-expression of the collagen IV subchains during the development of CSS. In addition, a strong correlation was found between the presence in the tubulointerstitium of alpha1(IV) and alpha2(IV) collagen chains, on the one hand, and the tubulointerstitial influx of R73+ and ED1+ cells, on the other, suggesting the involvement of inflammatory cells in the observed alterations in matrix production. Changes in the relative abundance of collagen IV chains in disease states may perturb the collagen IV network in the tubulointerstitial compartment and thereby play a role in the development of renal failure.

The purpose of this article is to review a set of recently obtained data concerning matrix and matrix adhesion molecules in renal disease. Our goal is not to cover the entire topic, but rather to focus on findings obtained with an experimental model for chronic lupus nephritis, evoked in mice by inducing graft-versus-host disease (GVHD). The overall aim of these studies was to investigate the role of adhesion molecules as targets for autoantibodies, in the recruitment of inflammatory cells, and in the accumulation of matrix in kidney disorders. In addition, we set out to discover how matrix proteins in renal diseases differ from normal matrix molecules both quantitatively, in their increased frequency, and qualitatively, in their intramolecular structure. The advances in understanding and methodology described in this review imply a substantial capability for greater insight into the pathogenesis of kidney disease; for making better use of renal biopsies, such as in applying competitive reverse-transcriptase-polymerase chain reaction (RT-PCR) in RNA analysis for matrix; and in developing more effective treatment strategies for patients with kidney disease.

Fibronectin accumulation in glomerulosclerotic lesions: Self-assembly sites and the heparin II binding domain. Background Glomerulosclerosis is a severe complication of many immunologically-mediated kidney diseases, eventually resulting in loss of renal function. In chronic graft-versus-host disease (GvHD) in mice, a model for human lupus nephritis, the end-stage sclerotic lesions were previously shown to contain large amounts of fibronectin (FN). This study investigated a domain-specific accumulation process of circulating plasma FN (pFN) in sclerotic lesions. Methods GvHD mice were injected with FITC-conjugated pFN or pFN-fragments, with or without heparin pre-incubation. pFN fragments were generated by digestion of pFN by cathepsin D, after which the fragments were separated on a heparin affinity column. Thus, two batches of fragments were obtained with either low or high affinity for heparin. Results FN accumulation was accompanied by an up-regulated expression of integrin α 5 β 1 , the FN receptor, in the periphery of sclerotic lesions. pFN-FITC injected into GvHD mice was trapped in sclerotic glomeruli within 24 hours. Both heparin and non-anti-coagulant heparin blocked the accumulation of pFN-FITC, indicating that the protective effect of heparin in the trapping of FN is independent of its anticoagulant properties, and probably results from preventing direct binding of FN in the sclerotic lesions. To investigate whether FN binds in the glomerulus via the heparin-binding regions, pFN fragments were generated and injected into GvHD mice. Whereas the fraction with high affinity for heparin did not accumulate in the sclerotic glomeruli, the fraction with low affinity for heparin did. Partial sequencing of the isolated peptides showed that in the glomerulus fibronectin does not bind via the heparin II binding region. Conclusions We hypothesize that the protective effect of heparin treatment may be the result of steric hindrance of the specific binding sites, that is, the I 1-5 and/or III 1 self-assembly sites of FN.