No abstract

There are about 2.5 million glomeruli in the kidneys each consisting of a barrel of glomerular basement membrane surrounded by glomerular endothelial cells on the inside and glomerular epithelial cells with established foot processes (podocytes) on the outside. Defects in this filtration apparatus lead to glomerular vascular leak or proteinuria. The role of vascular endothelial growth factor (VEGF) in the regulation of glomerular vascular permeability is still unclear. Recent studies indicate that patients receiving anti-VEGF antibody therapy may have an increased incidence of proteinuria. In a different setting, pregnancies complicated by preeclampsia are associated with elevated soluble VEGF receptor 1 protein (sFlt-1), endothelial cell dysfunction and proteinuria. These studies suggest that neutralization of physiologic levels of VEGF, a key endothelial survival factor, may lead to proteinuria. In the present study, we evaluated the potential of anti-VEGF neutralizing antibodies and sFlt-1 in the induction of proteinuria. Our studies demonstrate that anti-VEGF antibodies and sFlt-1 cause rapid glomerular endothelial cell detachment and hypertrophy, in association with down-regulation of nephrin, a key epithelial protein in the glomerular filtration apparatus. These studies suggest that down-regulation or neutralization of circulating VEGF may play an important role in the induction of proteinuria in various kidney diseases, some forms of cancer therapy and also in women with preeclampsia.

A mouse model for the autosomal form of Alport syndrome was produced. These mice develop a progressive glomerulonephritis with microhematuria and proteinuria, consistent with the human disease. End-stage renal disease develops at -14 weeks of age. TEM analysis of the glomerular basement membranes (GBM) during development of renal pathology revealed focal multilaminated thickening and thinning beginning in the external capillary loops at 4 weeks and spreading throughout the GBM by 8 weeks. By 14 weeks, half of the glomeruli were fibrotic with collapsed capillaries. Immunofluorescence analysis of the GBM showed the absence of type IV collagen ~-3, e~-4, and c~-5 chains and a persistence of ~-1 and ~-2 chains (these chains normally localize to the mesangial matrix). Northern blot analysis using probes specific for the collagen chains illustrate the absence of COL4A3 in the knockout, whereas mRNAs for the remaining chains are unchanged. An accumulation of fibronectin, heparan sulfate proteoglycan, laminin-1, and entactin was observed in the GBM of the affected animals. The temporal and spatial pattern of accumulation was consistent with that for thickening of the GBM as observed by TEM. Thus, expression of these basement membrane-associated proteins may be involved in the progression of Alport renal disease pathogenesis. The levels of mRNAs encoding the basement membrane-associated proteins at 7 weeks were unchanged.

Alport syndrome is a genetic disorder resulting from mutations in type IV collagen genes. The defect results in pathological changes in kidney glomerular and inner-ear basement membranes. In the kidney, progressive glomerulonephritis culminates in tubulointerstitial fibrosis and death. Using gene knockout-mouse models, we demonstrate that two different pathways, one mediated by transforming growth factor (TGF)-beta1 and the other by integrin alpha1beta1, affect Alport glomerular pathogenesis in distinct ways. In Alport mice that are also null for integrin alpha1 expression, expansion of the mesangial matrix and podocyte foot process effacement are attenuated. The novel observation of nonnative laminin isoforms (laminin-2 and/or laminin-4) accumulating in the glomerular basement membrane of Alport mice is markedly reduced in the double knockouts. The second pathway, mediated by TGF-beta1, was blocked using a soluble fusion protein comprising the extracellular domain of the TGF-beta1 type II receptor. This inhibitor prevents focal thickening of the glomerular basement membrane, but does not prevent effacement of the podocyte foot processes. If both integrin alpha1beta1 and TGF-beta1 pathways are functionally inhibited, glomerular foot process and glomerular basement membrane morphology are primarily restored and renal function is markedly improved. These data suggest that integrin alpha1beta1 and TGF-beta1 may provide useful targets for a dual therapy aimed at slowing disease progression in Alport glomerulonephritis.

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Type IV collagen is a predominant component of basement membranes, and glomeruli of a kidney filter Ϸ70 -90 liters of plasma every day through a specialized glomerular basement membrane (GBM). In Alport syndrome, a progressive disease primarily affecting kidneys, mutations in GBM-associated type IV collagen genes (COL4A3, COL4A4, or COL4A5) lead to basement membrane structural defects, proteinuria, renal failure, and an absence of all three GBM collagen triple helical chains because of obligatory posttranslational assembly requirements. Here, we demonstrate that transplantation of wild-type bone marrow (BM) into irradiated COL4A3 ؊/؊ mice results in a possible recruitment of BM-derived progenitor cells as epithelial cells (podocytes) and mesangial cells within the damaged glomerulus, leading to a partial restoration of expression of the type IV collagen ␣3 chain with concomitant emergence of ␣4 and ␣5 chain expression, improved glomerular architecture associated with a significant reduction in proteinuria, and improvement in overall kidney histology compared with untreated COL4A3 ؊/؊ mice or irradiated COL4A3 ؊/؊ mice with BM from adult COL4A3 ؊/؊ mice. The ␣3(IV) collagen produced by BM-derived podocytes integrates into the GBM and associates with other ␣-chains to form type IV collagen triple helical networks. This study demonstrates that BM-derived stem cells can offer a viable strategy for repairing basement membrane defects and conferring therapeutic benefit for patients with Alport syndrome.Alport syndrome ͉ bone marrow transplantation ͉ glomerular basement membrane ͉ type IV collogen ␣3 chain ͉ glomeruli

SummaryGenetic studies have indicated that susceptibility to rheumatoid arthritis (R.A) maps to the HLA-DP, locus of the major histocompatibility complex. Strong linkage disequilibrium between certain HLA-DQ genes and HLA-DP,. genes associated with RA, however, suggests that HLA-DQ molecules may also play a role in RA susceptibility. To examine the role of HLA-DQ molecules in arthritis, we generated transgenic mice expressing the DQA 1"0301 and DQB 1"0302 genes from an RA predisposing haplotype (DQ8/DR.4Dw4). The transgenes were introduced into mouse class II-deficient H-2Ab ~ mice, and their susceptibility to experimental collagen-induced arthritis was evaluated. The HLA-DQ8+,H-2Ab ~ mice displayed good expression of the DQ8 molecule, while no surface expression of endogenous murine class II molecules could be detected. The DQ8 molecule also induced the selection of CD4 + T cells expressing a normal repertoire of V~ T cell receptors. Immunization of HLA-DQ8+,H-2Ab ~ mice with bovine type II collagen (CII) induced a strong antibody response that was crossreactive to homologous mouse CII. Also, in vitro proliferative responses against bovine CII, which were blocked in the presence of an antibody specific for HLA-DQ and mouse CD4, were detected. Finally, a severe polyarthritis developed in a majority of HLA-DQ8+,H-2Ab ~ mice, which was indistinguishable from the disease observed in arthritis susceptible B10.T(6R) (H-2Aq) controls. In contrast, HLA-DQ8-,H-2Ab ~ fullsibs did not generate CII antibody and were completely resistant to arthritis. Therefore, these results strongly suggest that HLA-DQ8 molecules contribute to genetic susceptibility to arthritis and also estabhsh a novel animal model for the study of human arthritis.I t is widely accepted that a strong genetic component contributes to the susceptibility or resistance to certain human autoimmune diseases (1). Attempts to identify the particular genes involved in these disorders has been an area of major focus for many laboratories, and inroads have been clearly made. Among the numerous genes studied, one group that has garnered much attention are the genes encoding the class I and class II molecules of the HLA complex. Located on the short arm of chromosome 6, the primary function of HLA class I and II molecules is to bind and present processed antigenic peptides to T cells bearing receptors specific for the peptide-HLA complex. This presentation event plays a pivotal role in shaping the cellular immune repertoire and dictating the nature and scope of the immune response against a given antigen (2).A role for HLA molecules in the etiology of autoimmune disease derives from genetic studies showing a clear association between the presence or absence of certain HLA class I or II alleles, as well as increased or decreased susceptibility to a particular autoimmune disorder. A disease with a strong autoimmune foundation and HLA class II association is rheumatoid arthritis (RA) 1. In Caucasians, genetic studies initially showed a high prevalence of the HLA-DR4Dw4...

Severity of fibrosis after injury is determined by the nature of the injury and host genetic susceptibility. Metabolism of collagen, the major component of fibrotic lesions, is, in part, regulated by integrins. Using a model of glomerular injury by adriamycin, which induces reactive oxygen species (ROS) production, we demonstrated that integrin alpha1-null mice develop more severe glomerulosclerosis than wild-type mice. Moreover, primary alpha1-null mesangial cells produce more ROS both at baseline and after adriamycin treatment. Increased ROS synthesis leads to decreased cell proliferation and increased glomerular collagen IV accumulation that is reversed by antioxidants both in vivo and in vitro. Thus, we have identified integrin alpha1beta1 as a modulator of glomerulosclerosis. In addition, we showed a novel pathway where integrin alpha1beta1 modulates ROS production, which in turn controls collagen turnover and ultimately fibrosis. Because integrin alpha1beta1 is expressed in many cell types this may represent a generalized mechanism of controlling matrix accumulation, which has implications for numerous diseases characterized by fibrosis.