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Ephs and ephrins are a family of membrane-bound proteins that function as receptor–ligand pairs. Members of the Eph–ephrin-B family have recently been reported to regulate the paracellular permeability of epithelial cells. In this study, we analyzed the expression and the function of ephrin-B1 in glomeruli. Using immunofluorescence (IF), we found that ephrin-B1 was expressed along the glomerular capillary loop. Immunoelectron microscopy revealed that ephrin-B1 expression was restricted at the slit diaphragm. Dual labeled IF showed ephrin-B1 colocalized with the slit diaphragm proteins nephrin and CD2-associated protein. Ephrin-B1 colocalized with nephrin at the late capillary loop stage of kidney development. Additionally, injection of rats with a nephritogenic anti-nephrin antibody (ANA) reduced ephrin-B1 expression. When podocytes were cultured in vitro, they extruded processes that co-stained for ephrin-B1 and for CD2-associated protein. When these podocytes were treated in culture with small interfering RNA for ephrin-B1, CD2-associated protein was reduced in the processes, with a remaining faint perinuclear staining. We suggest that ephrin-B1 has a role in maintaining barrier function at the slit diaphragm.Kidney International (2007) 72, 954–964; doi:10.1038/sj.ki.5002454; published online 1 August 2007 [ABSTRACT FROM AUTHOR]

Most advanced glomerular diseases are characterized by abnormal extracellular matrix (ECM) accumulation in the glomeruli, and matrix metalloproteinases (MMPs) play a pivotal role in ECM remodeling in various glomerular diseases. The proto-oncogene, ets-1, is a transcription factor regulating the expression of various matrix proteinases, including MMP-1, MMP-3, and MMP-9. The goal of the present study was to characterize the role of Ets-1 in the progression of glomerular diseases. Overexpression of Ets-1 in cultured mesangial cells prevented transforming growth factor (TGF)-β-induced inhibition of DNA-binding activity and TGF-β-induced type I collagen production. In addition, exogenous Ets-1 abolished TGF-β-induced collagen gel contraction. The in vivo transfection of the ets-1 gene into nephritic kidney resulted in the increases in glomerular MMP-1, MMP-3, and MMP-9 mRNA, decreases in mesangial ECM deposition, and attenuation of fibronectin extradomain A (EDA) and type I collagen expression. In contrast, knockdown of Ets-1 in glomeruli resulted in severe ECM deposition in diseased glomeruli. In conclusion, Ets-1 promotes degradation of ECM proteins and is critical for integral glomerular reorganization.Kidney International (2006) 70, 298–305. doi:10.1038/sj.ki.5001541; published online 31 May 2006 [ABSTRACT FROM AUTHOR]

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that maintains the glomerular and peritubular capillary (PTC) network in the kidney. The soluble form of the VEGF receptor-1 (soluble fms-like tyrosine kinase 1 (sFlt-1)) is known to regulate VEGF activity by binding VEGF in the circulation. We hypothesized that VEGF may be beneficial for maintaining glomerular filtration barrier and vascular network in rats with progressive glomerulonephritis (GN). For blockade of VEGF activity in vivo, rats were transfected twice with plasmid DNA encoding the murine sFlt-1 gene into femoral muscle 3 days before and 2 weeks after the induction of antiglomerular basement membrane antibody-induced GN. Inhibition of VEGF with sFlt-1 resulted in massive urinary protein excretion, concomitantly with downregulated expression of nephrin in nephritic rats. Further, blockade of VEGF induced mild proteinuria in normal rats. Administration of sFlt-1 affected neither the infiltration of macrophages nor crescentic formation. In contrast, treatment of sFlt-1 accelerated the progression of glomerulosclerosis and interstitial fibrosis accompanied with renal dysfunction and PTC loss at day 56. VEGF may play a role in maintaining the podocyte function as well as renal vasculature, thereby protecting glomeruli and interstitium from progressive renal insults.Kidney International (2006) 69, 1986–1995. doi:10.1038/sj.ki.5000439; published online 26 April 2006 [ABSTRACT FROM AUTHOR]

A single intravenous injection of anti-Thy-1 monoclonal antibody (mAb) 1-22-3 is known to cause reversible mesangial proliferative glomerulonephritis. However, mAb 1-22-3 injection followed by unilateral nephrectomy leads to progressive glomerulosclerosis and tubulointerstitial change with an irreversible course. To identify genes that play an important role in the irreversible progression of renal injury, we used microarray technology to identify differences in gene expression between these models. Rats were intravenously injected with mAb 1-22-3 1 week after unilateral nephrectomy (irreversible model) or a sham operation (reversible model), and rats were killed on days 4, 7, 14, 42, and 56 after the injection. complementary DNA probes prepared from kidney messenger RNAs were hybridized with oligonucleotide microarrays containing 4854 rat genes. The microarray identified 189 differentially expressed genes, having at least a two-fold difference in expression level between the two models, and they were classified into five clusters. One of the clusters consisted of genes whose expression was markedly upregulated in the irreversible model. This cluster included the genes encoding osteopontin, kidney injury molecule-1, and thymosin β10. Increased expression of thymosin β10 was localized mainly in macrophages in the fibrotic interstitium, and upregulation of thymosin β10 expression was also observed in a unilateral ureteral obstruction model. The microarray analysis yielded information on the molecular mechanisms responsible for the difference in disease progression between the reversible and irreversible model of anti-Thy-1 nephritis. Thymosin β10 may play an important role in the progression of kidney disease.Kidney International (2006) 69, 996–1004. doi:10.1038/sj.ki.5000191; published online 25 January 2006 [ABSTRACT FROM AUTHOR]

Background: Slit diaphragms are intercellular junctions of podocytes of the renal glomerulus. The molecular composition of slit diaphragms is still elusive. Slit diaphragms are characterized by the presence of a wide intercellular space. The morphological feature is shared by desmosomes and adherens junctions, which contain members of the cadherin superfamily. Thus, we have hypothesized that some components of slit diaphragms belong to the cadherin superfamily. Consequently, we have isolated cDNA encoding FAT from reverse-transcribed (RT) glomerular cDNA by homology polymerase chain reaction (PCR) using primers based on conserved sequences in cadherin molecules. FAT is a novel member of the cadherin superfamily with 34 tandem cadherin-like extracellular repeats, and it closely resembles the Drosophila tumor suppressor fat.Methods: Expression of FAT was examined in glomeruli of the adult rat kidney by the ribonuclease protection assay and in situ hybridization. To localize the FAT protein in podocytes minutely, we prepared affinity-purified antibody against FAT by immunizing rabbits against an oligopeptide corresponding to the C-terminal 20 amino acids.Results: Expression of FAT mRNA was detected in total RNA from glomeruli. In situ hybridization revealed significant signals in podocytes. Western blot analysis using solubilized glomeruli showed a single band, in which the molecular weight was more than 500 kD. Immunostaining of cultured epithelial cells from rat kidney (NRK52E) revealed FAT accumulation in cell-cell contact sites. In the glomerulus, FAT staining was observed distinctly along glomerular capillary walls. Double-label immunostaining using monoclonal antibody against slit diaphragms (mAb 5-1-6) showed identical localization of anti-FAT antibody and mAb 5-1-6. Furthermore, the double-label immunogold technique with ultrathin cryosections demonstrated that gold particles for FAT cytoplasmic domain were located at the base of slit diaphragms labeled by mAb 5-1-6 and that the cytoplasmic domain of FAT colocalized with ZO-1, a cytoplasmic component associated with slit diaphragms.Conclusion: The molecular structure of FAT and its colocalization with 5-1-6 antigen and ZO-1 indicate that FAT is a component of slit diaphragms. [ABSTRACT FROM AUTHOR]