Hepatitis C virus (HCV) infection is frequentlyHepatitis C virus infection (HCV) is a major problem worldwide, frequently complicated by a virus-associated glomerulonephritis. During the course of infection, immune complexes and viral RNA reach the mesangium.

Shared transcription factor binding sites that are conserved in distance and orientation help control the expression of gene products that act together in the same biological context. New bioinformatics approaches allow the rapid characterization of shared promoter structures and can be used to find novel interacting molecules. Here, these principles are demonstrated by using molecules linked to the unique functional unit of the glomerular slit diaphragm. An evolutionarily conserved promoter model was generated by comparative genomics in the proximal promoter regions of the slit diaphragm-associated molecule nephrin. Phylogenetic promoter fingerprints of known elements of the slit diaphragm complex identified the nephrin model in the promoter region of zonula occludens-1 (ZO-1). Genome-wide scans using this promoter model effectively predicted a previously unrecognized slit diaphragm molecule, cadherin-5. Nephrin, ZO-1, and cadherin-5 mRNA showed stringent coexpression across a diverse set of human glomerular diseases. Comparative promoter analysis can identify regulatory pathways at work in tissue homeostasis and disease processes.bioinformatics ͉ gene regulation ͉ podocyte ͉ slit diaphragm

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IntroductionElevated serum levels of the proinflammatory cytokine tumor necrosis factor alpha (TNFα) correlate with an increased risk for atherothrombotic events and TNFα is known to induce prothrombotic molecules in endothelial cells. Based on the preexisting evidence for the impact of TNFα in the pathogenesis of autoimmune disorders and their known association with an acquired hypercoagulability, we investigated the effects of TNFα and the role of the TNF receptor subtypes TNFR1 and TNFR2 for arteriolar thrombosis in vivo.MethodsArteriolar thrombosis and platelet-rolling in vivo were investigated in wildtype, TNFR1-/-, TNFR2-/- and TNFR1-/R2-/- C57BL/6 mice using intravital microscopy in the dorsal skinfold chamber microcirculation model. In vitro, expression of prothrombotic molecules was assessed in human endothelial cells by real-time PCR and flow cytometry.ResultsIn wildtype mice, stimulation with TNFα significantly accelerated thrombotic vessel occlusion in vivo upon ferric chloride injury. Arteriolar thrombosis was much more pronounced in TNFR1-/- animals, where TNFα additionally led to increased platelet-endothelium-interaction. TNFα dependent prothrombotic effects were not observed in TNFR2-/- and TNFR1-/R2- mice. In vitro, stimulation of human platelet rich plasma with TNFα did not influence aggregation properties. In human endothelial cells, TNFα induced superoxide production, p-selectin, tissue factor and PAI-1, and suppressed thrombomodulin, resulting in an accelerated endothelial dependent blood clotting in vitro. Additionally, TNFα caused the release of soluble mediators by endothelial cells which induced prothrombotic and suppressed anticoagulant genes comparable to direct TNFα effects.ConclusionsTNFα accelerates thrombus formation in an in vivo model of arteriolar thrombosis. Its prothrombotic effects in vivo require TNFR2 and are partly compensated by TNFR1. In vitro studies indicate endothelial mechanisms to be responsible for prothrombotic TNFα effects. Our results support a more selective therapeutic approach in anticytokine therapy favouring TNFR2 specific antagonists.

Detection of multiple vascular endothelial growth factor splice isoforms in single glomerular podocytes. Glomerular podocytes are major determinants of filtration permselectivity in the glomerulus. Although the molecular mechanisms determining the characteristics of the glomerular filtration unit are incompletely understood, vascular endothelial growth factor (VEGF) has been implicated. To analyze this process in situ, we established a method that allows exploration of in vivo mRNA expression of podocytes using single-cell reverse transcriptase-polymerase chain reaction (RT-PCR). Microdissected mouse glomeruli were held in a patch-clamp apparatus, and single podocytes were harvested by aspiration. After lysis, the cells were reverse transcribed, and PCR was performed (45 cycles). The podocyte nature of the material was confirmed by detection of podocyte-specific mRNA (glomerular epithelial protein 1 and Wilms' tumor protein 1). Using specific oligonucleotide primers, VEGF was detected in mRNA obtained from renal cortex, single microdissected glomeruli, cultured murine podocytes, and single podocytes in situ. All cells examined expressed three VEGF isoforms (121, 165, and 189). These differ in their capacity for binding to extracellular matrix and could have different potencies regulating glomerular endothelial permeability. Our approach should allow a semiquantitative, isoform-specific evaluation of VEGF mRNA expression in podocytes during nephrogenesis and in glomerular disease. SINGLE PODOCYTE REVERSE TRANSCRIPTASE-POLYMERASE CHAIN REACTIONSingle-cell RT-PCR was performed as described [5]. In brief, microdissected mouse glomeruli were fixed via a

BackgroundProliferation and apoptosis of mesangial cells (MC) are important mechanisms during nephrogenesis, for the maintenance of glomerular homeostasis as well as in renal disease and glomerular regeneration. Expression of chemokines and chemokine receptors by intrinsic renal cells, e.g. SLC/CCL21 on podocytes and CCR7 on MC is suggested to play a pivotal role during these processes. Therefore the effect of selected chemokines on MC proliferation and apoptosis was studied.MethodsProliferation assays, cell death assays including cell cycle analysis, hoechst stain and measurement of caspase-3 activity were performed.ResultsA dose-dependent, mesangioproliferative effect of the chemokine SLC/CCL21, which is constitutively expressed on human podocytes was seen via activation of the chemokine receptor CCR7, which is constitutively expressed on MC. In addition, in cultured MC SLC/CCL21 had a protective effect on cell survival in Fas-mediated apoptosis. The CXCR3 ligands IP-10/CXCL10 and Mig/CXCL9 revealed a proproliferative effect but did not influence apoptosis of MC. Both the CCR1 ligand RANTES/CCL5 and the amino-terminally modified RANTES analogue Met-RANTES which blocks CCR1 signalling had no effect on proliferation and apoptosis.ConclusionsThe different effects of chemokines and their respective receptors on proliferation and apoptosis of MC suggest highly regulated, novel biological functions of chemokine/chemokine receptor pairs in processes involved in renal inflammation, regeneration and glomerular homeostasis.

Activation of CCR7 on mesangial cells by SLC/CCL21 enhances the degree and firmness of cell adhesion and increases cell spreading and the formation of cell-cell contacts. This includes integrin-linked kinase activation and F-actin rearrangements. Thus, local chemokine generation and chemokine receptor expression on mesangial cells may play an important role in the maintenance of glomerular homeostasis and in local remodeling processes.

ILK regulates podocyte cell matrix interaction, proliferation, and slit membrane gene expression in podocyte damage. As this pathway is amendable to pharmacologic intervention, further detailed studies of in vivo ILK function in glomerular disease appear justified.