Cyclosporine (CsA) nephrotoxicity is a severe complication in organ transplantation because it leads to impaired renal function and chronic allograft nephropathy, which is a major predictor of graft loss. Animal models and in vivo studies indicate that the transmembrane efflux pump P-glycoprotein contributes substantially to CsA nephrotoxicity. It was hypothesized that the TT genotype at the ABCB1 3435C3 T polymorphism, which is associated with decreased expression of P-glycoprotein in renal tissue, is a risk factor for developing CsA nephrotoxicity. In a case-control study, 18 of 97 patients developed CsA nephrotoxicity and showed complete recovery of renal function in all cases when switched to a calcineurin inhibitor-free regimen. Both recipients and donors were genotyped for ABCB1 polymorphisms at the positions 3435C3 T and 2677G3 T/A. For controlling for population stratification, two additional polymorphisms, CYP2D6*4 and CYP3A5*3, with intermediate allelic frequencies were studied. The P-glycoprotein low expressor genotype 3435TT only of renal organ donors but not of the recipients was overrepresented in patients with CsA nephrotoxicity as compared with patients without toxicity ( 2 ‫؍‬ 10.5; P ‫؍‬ 0.005). CsA dosage, trough levels, and the concentration per dose ratio were not different between the patient groups. In a multivariate model that included several other nongenetic covariates, only the donor's ABCB1 3435TT genotype was strongly associated with CsA nephrotoxicity (odds ratio, 13.4; 95% confidence interval, 1.2 to 148; P ‫؍‬ 0.034). A dominant role of the donor's ABCB1 genotype was identified for development of CsA nephrotoxicity. This suggests that P-glycoprotein is an important factor in CsA nephrotoxicity.

The chloride intracellular channel 5A (CLIC5A) protein, one of two isoforms produced by the CLIC5 gene, was isolated originally as part of a cytoskeletal protein complex containing ezrin from placental microvilli. Whether CLIC5A functions as a bona fide ion channel is controversial. We reported previously that a CLIC5 transcript is enriched approximately 800-fold in human renal glomeruli relative to most other tissues. Therefore, this study sought to explore CLIC5 expression and function in glomeruli. RT-PCR and Western blots show that CLIC5A is the predominant CLIC5 isoform expressed in glomeruli. Confocal immunofluorescence and immunogold electron microscopy reveal high levels of CLIC5A protein in glomerular endothelial cells and podocytes. In podocytes, CLIC5A localizes to the apical plasma membrane of foot processes, similar to the known distribution of podocalyxin and ezrin. Ezrin and podocalyxin colocalize with CLIC5A in glomeruli, and podocalyxin coimmunoprecipitates with CLIC5A from glomerular lysates. In glomeruli of jitterbug (jbg/jbg) mice, which lack the CLIC5A protein, ezrin and phospho-ERM levels in podocytes are markedly lower than in wild-type mice. Transmission electron microscopy reveals patchy broadening and effacement of podocyte foot processes as well as vacuolization of glomerular endothelial cells. These ultrastructural changes are associated with microalbuminuria at baseline and increased susceptibility to adriamycin-induced glomerular injury compared with wild-type mice. Together, the data suggest that CLIC5A is required for the development and/or maintenance of the proper glomerular endothelial cell and podocyte architecture. We postulate that the interaction between podocalyxin and subjacent filamentous actin, which requires ezrin, is compromised in podocytes of CLIC5A-deficient mice, leading to dysfunction under unfavorable genetic or environmental conditions.

TIMAP (TGF-␤1 inhibited, membrane-associated protein) is a prenylated, endothelial cell-predominant protein phosphatase 1 (PP1c) regulatory subunit that localizes to the plasma membrane of filopodia. Here, we determined whether phosphorylation regulates TIMAP-associated PP1c function. Phosphorylation of TIMAP was observed in cells metabolically labeled with [ 32 P]orthophosphate and was reduced by inhibitors of protein kinase A (PKA) and glycogen synthase kinase-3 (GSK-3). In cell-free assays, immunopurified TIMAP was phosphorylated by PKA and, after PKA priming, by GSK-3␤. Site-specific Ser to Ala substitution identified amino acid residues Ser 333 /Ser 337 as the likely PKA/GSK-3␤ phosphorylation site. Substitution of Ala for Val and Phe in the KVSF motif of TIMAP (TIMAP V64A/F66A ) abolished PP1c binding and TIMAPassociated PP1c activity. TIMAP V64A/F66A was hyper-phosphorylated in cells, indicating that TIMAP-associated PP1c auto-dephosphorylates TIMAP. Constitutively active GSK-3␤ stimulated phosphorylation of TIMAP V64A/F66A , but not wild-type TIMAP, suggesting that the PKA/GSK-3␤ site may be subject to dephosphorylation by TIMAP-associated PP1c. Substitution of Asp or Glu for Ser at amino acid residues 333 and 337 to mimic phosphorylation reduced the PP1c association with TIMAP. Conversely, GSK-3 inhibitors augmented PP1c association with TIMAP-PP1c in cells. The 333/337 phosphomimic mutations also increased TIMAP-associated PP1c activity in vitro and against the non-integrin laminin receptor 1 in cells. Finally, TIMAP mutants with reduced PP1c activity strongly stimulated endothelial cell filopodia formation, an effect mimicked by the GSK-3 inhibitor LiCl. We conclude that TIMAP is a target for PKA-primed GSK-3␤-mediated phosphorylation. This phosphorylation controls TIMAP association and activity of PP1c, in turn regulating extension of filopodia in endothelial cells.PP1c 2 belongs to the family of serine/threonine phosphatases (1) that includes PP2A, PP4-PP7, and calcineurin (PP2B).Ser/Thr phosphatases counteract essentially every signal involving Ser/Thr kinases, and like the kinases their activity is tightly regulated. Because of extreme sequence conservation (Ͼ90%) among PP1c isoforms, substrate-and location-specific actions are not inherent in PP1c itself but dictated by more than 50 PP1c regulatory (targeting) subunits (1). Among these, the myosin phosphatase-targeting (MYPT) subunits regulate muscle and non-muscle cell contraction through dephosphorylation of regulatory myosin light chains (2). Most PP1c regulatory subunits share the PP1c binding motif RVXF, but their structures are otherwise diverse, serving in each case highly specific and localized functions (1).The function of PP1c targeting subunits is regulated by changes in expression, by allosteric effects of substrates, and by phosphorylation (1). For example, phosphorylation at or near the RVXF motif of NIPP-1 reduces PP1c binding, and phosphorylation elsewhere in the molecule can increase or decrease PP1c activity (3). The activity of...

. A SAGEbased comparison between glomerular and aortic endothelial cells.

In this study we demonstrated for the first time an inhibitory effect of MPA on the stimulated IL-6 expression of renal tubular epithelial cells. In contrast to older data, which showed a synergistic upregulation of the expression of a CC-chemokine by a combination of cytokines and MPA, in the present study we could demonstrate an immunosuppressive effect of MPA on the expression of an important cytokine.