Histochemical detection of ischemia-like alterations induced in kidney tissue in vitro--different sensitivity to oxidant stress of glomerular ENTPD1 versus E5NT.

The expression of ENTPD1 (ecto-nucleoside triphosphate diphosphohydrolase) along the glomerular microvasculature of the kidney is downregulated in ischemic conditions, in contrast to E5NT (ecto-5'-nucleotidase), which may explain the increased tendency for intraglomerular microthrombus formation in vivo. It has been suggested that in ischemia, reactive oxygen species (ROS) affect glomerular ENTPD1, whereas E5NT seems less sensitive to oxidant stress. To test this hypothesis, a soluble ATP and ADP hydrolyzing enzyme solution (apyrase) [0.4 U/ml] or 5'-nucleotidase solution [0.33 U/ml] as well renal tissue were exposed to ROS, generated by gamma-irradiation in vitro. The enzymes diluted in distilled water or cryostat rat kidney sections were exposed to gamma-irradiation (0.037 Gy/s) for 0, 2, 5, 10, or 15 min, with or without supplementation of the ROS scavenger dimethylsulfoxide (DMSO). The enzyme activity of the samples was biochemically tested using standard methods, before and after irradiation. The reaction product of irradiated versus nonirradiated kidney sections was semiquantitatively evaluated after histochemical staining for either glomerular ENTPD1 or glomerular E5NT expression. The results show that the enzyme activity in samples of soluble apyrase was significantly decreased after irradiation. This effect was inhibited by DMSO. In contrast, 5'-nucleotidase activity showed only a limited decline of the activity curve after irradiation, which could also be restored following supplementation of DMSO. Glomerular ENTPD1 expression showed significant decrease after irradiation of kidney sections; again, this was inhibitable by DMSO. Glomerular E5NT activity was not altered by irradiation and DMSO supplementation did not affect its activity. It is concluded that soluble apyrase as well as the glomerular ENTPD1 are sensitive to oxidant stress, which may explain their downregulation in the ischemic condition in vivo. However, soluble 5'-nucleotidase and E5NT seem much less sensitive to ROS. This relative insensitivity of E5NT to oxidant injury may counteract ischemic injury by promoting local generation of adenosine in the ischemic micro-environment.

被引量：2 发表：1970

Rho/Rho kinase pathway regulates maintenance of the differentiated tubular epithelial cell phenotype on laminin-1.

Maintenance of a polarized tubular epithelium by appropriate intracellular signaling and extracellular matrix is critical both in normal renal function as well as in acute and chronic tubular injury. We examined the hypothesis that maintenance of a differentiated epithelial phenotype on the basement membrane glycoprotein laminin-1 is controlled by the Rho/Rho kinase pathway. Using the tubular epithelial cell lines LLC-PK1 and MDCK which were cultured on laminin-1 vs. collagen IV, we analyzed cell morphology and motility (cohort migration assay) as well as expression of differentiation and dedifferentiation markers (immunofluorescence microscopy). Cohort migration of LLC-PK1 cells was significantly slowed down on laminin-1 (10.7 +/- 2.2 m.u. (migratory units)) compared with collagen IV (16.6 +/- 2.3 m.u.; BSA control: 2.8 +/- 2.5 m.u.). Inhibition of the Rho/Rho kinase pathway by C3 exotoxin (1 mug/ml) or the Rho kinase inhibitor Y27632 (10 microM) significantly augmented cohort migration on laminin-1 (14.5 +/- 1.4 and 16.0 +/- 1.8 m.u. vs. 10.7 +/- 2.2 m.u.). In parallel to the increased migratory activity, inhibition of the Rho/Rho kinase pathway resulted in a more mesenchymal phenotype of LLC-PK1 cells on laminin-1 with increased formation of lamellopodia and filopodia, distinct loss of focal contacts and stress fibers, upregulation of the dedifferentiation marker vimentin, and loss of cell-cell contacts with translocation of beta-catenin from the adherens junctions to the cytosol and nucleus. Similarly, cohort migration of MDCK cells was retarded on laminin-1 when compared with collagen IV, and addition of the Rho kinase inhibitor Y27632 resulted in enhanced motility and a change in cell morphology. The study demonstrates that the Rho/Rho kinase pathway is required to maintain a non-migratory epithelial phenotype of cultured renal tubular LLC-PK1 and MDCK cells on the basement membrane glycoprotein laminin-1.

被引量：4 发表：1970

The N- and L-type calcium channel blocker cilnidipine suppresses renal injury in dahl rats fed a high-sucrose diet, an experimental model of metabolic syndrome.

被引量：15 发表：1970

Increased urinary TGF-beta1 and cortical renal GLUT1 and GLUT2 levels: additive effects of hypertension and diabetes.

被引量：5 发表：1970

Chloride channels and endocytosis: new insights from Dent's disease and ClC-5 knockout mice.

Dent's disease is a hereditary renal tubular disorder characterized by low-molecular weight (LMW) proteinuria, hypercalciuria and nephrolithiasis. The disease is due to mutations of ClC-5, a member of the family of voltage-gated CLC chloride channels. ClC-5 is expressed in part in cells lining the proximal tubule (PT) of the kidney, where it colocalizes with albumin-containing endocytic vesicles belonging to the receptor-mediated endocytic pathway that ensures efficient reabsorption of ultrafiltrated LMW proteins. Since progression along the endocytic apparatus requires endosomal acidification, it has been suggested that dysfunction of ClC-5 in endosomes may lead to inefficient reabsorption of LMW proteins and dysfunction of PT cells. Analysis of a ClC-5 knockout (KO) mouse model, displaying all the characteristic renal tubular defects of Dent's disease, showed evidence of a severe LMW proteinuria. Cytochemical studies with the endocytic tracer, peroxidase, showed poor transfer into early endocytic vesicles, suggesting that impairment of receptor-mediated endocytosis in PT cells is the basis for the defective uptake of LMW proteins in patients with Dent's disease. Endocytosis and processing of LMW proteins involve the multiligand tandem receptors, megalin and cubilin, that are abundantly expressed at the brush border of PT cells. Characterization of the endocytic defect in ClC-5 KO mice revealed that ligands of both megalin and cubilin were affected. The total kidney content of megalin and especially cubilin at the protein level was decreased but, more importantly, using analytical subcellular fractionation and quantitative immunogold labelling we demonstrated a selective disappearance of megalin and cubilin at the brush border of PT cells. These observations allowed us to conclude that defective protein endocytosis linked to ClC-5 inactivation is due at least in part to a major and selective loss of megalin and cubilin at the brush border, reflecting a trafficking defect in renal PT cells. These results improve our understanding of Dent's disease, taken as a paradigm for renal Fanconi syndrome and nephrolithiasis, and demonstrate multiple roles for ClC-5 in the kidney. These studies also provided insights into important functions such as apical endocytosis, handling of proteins by renal tubular cells, calcium metabolism, and urinary acidification.

被引量：18 发表：2005