S X Wang1, P Menè, H Holthöfer. 1. Haartman Institute, Department of Bacteriology and Immunology, University of Helsinki, Helsinki, Finland.
Abstract
BACKGROUND: Mutations in the recently cloned NPHS1 gene result in congenital nephrotic syndrome of the Finnish type (CNF). The protein product of NPHS1, nephrin, is expressed uniquely in kidney glomerular podocytes, and is the first true component of the interpodocyte slit membrane. The precise functions of nephrin remain unknown, but the presence of several tyrosine residues in the intracellular domain suggest a role in signalling. We searched for nephrin expressing cell line for use in signal transduction studies and also characterized the main features of calcium signalling in nephrin-deficient cultural glomerular epithelial cells. METHODS: We used A293 cell line, found to naturally express nephrin, as well as cultured CNF glomerular cells using reverse-transcription PCR, immunocytochemistry and intracellular Ca2+ measurements. RESULTS: Phorbol-12-myristate-13-acetate significantly upregulated the nephrin expression in A293 cells, while no change was found after treatment with additional stimulants for other main signalling pathways, e.g. okadaic acid, lysophosphatidic acid, bradykinin, angiotensin II (ANG II) and arginine vasopressin (AVP). No changes in basal or ANG II- or AVP-stimulated intracellular Ca2+ fluxes in CNF glomerular cells were observed. CONCLUSIONS: Protein kinase C may be the key intracellular signalling system in the regulation of nephrin.
BACKGROUND: Mutations in the recently cloned NPHS1 gene result in congenital nephrotic syndrome of the Finnish type (CNF). The protein product of NPHS1, nephrin, is expressed uniquely in kidney glomerular podocytes, and is the first true component of the interpodocyte slit membrane. The precise functions of nephrin remain unknown, but the presence of several tyrosine residues in the intracellular domain suggest a role in signalling. We searched for nephrin expressing cell line for use in signal transduction studies and also characterized the main features of calcium signalling in nephrin-deficient cultural glomerular epithelial cells. METHODS: We used A293 cell line, found to naturally express nephrin, as well as cultured CNF glomerular cells using reverse-transcription PCR, immunocytochemistry and intracellular Ca2+ measurements. RESULTS:Phorbol-12-myristate-13-acetate significantly upregulated the nephrin expression in A293 cells, while no change was found after treatment with additional stimulants for other main signalling pathways, e.g. okadaic acid, lysophosphatidic acid, bradykinin, angiotensin II (ANG II) and arginine vasopressin (AVP). No changes in basal or ANG II- or AVP-stimulated intracellular Ca2+ fluxes in CNF glomerular cells were observed. CONCLUSIONS: Protein kinase C may be the key intracellular signalling system in the regulation of nephrin.
Authors: Mervi Ristola; Satu Arpiainen; Moin A Saleem; Peter W Mathieson; Gavin I Welsh; Sanna Lehtonen; Harry Holthöfer Journal: BMC Mol Biol Date: 2009-08-24 Impact factor: 2.946