Nitric oxide increases albumin permeability of isolated rat glomeruli via a phosphorylation-dependent mechanism

Nitric oxide (NO) has been implicated in the induction of proteinuria in ac ute inflammatory glomerulonephritis and in the increased vascular permeabil ity seen in various other disease conditions. The complicated interactions of NO with other factors in vivo hinder analysis of the mechanisms involved . By use of a recently introduced method for measuring, albumin permeabilit y (P-a) in isolated glomeruli, the question of whether NO has a direct effe ct on the permeability barrier of glomerular tufts was examined and the pot ential mechanisms were explored. Exposure of isolated glomeruli to three NO donors, s-nitroso-N-acetyl-penicillamine (SNAP), (Z)-1-[-2-(aminoethyl) -N -(2-ammonioethyl) amino] diazen-1-ium-1,2-diolate (DETA-NONOate), and sodiu m nitroprusside, all increased the P-a. This action of NO was time- and con centration-dependent and could be mimicked by 8-bromoguanosine 3', 5'-cycli c monophosphate. Western blot analysis of the proteins from NO donor-treate d glomeruli revealed an increase of phosphotyrosine levels of proteins of m olecular mass about 120 and 70 kD. The demonstration that pretreatment of g lomeruli with the tyrosine kinase inhibitor, genistein, could largely preve nt the effect of SNAP and DETA-NONOate confirmed the crucial role of tyrosi ne phosphorylation in the NO-induced increase of P-a. Furthermore, the tyro sine phosphatase inhibitor, phenylarsine oxide (PAO), could mimic the actio n of NO on P-a. NO-enhanced tyrosine phosphorylation was further confirmed by immunofluorescence staining. where positive cells in SNAP- and PAO-treat ed glomeruli were much more frequent than that in controls. By use of dual- label staining in combination with podocyte specific marker, nephrin, it wa s observed that most of the phosphorylated positive cells corresponded to p odocytes. These results suggest that NO impairs the glomerular permeability barrier through a tyrosine phosphorylation-dependent mechanism.