Glomerular proteins related to slit diaphragm and matrix adhesion in the foot processes are highly tyrosine phosphorylated in the normal rat kidney.

Background: Tyrosine phosphorylation of proteins has been a focus of extensive studies since it plays crucial roles in regulation of diverse biological reactions. To understand the involvement of tyrosine phosphorylation in kidney functions, a comprehensive proteomic study for tyrosine-phosphorylated proteins was performed in the normal rat kidney.

Methods: Two-dimensional gel electrophoresis and immunoprecipitation using anti-phosphotyrosine antibodies were employed to detect tyrosine-phosphorylated proteins. The proteins were analysed by mass spectrometry and validated by immunological analyses using specific antibodies.

Results: Most of tyrosine-phosphorylated proteins were confined to the glomerulus and predominantly localized along the glomerular capillary wall, especially in the foot processes of podocytes. Our systematic proteomic analysis identified nephrin, SHPS-1 (tyrosine-protein phosphatase non-receptor-type substrate 1), FAK1 and paxillin as major tyrosine-phosphorylated proteins and Neph1, talin and vinculin as minor tyrosine-phosphorylated proteins. In the present study, SHPS-1 was identified as a novel tyrosine-phosphorylated protein in the glomerulus and was also predominantly localized at the foot processes. Mass spectrometric analysis identified in vivo phosphorylation sites of SHPS-1 on Y460, Y477 and Y501.

Conclusion: This study identified tyrosine-phosphorylated proteins in normal rat kidney, which were prominently rich in the glomerulus and localized at the podocyte foot processes. These proteins were categorized as cell-to-cell or cell-to-matrix adhesion complex-related molecules, suggesting their pivotal roles in the glomerular ultrafiltration.