Localization of Tyrosine-Phosphorylated Proteins in the Normal Rat Kidney

Protein tyrosine phosphorylation is responsible for activating or repressing the activity of proteins involved in numerous signal transduction pathways in a living organism. Using immunochemical methods with two well characterized monoclonal anti-phosphotyrosine antibodies, we examined the distribution and localization of tyrosinephosphorylated proteins expressed in the three major compartments of the normal rat kidney: the glomerulus, cortex, and medulla. Western blotting analysis revealed that tyrosine-phosphorylated proteins were predominantly expressed in the glomerulus, compared with the cortex and medulla. The intensity of tyrosine-phosphorylated protein bands was augmented by using orthovanadate - an inhibitor of protein tyrosine phosphatase - during sample preparation, and the bands were undetectable when the antibodies were absorbed with phosphotyrosine but not with phosphoserine or phosphothreonine. Immunofluorescence microscopy indicated that the phosphotyrosine staining was intense along glomerular capillary walls and sparse along parts of the tubuli. Immunoelectron microscopy further showed that phosphotyrosine immunoreactivity was predominantly localized at the basal membranes of podocyte foot processes. In addition, some immunogold labeling was also observed at cell-matrix attachment sites of glomerular endothelial cells and mesangial cells as well as at basal membranes of epithelial cells of proximal tubules, distal tubules, and collecting ducts. In conclusion, tyrosine-phosphorylated proteins are predominantly and constitutively expressed in the normal rat glomerulus, especially in the basal membranes of podocyte foot processes, suggesting tyrosinephosphorylated proteins could play an important role in maintaining the unique organization of foot processes and in the glomerular ultra filtration there.