CYCLOSPORINE TREATMENT ALTERS PROTEIN-PHOSPHORYLATION IN KIDNEY MEMBRANES

Phosphorylation, protein carboxyl methylation, and ADP-ribosylation we re assayed in renal basolateral membranes and brush border membranes i solated from rats treated by subcutaneous administration of 5 or 10 mg /(kg day) of cyclosporin A (CsA) for 10 days to investigate potential alterations in signal transduction in kidney cortex. Protein carboxyl methylation of class II measured in membranes and in cytosolic fractio n was not affected by CsA treatment. ADP-ribosylation performed in the presence of pertussis or cholera toxin was also similar in control an d treated rats. However, changes in phosphorylation of endogenous subs trates were observed in membranes and cytosol isolated from rats treat ed with 10 mg/(kg day) of CsA. Phosphorylation was increased for two b rush border membrane proteins (56 and 77 kilodaltons (kDa)) by 47 and 24% and for two basolateral membrane proteins (51 and 80 kDa) by 28 an d 29%, respectively. In the cytosolic fraction, phosphorylation of two proteins (31 and 65 kDa) was increased by 37% and that of 25- and 43- kDa proteins was reduced by 29%. Protein kinase A, protein kinase C, a nd tyrosine protein kinase activities were also determined in membrane s. Increases in protein kinase C and tyrosine protein kinase activitie s were observed in basolateral membranes, but not in brush border memb ranes after cyclosporin A administration. Endogenous substrates for ty rosine kinase were also detected with an antiphosphotyrosine (PY20) mo noclonal antibody. Densitometric analysis indicated that tile phosphor ylation of three proteins of high molecular masses (61, 132, and 183 k Da) was stimulated by CsA in basolateral membranes. Immunodetection of calcineurin with a monoclonal antibody directed against the beta-subu nit was similar in control and treated rats. These results suggest tha t cyclosporin A treatment may bring about changes in covalent modifica tions as an adaptative response in signal transduction in the kidney p roximal tubule.