Mechanisms of PKC-Dependent Na+ K+ ATPase Phosphorylation in the Rat Kidney with Chronic Renal Failure

Abstract

The present work was designed to study Na+ K+ ATPase α1-subunit phosphorylation in rats with chronic renal failure (CRF) in comparison with normal rats. Na+ K+ ATPase α1-subunit phosphorylation degree was measured by binding the McK-1 antibody to dephosphorylated Ser-23 in microdissected medullary thick ascending limb of Henle (mTAL) segments. In addition, the total Na+ K+ ATPase α1-subunit expression and activity were also measured in the outer renal medulla homogenates and membranes.CRF rats showed a higher Na+ K+ ATPase activity, as compared with control rats (18.95 ± 2.4 vs. 11.21 ± 1.5 μmol Pi/mg prot/h, p < 0.05), accompanied by a higher total Na+ K+ ATPase expression (0.54 ± 0.04 vs. 0.27 ± 0.02 normalized arbitrary units (NU), p < 0.05). When McK-1 antibody was used, a higher immunosignal in mTAL of CRF rats was observed, as compared with controls (6.3 ± 0.35 vs.4.1 ± 0.33 NU, p < 0.05). The ratio Na+ K+ ATPase α1-subunit phosphorylation / total Na+ K+ ATPase α1-subunit expression per μg protein showed a non-significant difference between CRF and control rats in microdissected mTAL segments (2.11 ± 0.12 vs.2.26 ± 0.18 NU, p = NS). The PKC inhibitor RO-318220 10−6M increased immunosignal (lower phosphorylation degree) in mTAL of CRF rats to 128.43 ± 7.08% (p < 0.05) but did not alter McK1 binding in control rats. Both phorbol 12-myristate 13-acetate (PMA) 10−6M and dopamine 10−6M decreased immunosignal in CRF rats, corresponding to a higher Na+ K+ ATPase α1-subunit phosphorylation degree at Ser-23 (55.26 ± 11.17% and 53.27 ± 7.12% compared with basal, p < 0.05). In mTAL of CRF rats, the calcineurin inhibitor FK-506 10−6M did not modify phosphorylation degree at Ser-23 of Na+ K+ ATPase α1-subunit (100.21 ± 3.00% compared with basal CRF). In control rats, FK 506 10−6M decreased the immunosignal, which corresponds to a higher Na+ K+ ATPase α1-subunit phosphorylation degree at Ser-23. The data suggest that the regulation of basal Na+ K+ ATPase α1-subunit phosphorylation degree at Ser-23 in mTAL segments of CRF rats was primarily dependent on PKC activation rather than calcineurin dependent mechanisms.