Mechanisms of PKC-dependent Na+K+ATPase phosphorilation in the rat kidney with chronic renal failure

CLAUDIA BERTUCCIO, ELVIRA ARRIZURIETA, FERNANDO R IBARRA, RODOLFO S MARTIN

RENAL FAILURE

Año: 2007 vol. 29 p. 13 - 22

0886-022X

The present wwk was designed to study Na+ K+ ATPase al-subunit phosphorylation in rats with chronic renal failure CRF) in comparison with normal rats. Na+ K+ ATPase al-sub- unit phosphorylation degree was measured by binding the McK-1 antibody to dephosphorylated Ser-23 in midssected medul- lary thick ascending limb of Henle (mTAL) segments. In addi- tion, the total Na+ K+ ATPase al-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 com- pared with control rats (18.95 f 2.4 vs. 11.21 f 1.5 pmol Wmg proth, p < 0.05), accompanied by a higher total Na+ K+ ATPase expression (0.54 f 0.04 vs. 0.27 f 0.02 normalized arbitrary unitsin the outer renal medulla homogenates and membranes. CRF rats showed a higher Na+ K+ ATPase activity, as com- pared with control rats (18.95 f 2.4 vs. 11.21 f 1.5 pmol Wmg proth, p < 0.05), accompanied by a higher total Na+ K+ ATPase expression (0.54 f 0.04 vs. 0.27 f 0.02 normalized arbitrary unitsf 2.4 vs. 11.21 f 1.5 pmol Wmg proth, p < 0.05), accompanied by a higher total Na+ K+ ATPase expression (0.54 f 0.04 vs. 0.27 f 0.02 normalized arbitrary unitsp < 0.05), accompanied by a higher total Na+ K+ ATPase expression (0.54 f 0.04 vs. 0.27 f 0.02 normalized arbitrary unitsf 0.04 vs. 0.27 f 0.02 normalized arbitrary units m], p < 0.05). When McK-1 antibody was sed, a higher immunosignal in mTAL of CRF rats was o b r v d , as c o m mp < 0.05). When McK-1 antibody was sed, a higher immunosignal in mTAL of CRF rats was o b r v d , as c o m min mTAL of CRF rats was o b r v d , as c o m m with controls (6.3 f0.35 vs.4.1 f 0.33 NU, p < 0.05). The ratio Na+ K+ ATPase al-subunit phosphorylation 1 total Na+ IC+f 0.33 NU, p < 0.05). The ratio Na+ K+ ATPase al-subunit phosphorylation 1 total Na+ IC+1 total Na+ IC+ ATPase al-subunit expression per ~g pmtein showed a non- significant difference between CRF and control rats in rniaodis- sected mTAL segments (2.1 1 f 0.12 vs.2.26 f 0.18 NU,p = NS). The PKC inhibitor RO-3 18220 10% increased immunosignal lower phosphorylation degree) in mTAL of CRF mts to 128.43 ff 0.12 vs.2.26 f 0.18 NU,p = NS). The PKC inhibitor RO-3 18220 10% increased immunosignal lower phosphorylation degree) in mTAL of CRF mts to 128.43 fCRF mts to 128.43 f 7.08% ( p < 0.05) but did not alter McKl binding in control rats. Both phorbol 12-myristate 13-acetate (PMA) 10% and doparn- ine I O ~ M decreased immunosignal in CRF rats, corresponding to a higher Na+ K+ ATPase al-subunit phosphorylation degree at Ser-23 (55.26 f 11.17% and 53.27 f 7.12% compared with basal,( p < 0.05) but did not alter McKl binding in control rats. Both phorbol 12-myristate 13-acetate (PMA) 10% and doparn- ine I O ~ M decreased immunosignal in CRF rats, corresponding to a higher Na+ K+ ATPase al-subunit phosphorylation degree at Ser-23 (55.26 f 11.17% and 53.27 f 7.12% compared with basal,(PMA) 10% and doparn- ine I O ~ M decreased immunosignal in CRF rats, corresponding to a higher Na+ K+ ATPase al-subunit phosphorylation degree at Ser-23 (55.26 f 11.17% and 53.27 f 7.12% compared with basal,CRF rats, corresponding to a higher Na+ K+ ATPase al-subunit phosphorylation degree at Ser-23 (55.26 f 11.17% and 53.27 f 7.12% compared with basal,f 11.17% and 53.27 f 7.12% compared with basal, < 0.05). In mTAL of CRF rats, the calcinemin inhibitor FK-0.05). In mTAL of CRF rats, the calcinemin inhibitor FK- 506 10% did not modify phosphorylation degree at Ser-23 of basal CRF). in control rats, FK 506 10% decreased the irnmun- osignal, which corresponds to a higher Na+ K+ ATPase al-sub- unit phosphorylation degree at Ser-23. The data suggest that the regulation of basal Na+ K+ ATPase al-subunit phosphorylation degree at Ser-23 in mTAL segments of CRF rats was primarily dependent on PKC activation rather than calcineurin dependent mechanisms.FK 506 10% decreased the irnmun- osignal, which corresponds to a higher Na+ K+ ATPase al-sub- unit phosphorylation degree at Ser-23. The data suggest that the regulation of basal Na+ K+ ATPase al-subunit phosphorylation degree at Ser-23 in mTAL segments of CRF rats was primarily dependent on PKC activation rather than calcineurin dependent mechanisms.CRF rats was primarily dependent on PKC activation rather than calcineurin dependent mechanisms. Keywords Na+ K+ ATPase phosphorylation, Na+ K+ ATPase al-subunit PKC site, McK-1 antibody, mTAL, CRF ratsNa+ K+ ATPase phosphorylation, Na+ K+ ATPase al-subunit PKC site, McK-1 antibody, mTAL, CRF rats Sodium homeostasis in chronic renal failure (CRF) is maintained until late stages of the disease by mechanisrns that have only been partially characterized. Thus, when total renal mass is reduced, single nephron glomerular fil- tration rate in the remaining nephrons augments[ll and is paralIeled by an increase in fractional sodium excretion FE,^).[',^] Several authors have found a reduction in prox- imal tubule fractional reabsorption of sodium in CRF,[~-~'Several authors have found a reduction in prox- imal tubule fractional reabsorption of sodium in CRF,[~-~'CRF,[~-~' which results in an increased delivery to both loop seg- ments and distal tu bu le^.[^'^] The increaseú sodiurn deliv- ery to the loop of Henle is associated with a compensatory inmment in sodium reabsorption in this segment, though