Acute calcineurin inhibition with tacrolimus increases phosphorylated UT-A1.
UT-A1, the urea transporter present in the apical membrane of the inner medullary collecting duct, is crucial to the kidney's ability to concentrate urine. Phosphorylation of UT-A1 on serines 486 and 499 is important for plasma membrane trafficking. The effect of calcineurin on dephosphorylation of UT-A1 was investigated. Inner medullary collecting ducts from Sprague-Dawley rats were metabolically labeled and treated with tacrolimus to inhibit calcineurin or calyculin to inhibit protein phosphatases 1 and 2A. UT-A1 was immunoprecipitated, electrophoresed, blotted, and total UT-A1 phosphorylation was assessed by autoradiography. Total UT-A1 was determined by Western blotting. A phospho-specific antibody to pser486-UT-A1 was used to determine whether serine 486 can be hyperphosphorylated by inhibiting phosphatases. Inhibition of calcineurin showed an increase in phosphorylation per unit protein at serine 486. In contrast, inhibition of phosphatases 1 and 2A resulted in an increase in UT-A1 phosphorylation but no increase in pser486-UT-A1. In vitro perfusion of inner medullary collecting ducts showed tacrolimus-stimulated urea permeability consistent with stimulated urea transport. The location of phosphorylated UT-A1 in rats treated acutely and chronically with tacrolimus was determined using immunohistochemistry. Inner medullary collecting ducts of the acutely treated rats showed increased apical membrane association of phosphorylated UT-A1 while chronic treatment reduced membrane association of phosphorylated UT-A1. We conclude that UT-A1 may be dephosphorylated by multiple phosphatases and that the PKA-phosphorylated serine 486 is dephosphorylated by calcineurin. This is the first documentation of the role of phosphatases and the specific site of phosphorylation of UT-A1, in response to tacrolimus.
Renal Division, Dept. of Medicine, Emory University, 1639 Pierce Dr., Atlanta, GA 30322, USA. tilori@emory.edu
Titilayo O
TO
Ilori
Yanhua
Y
Wang
Mitsi A
MA
Blount
Christopher F
CF
Martin
Jeff M
JM
Sands
Janet D
JD
Klein
D000818:Animals; D065095:Calcineurin Inhibitors; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D007685:Kidney Tubules, Collecting / Q000187:drug effects* / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000032:analysis / Q000378:metabolism*; D010080:Oxazoles / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D012694:Serine / Q000378:metabolism; D016559:Tacrolimus / Q000494:pharmacology*; D000096982:Urea Transporters
D065095:Calcineurin Inhibitors; D004791:Enzyme Inhibitors; D007166:Immunosuppressive Agents; D026901:Membrane Transport Proteins; D010080:Oxazoles; D010749:Phosphoprotein Phosphatases; D012694:Serine; D016559:Tacrolimus; D000096982:Urea Transporters