Abstract
Renal excretion of citrate, an inhibitor of calcium stone formation, is controlled mainly by reabsorption via the apical Na(+)-dicarboxylate cotransporter NaDC1 (SLC13A2) in the proximal tubule. Recently, it has been shown that the protein phosphatase calcineurin inhibitors cyclosporin A (CsA) and FK-506 induce hypocitraturia, a risk factor for nephrolithiasis in kidney transplant patients, but apparently through urine acidification. This suggests that these agents up-regulate NaDC1 activity. Using the Xenopus lævis oocyte and HEK293 cell expression systems, we examined first the effect of both anti-calcineurins on NaDC1 activity and expression. While FK-506 had no effect, CsA reduced NaDC1-mediated citrate transport by lowering heterologous carrier expression (as well as endogenous carrier expression in HEK293 cells), indicating that calcineurin is not involved. Given that CsA also binds specifically to cyclophilins, we determined next whether such proteins could account for the observed changes by examining the effect of selected cyclophilin wild types and mutants on NaDC1 activity and cyclophilin-specific siRNA. Interestingly, our data show that the cyclophilin isoform B is likely responsible for down-regulation of carrier expression by CsA and that it does so via its chaperone activity on NaDC1 (by direct interaction) rather than its rotamase activity. We have thus identified for the first time a regulatory partner for NaDC1, and have gained novel mechanistic insight into the effect of CsA on renal citrate transport and kidney stone disease, as well as into the regulation of membrane transporters in general.
Highlights
The availability of citrate2Ϫ increases as pH lowers, due to protonation of citrate3Ϫ, thereby increasing the activity of NaDC1
Recent observations with cyclosporin A (CsA) and FK-506, which exert their normal effects by indirectly inhibiting the protein phosphatase calcineurin, suggested to us that the CsA-sensitive cyclophilins might be implicated in NaDC1 regulation
Because CsA is toxic at high doses, we examined the viability of NaDC1-expressing oocytes following 72 h CsA treatment
Summary
CDNA Constructs—Nine different cDNA constructs were used in this study. The vectors Pol and pNWP were used for protein expression in X. laevis oocytes, whereas the vector pcDNA3.1 (ϩ) (Invitrogen) was used for protein expression in HEK293 cells. The huNaDC1 FLAG N-ter/Pol construct was generated by the N-terminal insertion of a prehybridized complementary oligonucleotide fragment possessing cohesive compatible ends and encoding the FLAG tag (supplemental Table S1), into a digested wt huNaDC1/Pol fragment. The mutant huCypB⌬C-ter(204 –208) was generated by the insertion of a prehybridized complementary oligonucleotide fragment, which possesses cohesive compatible ends and in which the 15 nucleotides encoding the C-terminal ER retention sequence AIAKE (corresponding to the amino acids 204 to 208) have been deleted (supplemental Table S1), into a digested wt huCypB fragment. Various Treatments in Oocytes and HEK293 Cells—In this study, “24 h” and “72 h” are used to mean that the oocytes were treated with CsA and FK-506 (Sigma), and salubrinal (Tocris Bioscience) for the last 24 h of the 72 h incubation or for the entire time, respectively. Counts from 5 to 12 oocytes, assuming equal membrane surface area of stage V-VI oocytes, or counts from one well of HEK293 cells, for which the protein concentration was determined, were averaged, transposed into influx rate, normalized to a control, and expressed as % Ϯ S.E. in Equation 1
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