The Calcium‐sensing Receptor Increases Activity of the Renal Na‐Cl Cotransporter Through the WNK4‐SPAK Pathway
Authors: Bazúa‐Valenti Silvana , Rojas‐Vega Lorena , Castañeda‐Bueno María , Barrera‐Chimal Jonatan , Bautista Rocío , Vázquez Norma , Ellison David H. , Riccardi Daniela , Bobadilla Norma A. , Gamba Gerardo 
Published: June 2021

BackgroundExtracellular calcium inhibits Na‐Cl reabsorption in the thick ascending limb of Henle's loop (TALH) to promote hypercalciuria by activating the Calcium‐sensing Receptor (CaSR) in the basolateral membrane. CaSR is also expressed in the apical membrane of the distal convoluted tubule (DCT), where we hypothesized that it could play a role in activating the Na‐Cl Cotransporter (NCC) to prevent salt loss, while further permitting calcium excretion. Here we show, using in vitro and in vivo models, that activation of CaSR increases NCC phosphorylation, and concomitant activation, via the WNK4‐SPAK pathway.MethodsNCC activity (thiazide‐sensitive 22Na+ uptake) and the effect of the type I CaSR agonist, Gd3+, were analyzed in X. leavis oocytes microinjected with NCC and CaSR cRNA alone or together with wild‐type WNK1 or WNK4. Expression and phosphorylation of SPAK and WNK4 were assessed in HEK‐293 cells co‐transfected with these proteins + wild‐type or mutant CaSR harboring the activating mutation E228K (CaSR‐E228K), as well as in the presence or absence of the type II agonist, R‐568. The WNK4‐NCC pathway was also studied with western blot analysis of kidneys of wild‐type mice administered with an acute dose of R‐568 (0.3μg/g for 3 h) or furosemide (15 mg/kg). Lastly, we used an ex vivo model where we perfused rat kidneys with R‐568 (0.60 μg/ml/min) using a Langendorff system.ResultsIn oocytes, CaSR activation with Gd3+ increased NCC activity two‐fold only in the presence of WNK4. In R‐568‐stimulated HEK‐293 cells, SPAK phosphorylation increased in a dose and time‐dependent manner. Using a specific WNK4 inhibitor or the absence of WNK4 precluded this effect. CaSR‐E228K markedly protected WNK4 from Kelch3‐induced degradation, resulting in increased WNK4 abundance. Moreover, R‐568‐activated CaSR was found to promote WNK4 phosphorylation. In vivo, R‐568 administration to wild‐type mice resulted in increased NCC phosphorylation and WNK4 protein (five‐fold p<0.05). Acute furosemide administration, which increases tubular Ca2+ to the DCT, also resulted in NCC phosphorylation. Lastly, ex vivo kidney perfusion with R‐568 resulted in a clear and significant increase in SPAK and NCC phosphorylation.DiscussionOur results suggest that activation of CaSR can increase NCC activity through the WNK4‐SPAK pathway. It is possible that activation of CaSR by calcium in the apical membrane of DCT increases Na‐Cl reabsorption via NCC to prevent Na‐Cl loss and further decrease calcium reabsorption in this nephron segment.Support or Funding InformationThis work was supported by the CONACyT Grant No.23 to GG from the “Fronteras de la ciencia” and No. 188712 to GG and No. 257726 to MC‐B. And the NIDDK RO1 grant No. DK051496 to DHE and GG.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.