High dietary potassium causes ubiquitin-dependent degradation of the kidney sodium-chloride cotransporter

Abstract

The thiazide-sensitive sodium-chloride cotransporter (NCC) in the renal distal convoluted tubule (DCT) plays a critical role in regulating blood pressure (BP) and K+ homeostasis. During hyperkalemia, reduced NCC phosphorylation and total NCC abundance facilitate downstream electrogenic K+ secretion and BP reduction. However, the mechanism for the K+-dependent reduction in total NCC levels is unknown. Here, we show that NCC levels were reduced in ex vivo renal tubules incubated in a high-K+ medium for 24–48 h. This reduction was independent of NCC transcription, but was prevented using inhibitors of the proteasome (MG132) or lysosome (chloroquine). Ex vivo, high K+ increased NCC ubiquitylation, but inhibition of the ubiquitin conjugation pathway prevented the high K+-mediated reduction in NCC protein. In tubules incubated in high K+ media ex vivo or in the renal cortex of mice fed a high K+ diet for 4 days, the abundance and phosphorylation of heat shock protein 70 (Hsp70), a key regulator of ubiquitin-dependent protein degradation and protein folding, were decreased. Conversely, in similar samples the expression of PP1α, known to dephosphorylate Hsp70, was also increased. NCC coimmunoprecipitated with Hsp70 and PP1α, and inhibiting their actions prevented the high K+-mediated reduction in total NCC levels. In conclusion, we show that hyperkalemia drives NCC ubiquitylation and degradation via a PP1α-dependent process facilitated by Hsp70. This mechanism facilitates K+-dependent reductions in NCC to protect plasma K+ homeostasis and potentially reduces BP. The thiazide-sensitive sodium-chloride cotransporter (NCC) in the renal distal convoluted tubule (DCT) plays a critical role in regulating blood pressure (BP) and K+ homeostasis. During hyperkalemia, reduced NCC phosphorylation and total NCC abundance facilitate downstream electrogenic K+ secretion and BP reduction. However, the mechanism for the K+-dependent reduction in total NCC levels is unknown. Here, we show that NCC levels were reduced in ex vivo renal tubules incubated in a high-K+ medium for 24–48 h. This reduction was independent of NCC transcription, but was prevented using inhibitors of the proteasome (MG132) or lysosome (chloroquine). Ex vivo, high K+ increased NCC ubiquitylation, but inhibition of the ubiquitin conjugation pathway prevented the high K+-mediated reduction in NCC protein. In tubules incubated in high K+ media ex vivo or in the renal cortex of mice fed a high K+ diet for 4 days, the abundance and phosphorylation of heat shock protein 70 (Hsp70), a key regulator of ubiquitin-dependent protein degradation and protein folding, were decreased. Conversely, in similar samples the expression of PP1α, known to dephosphorylate Hsp70, was also increased. NCC coimmunoprecipitated with Hsp70 and PP1α, and inhibiting their actions prevented the high K+-mediated reduction in total NCC levels. In conclusion, we show that hyperkalemia drives NCC ubiquitylation and degradation via a PP1α-dependent process facilitated by Hsp70. This mechanism facilitates K+-dependent reductions in NCC to protect plasma K+ homeostasis and potentially reduces BP. Hypertension is a worldwide public-health challenge because of its high frequency and concomitant risks of cardiovascular or kidney disease (1Kearney P.M. Whelton M. Reynolds K. Whelton P.K. He J. Worldwide prevalence of hypertension: A systematic review.J. Hypertens. 2004; 22: 11-19Crossref PubMed Scopus (835) Google Scholar). The kidney's ability to adjust NaCl excretion plays a critical role in blood pressure (BP) control (2Guyton A.C. Blood pressure control--special role of the kidneys and body fluids.Science. 1991; 252: 1813-1816Crossref PubMed Scopus (712) Google Scholar). The thiazide-sensitive sodium-chloride cotransporter (NCC), expressed in the distal convoluted tubule (DCT), is essential for BP control. This is highlighted by loss-of-function NCC mutations underlying hypotensive Gitelman's syndrome or activation of NCC in hypertensive pseudohypoaldosteronism type II (PHAII or Gordon syndrome) (3Simon D.B. Nelson-Williams C. Bia M.J. Ellison D. Karet F.E. Molina A.M. Vaara I. Iwata F. Cushner H.M. Koolen M. Gainza F.J. Gitleman H.J. Lifton R.P. Gitelman's variant of Bartter's syndrome, inherited hypokalaemic alkalosis, is caused by mutations in the thiazide-sensitive Na-Cl cotransporter.Nat. Genet. 1996; 12: 24-30Crossref PubMed Scopus (989) Google Scholar, 4Wilson F.H. Disse-Nicodeme S. Choate K.A. Ishikawa K. Nelson-Williams C. Desitter I. Gunel M. Milford D.V. Lipkin G.W. Achard J.M. Feely M.P. Dussol B. Berland Y. Unwin R.J. Mayan H. et al.Human hypertension caused by mutations in WNK kinases.Science. 2001; 293: 1107-1112Crossref PubMed Scopus (1146) Google Scholar, 5Yang C.L. Angell J. Mitchell R. Ellison D.H. WNK kinases regulate thiazide-sensitive Na-Cl cotransport.J. Clin. Invest. 2003; 111: 1039-1045Crossref PubMed Scopus (378) Google Scholar). Dietary K+ intake inversely associates with BP, with low dietary K+ intake increasing the risk of death and cardiovascular events, and a high dietary K+ intake associated with lower BP (6Mente A. O'Donnell M.J. Rangarajan S. McQueen M.J. Poirier P. Wielgosz A. Morrison H. Li W. Wang X. Di C. Mony P. Devanath A. Rosengren A. Oguz A. Zatonska K. et al.Association of urinary sodium and potassium excretion with blood pressure.N. Engl. J. Med. 2014; 371: 601-611Crossref PubMed Scopus (493) Google Scholar, 7O'Donnell M. Mente A. Rangarajan S. McQueen M.J. Wang X. Liu L. Yan H. Lee S.F. Mony P. Devanath A. Rosengren A. Lopez-Jaramillo P. Diaz R. Avezum A. Lanas F. et al.Urinary sodium and potassium excretion, mortality, and cardiovascular events.N. Engl. J. Med. 2014; 371: 612-623Crossref PubMed Scopus (556) Google Scholar, 8Akita S. Sacks F.M. Svetkey L.P. Conlin P.R. Kimura G. DASH-Sodium Trial Collaborative Research GroupEffects of the dietary approaches to stop hypertension (DASH) diet on the pressure-natriuresis relationship.Hypertension. 2003; 42: 8-13Crossref PubMed Scopus (82) Google Scholar). The effects of K+ on BP are blunted in NCC knockout mice, highlighting that NCC plays an essential role in the antihypertensive effects of dietary K+ (9Terker A.S. Zhang C. McCormick J.A. Lazelle R.A. Zhang C. Meermeier N.P. Siler D.A. Park H.J. Fu Y. Cohen D.M. Weinstein A.M. Wang W.H. Yang C.L. Ellison D.H. Potassium modulates electrolyte balance and blood pressure through effects on distal cell voltage and chloride.Cell Metab. 2015; 21: 39-50Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). Patients with Gitelman's syndrome suffer from hypokalemia, with suffer from hyperkalemia, that NCC is also essential for K+ homeostasis (3Simon D.B. Nelson-Williams C. Bia M.J. Ellison D. Karet F.E. Molina A.M. Vaara I. Iwata F. Cushner H.M. Koolen M. Gainza F.J. Gitleman H.J. Lifton R.P. Gitelman's variant of Bartter's syndrome, inherited hypokalaemic alkalosis, is caused by mutations in the thiazide-sensitive Na-Cl cotransporter.Nat. Genet. 1996; 12: 24-30Crossref PubMed Scopus (989) Google Scholar, 4Wilson F.H. Disse-Nicodeme S. Choate K.A. Ishikawa K. Nelson-Williams C. Desitter I. Gunel M. Milford D.V. Lipkin G.W. Achard J.M. Feely M.P. Dussol B. Berland Y. Unwin R.J. Mayan H. et al.Human hypertension caused by mutations in WNK kinases.Science. 2001; 293: 1107-1112Crossref PubMed Scopus (1146) Google Scholar). During hyperkalemia, a reduction in NCC phosphorylation and total NCC abundance to downstream of the renal tubule to facilitate electrogenic K+ secretion and plasma K+ levels. reductions in total and NCC levels are with the ability of a high K+ diet to lower BP. NCC phosphorylation high dietary K+ intake by in the plasma via the potassium of and and of the pathway R. He H. by inhibition of 2014; PubMed Scopus Google Scholar, S. M. L. X. A. C. Ellison D.H. J. J. G. The of on the cotransporter is by 2015; PubMed Scopus Google Scholar, Wang A.S. D.H. McCormick J.A. Yang C.L. Ellison D.H. Wang W.H. Potassium by renal distal tubules PubMed Scopus Google Scholar, P. Zhang Wang W.H. D.H. of renal ability to potassium increased dietary potassium PubMed Scopus Google Scholar). However, a mechanism for a reduction in total NCC high dietary K+ the of was to the mechanism of high K+ total NCC that high K+ ubiquitin-dependent NCC degradation in a mechanism facilitated by protein effects on heat shock protein This mechanism K+ secretion hyperkalemia and is a for high dietary K+ BP. renal NCC expression and its total renal NCC expression and phosphorylation are lower a high-K+ levels are increased J. F. D. S. and phosphorylation of the cotransporter NCC in vivo is by dietary and J. PubMed Scopus Google Scholar, G. of dietary on expression of renal and J. PubMed Scopus Google Scholar, S. R. Mitchell C. The thiazide-sensitive Na-Cl cotransporter is an S. A. PubMed Scopus Google Scholar). is that reduction in NCC is by an in plasma K+ to the high-K+ diet (9Terker A.S. Zhang C. McCormick J.A. Lazelle R.A. Zhang C. Meermeier N.P. Siler D.A. Park H.J. Fu Y. Cohen D.M. Weinstein A.M. Wang W.H. Yang C.L. Ellison D.H. Potassium modulates electrolyte balance and blood pressure through effects on distal cell voltage and chloride.Cell Metab. 2015; 21: 39-50Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). the ability of in K+ to NCC abundance of we an ex vivo that renal tubules from of in of for or was to balance and K+ that to control NCC levels were in K+ and in K+ media A and with NaCl of similar A K+ the effects on NCC the with NCC levels increased in K+ lower but in K+ the of K+ is by the similar tubule were using or and the expression of NCC that the effects of K+ in are independent of the that the effects of high K+ to NCC abundance are to cell death of renal tubules or effects of the high the of the renal tubule was in cell or was in the tubules the to incubated in control or high K+ medium and and plasma K+ the of from the the effects of high K+ on NCC expression were with tubules were incubated in high K+ medium with or for h. high K+ independent of the of In abundance increased with or high K+ K+ to from B. P. Wang W.H. D. Potassium through to regulate its Scholar). abundance is by K+ in the were incubated in low or high K+ medium for or h. to control low K+ medium the protein expression of high K+ medium were in NCC expression of tubules for in low or high K+ that the of K+ to NCC levels was independent of in NCC and to NCC a the effects of high K+ on NCC were tubules were incubated for with the or the and with also the degradation of via K.A. of are to ubiquitin by proteasome Google in of high NCC degradation via the or The ubiquitylation plays an role in and protein and NCC F. L. G. R.A. The sodium NCC is by PubMed Scopus Google Scholar). a role of K+ to protein ubiquitylation, tubules incubated for or in K+ media protein ubiquitylation, tubules incubated in K+ increased ubiquitylation levels K+ the of tubules incubated in high K+ medium with or to NCC degradation were using a ubiquitin and NCC and ubiquitin levels were In with a role of K+ to protein ubiquitylation, high K+ to a in the total of high K+ in the of increased the of with A and K+ the of NCC A and NCC is However, in the of to NCC high K+ increased the abundance of NCC with A and using an NCC similar increased ubiquitylation to the high K+-mediated reduction in total NCC tubules were incubated for in control or high K+ media with a of the Y. J. P. Li J.A. A.M. of a of PubMed Scopus Google Scholar). In and prevented the in NCC with high with the increasing NCC levels control and high K+ that high K+ reduces total NCC levels via an in K+ NCC renal tubules were incubated in control or high K+ medium with or of the were to using a ubiquitin samples and samples were for levels of NCC and and of NCC and ubiquitin to control are tubules were incubated in control or high K+ medium with or a of the and NCC protein levels were are are were using a by a The balance ubiquitin-dependent degradation and protein is by the heat shock protein 70 and A for degradation by the 2015; PubMed Scopus Google Scholar). In the abundance of and heat shock was increased in the a high dietary K+ intake L. R.A. in vivo protein of the high dietary or low dietary J. PubMed Scopus Google an role in NCC levels a high K+ The role of Hsp70 and on their with the of protein by and the protein are to the degradation of the protein P. P. R. B. phosphorylation of Hsp70 and to and to protein PubMed Scopus Google Scholar). in cell that NCC with the Hsp70, and heat shock 70 with and and that a NCC ubiquitylation and degradation A.C. A. S. Hsp70 and regulate thiazide-sensitive cotransporter degradation and Full Text Full Text PDF PubMed Scopus Google Scholar, K. P. S. A.C. The thiazide-sensitive NaCl cotransporter is for Full Text Full Text PDF PubMed Scopus Google Scholar). In in from tubule in control media Hsp70 and coimmunoprecipitated a heat shock are in K+-mediated NCC tubules were incubated for with a of In reduced with increased NCC expression In the of the ability of high K+ to NCC abundance was also the effects of were that are in the of NCC and an essential role in high NCC in tubules incubated in of K+ for 24–48 levels increased in with increasing K+ levels A and in are in on transcription, expression increased with increasing K+ levels shock protein expression and phosphorylation are in to high tubules were incubated in low K+ control or high K+ medium and levels or h. The samples were for for the for the are the of to control. with control media were using a by a similar levels using are and expressed to control with control. were using a by a tubules were incubated in control or high K+ medium for or h. were and to for or for NCC and show to control and are are were using an with in ubiquitin-dependent degradation on their with and to degradation of the protein P. P. R. B. phosphorylation of Hsp70 and to and to protein PubMed Scopus Google Scholar). In tubules incubated in high K+ media for and was with tubules in control media A reduction in NCC abundance was that the reduction in the high K+-mediated NCC and dephosphorylate of the Hsp70 P. P. R. B. phosphorylation of Hsp70 and to and to protein PubMed Scopus Google Scholar, R. R. J. to of a of and protein PubMed Google Scholar, G. Liu R. A for protein for protein in cell control and PubMed Scopus Google Scholar). dephosphorylate to of NCC with and for to of NCC via the protein K. Yang C.L. M. D. R.A. Ellison D.H. J. reduces phosphorylation of renal NaCl cotransporter and 2014; PubMed Scopus Google Scholar, D. S. A. J. F. R.A. D. J. the of the renal NaCl PubMed Scopus Google and the abundance of the of the in the of mice fed a high K+ diet for 4 L. R.A. in vivo protein of the high dietary or low dietary J. PubMed Scopus Google Scholar). However, a role of for NCC abundance K+ a on tubules were in of expression increased high K+ and low K+ in K+ on levels of the or In with the in protein levels were lower 24–48 in K+ and increased in K+ to control media and a of was with NCC with NaCl of similar levels the high K+-mediated in In from tubule in control NCC and also coimmunoprecipitated a The of NCC with and was by tubules in high K+ or control medium and using an for total In NCC expression in with a of The coimmunoprecipitated samples show that the with NCC is by in high K+ that is independent of K+ a role of for the effects of high K+ on tubules were incubated for in control or high K+ media with or the The in total NCC in high K+ medium was by and that is in the effects of K+ on In with the of the A increased total NCC expression and the in total NCC in high K+ medium A role for in the effects of high K+ on NCC W. F. Y. S. inhibitors sodium-chloride cotransporter in to high potassium Full Text Full Text PDF PubMed Scopus Google Scholar). However, inhibiting with or the in NCC of high high and A S. M. H. H. K. is a and of protein type 2001; PubMed Scopus Google Scholar). However, the A. an with protein and is for PubMed Scopus Google total NCC levels to control or the in NCC with high K+ increased a known S. J.M. protein to dephosphorylate 2014; Full Text Full Text PDF PubMed Scopus Google inhibition of that increased high K+ plays a critical role in NCC levels. to of NCC via the K. Yang C.L. M. D. R.A. Ellison D.H. J. reduces phosphorylation of renal NaCl cotransporter and 2014; PubMed Scopus Google Scholar, D. S. A. J. F. R.A. D. J. the of the renal NaCl PubMed Scopus Google Scholar). of tubules for in K+ expression with control high K+ This of low K+ on was but levels were also increased by high K+ The increased expression with high K+ the increased in tubules in vivo, mice were fed a high K+ or control diet for 4 of the mice high dietary K+ intake increased plasma levels and urinary excretion of K+ In kidney cortex samples from mice, expression and of the and S. Mitchell C. of and in Clin. Invest. PubMed Scopus Google Scholar, G. of and of in the J. PubMed Scopus Google were increased by increased dietary NCC and levels were levels were increased in the renal cortex of the high mice In levels were was in the renal cortex of mice high dietary K+ intake was in the expression of in vivo in ex vivo A high dietary K+ intake is associated with lower BP and a reduction in cardiovascular disease (6Mente A. O'Donnell M.J. Rangarajan S. McQueen M.J. Poirier P. Wielgosz A. Morrison H. Li W. Wang X. Di C. Mony P. Devanath A. Rosengren A. Oguz A. Zatonska K. et al.Association of urinary sodium and potassium excretion with blood pressure.N. Engl. J. Med. 2014; 371: 601-611Crossref PubMed Scopus (493) Google Scholar, 7O'Donnell M. Mente A. Rangarajan S. McQueen M.J. Wang X. Liu L. Yan H. Lee S.F. Mony P. Devanath A. Rosengren A. Lopez-Jaramillo P. Diaz R. Avezum A. Lanas F. et al.Urinary sodium and potassium excretion, mortality, and cardiovascular events.N. Engl. J. Med. 2014; 371: 612-623Crossref PubMed Scopus (556) Google Scholar, 8Akita S. Sacks F.M. Svetkey L.P. Conlin P.R. Kimura G. DASH-Sodium Trial Collaborative Research GroupEffects of the dietary approaches to stop hypertension (DASH) diet on the pressure-natriuresis relationship.Hypertension. 2003; 42: 8-13Crossref PubMed Scopus (82) Google Scholar). The actions of NCC are to a role in the renal to dietary K+ with increased NCC abundance low K+ but lower NCC levels high K+ intake J. F. D. S. and phosphorylation of the cotransporter NCC in vivo is by dietary and J. PubMed Scopus Google Scholar, G. of dietary on expression of renal and J. PubMed Scopus Google Scholar, M. R.A. of the renal NaCl cotransporter and its role in potassium PubMed Scopus Google Scholar). The mechanism of dietary K+-dependent NCC is to the mechanism for total NCC levels high dietary K+ A is that high K+ NCC transcription, but ubiquitin-dependent degradation to NCC via is critical for A in protein ubiquitylation in ex vivo tubule incubated in high The mechanism for is but the of was associated with cell cell or in in NCC also F. L. G. R.A. The sodium NCC is by PubMed Scopus Google plays an role in NCC degradation by the proteasome via degradation and by the lysosome A.C. A. S. Hsp70 and regulate thiazide-sensitive cotransporter degradation and Full Text Full Text PDF PubMed Scopus Google Scholar, Liu J. Ellison D.H. the thiazide-sensitive NaCl cotransporter to the lysosome and Full Text Full Text PDF PubMed Scopus Google Scholar). a mechanism by high K+ ubiquitin-dependent degradation of high K+ increased NCC ubiquitylation inhibition of the or the but NCC levels were decreased. The of NCC degradation was by the of high K+ effects on NCC the was is the mechanism for the high ubiquitylation and degradation of is to we that the pathway to the effects of increased of on the of heat shock protein 70 The protein Hsp70 plays an essential role in protein and ubiquitin-dependent protein degradation A for degradation by the 2015; PubMed Scopus Google Scholar). of the the expressed and Hsp70, by the of with the ubiquitylation and degradation of P. P. R. B. phosphorylation of Hsp70 and to and to protein PubMed Scopus Google Scholar, R. R. J. to of a of and protein PubMed Google Scholar, G. Liu R. A for protein for protein in cell control and PubMed Scopus Google Scholar). This process is for degradation of via and for control are and for degradation P.M. control the plasma PubMed Scopus Google Scholar). levels were in the renal cortex of mice a high K+ and levels were increased. in PP1α, and levels in tubules by increasing K+ levels. facilitate a in balance the degradation of mechanism PP1α, and were to a ex vivo, and that a NCC ubiquitylation and degradation A.C. A. S. Hsp70 and regulate thiazide-sensitive cotransporter degradation and Full Text Full Text PDF PubMed Scopus Google Scholar, K. P. S. A.C. The thiazide-sensitive NaCl cotransporter is for Full Text Full Text PDF PubMed Scopus Google Scholar). in tubule the in levels the reduction in total NCC that is a for effects on and inhibition of or prevented the high degradation of high K+ or or activation of in vivo the effects of high K+ is unknown. The role of and ubiquitin D. C. L. D. P. R. Yang B. G. modulates renal cotransporter via the 22: PubMed Scopus Google in the to high K+ is the of but that dietary NCC abundance via and that the of dietary K+ on NCC A. A.S. D.H. McCormick J.A. NCC through and its activation by J. PubMed Scopus Google Scholar). is the K+ the in the and of or inhibition of the A. Wang W.H. Yang C.L. Ellison D.H. convoluted tubule is via and J. PubMed Scopus Google Scholar). in the K+ are to the plasma via in levels and of the protein and the pathway and NCC phosphorylation R. He H. by inhibition of 2014; PubMed Scopus Google Scholar, S. M. L. X. A. C. Ellison D.H. J. J. G. The of on the cotransporter is by 2015; PubMed Scopus Google Scholar). In K+ and in in the in is the WNK WNK in C. J. R.J. Yang C.L. McCormick J.A. J. Ellison D.H. S. K. WNK and to NCC activation in J. PubMed Scopus Google Scholar). The ability of high K+ to NCC abundance is also reduced in mice Wang A.S. D.H. McCormick J.A. Yang C.L. Ellison D.H. Wang W.H. Potassium by renal distal tubules PubMed Scopus Google Scholar, P. Zhang Wang W.H. D.H. of renal ability to potassium increased dietary potassium PubMed Scopus Google Scholar). is an NCC phosphorylation and NCC ubiquitylation R.A. ubiquitylation of the thiazide-sensitive cotransporter NCC and 2014; Full Text Full Text PDF PubMed Scopus Google Scholar, A. M. Y. K. M. K. S. S. of Na-Cl cotransporter by and kinases its PubMed Scopus Google that total NCC levels in to high K+ in mice because NCC phosphorylation is that the of is to the NCC degradation mechanism In mice, high dietary K+ increased and and in is a of increased of from the to increased plasma K+ S. Mitchell C. of and in Clin. Invest. PubMed Scopus Google Scholar, G. of and of in the J. PubMed Scopus Google Scholar). In K+ of in B. P. Wang W.H. D. Potassium through to regulate its Scholar). In with in low K+ abundance in high K+ Hsp70 also to expression by increasing its with II and its from the R.A. A. C. L. Hsp70 sodium expression by increasing its with II and its from Full Text Full Text PDF PubMed Scopus Google Scholar). in Hsp70 also a role in the of K+ on However, and Hsp70 and effects with to the of in with but Hsp70 increasing the and expression of L. Yan W. effects of and Hsp70 on the and expression of sodium S. A. PubMed Scopus Google Scholar). or Hsp70 plays a role in In conclusion, we a mechanism for NCC reduction a high K+ protein to high K+ intake drives ubiquitin-dependent NCC degradation in a mechanism facilitated by heat shock protein This mechanism K+ secretion hyperkalemia and is a for high dietary K+ BP. of the was on that a on an to the or the on the was the of similar The were a NCC NCC J. R.A. phosphorylation of the thiazide-sensitive sodium cotransporter in the distal convoluted Full Text Full Text PDF PubMed Scopus Google cell the of S. M. G. A. D. J. of the renal sodium cotransporter in to potassium intake in Full Text Full Text PDF PubMed Scopus Google and S. Mitchell C. of and in Clin. Invest. PubMed Scopus Google Scholar). was by of mice with the NCC S. R. F. R. K. P. H. to and distal convoluted tubule of and J. PubMed Scopus Google Scholar). was for and in of NCC in kidney was by on from and mice P. M. Gitelman's syndrome in mice the cotransporter of the distal convoluted Full Text Full Text PDF PubMed Scopus Google by In were with F. R.A. of sodium cotransporter NCC in J. 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