Calcineurin inhibitors block sodium-chloride cotransporter dephosphorylation in response to high potassium intake
Authors: Wakana Shoda, Naohiro Nomura, Fumiaki Ando, Yutaro Mori, Takayasu Mori, Eisei Sohara, Tatemitsu Rai, Shinichi Uchida
Journal: Kidney International (2016)

Dietary potassium intake is inversely related to blood pressure and mortality. Moreover, the sodium-chloride cotransporter (NCC) plays an important role in blood pressure regulation and urinary potassium excretion in response to potassium intake. Previously, it was shown that NCC is activated by the WNK4-SPAK cascade and dephosphorylated by protein phosphatase. However, the mechanism of NCC regulation with acute potassium intake is still unclear. To identify the molecular mechanism of NCC regulation in response to potassium intake, we used adult C57BL/6 mice fed a 1.7% potassium solution by oral gavage. We confirmed that acute potassium load rapidly dephosphorylated NCC, which was not dependent on the accompanying anions. Mice were treated with tacrolimus (calcineurin inhibitor) and W7 (calmodulin inhibitor) before the oral potassium loads. Dephosphorylation of NCC induced by potassium was significantly inhibited by both tacrolimus and W7 treatment. There was no significant difference in WNK4, OSR1, and SPAK expression after high potassium intake, even after tacrolimus and W7 treatment. Another phosphatase, protein phosphatase 1, and its endogenous inhibitor I-1 did not show a significant change after potassium intake. Hyperkaliuria, induced by high potassium intake, was significantly suppressed by tacrolimus treatment. Thus, calcineurin is activated by an acute potassium load, which rapidly dephosphorylates NCC, leading to increased urinary potassium excretion. Dietary potassium intake is inversely related to blood pressure and mortality. Moreover, the sodium-chloride cotransporter (NCC) plays an important role in blood pressure regulation and urinary potassium excretion in response to potassium intake. Previously, it was shown that NCC is activated by the WNK4-SPAK cascade and dephosphorylated by protein phosphatase. However, the mechanism of NCC regulation with acute potassium intake is still unclear. To identify the molecular mechanism of NCC regulation in response to potassium intake, we used adult C57BL/6 mice fed a 1.7% potassium solution by oral gavage. We confirmed that acute potassium load rapidly dephosphorylated NCC, which was not dependent on the accompanying anions. Mice were treated with tacrolimus (calcineurin inhibitor) and W7 (calmodulin inhibitor) before the oral potassium loads. Dephosphorylation of NCC induced by potassium was significantly inhibited by both tacrolimus and W7 treatment. There was no significant difference in WNK4, OSR1, and SPAK expression after high potassium intake, even after tacrolimus and W7 treatment. Another phosphatase, protein phosphatase 1, and its endogenous inhibitor I-1 did not show a significant change after potassium intake. Hyperkaliuria, induced by high potassium intake, was significantly suppressed by tacrolimus treatment. Thus, calcineurin is activated by an acute potassium load, which rapidly dephosphorylates NCC, leading to increased urinary potassium excretion. Potassium (K+) intake is important for human health, and it is well-known that higher K+ intake is associated with lower mortality and blood pressure (BP).1Mente A. O'Donnell M.J. Rangarajan S. et al.Association of urinary sodium and potassium excretion with blood pressure.N Engl J Med. 2014; 371: 601-611Crossref PubMed Scopus (566) Google Scholar, 2Gijsbers L. Dower J.I. Schalkwijk C.G. et al.Effects of sodium and potassium supplementation on endothelial function: a fully controlled dietary intervention study.Br J Nutr. 2015; 114: 1419-1426Crossref PubMed Scopus (27) Google Scholar, 3Skrabal F. Gasser R.W. Finkenstedt G. et al.Low-sodium diet versus low-sodium/high-potassium diet for treatment of hypertension.Klin Wochenschr. 1984; 62: 124-128Crossref PubMed Scopus (13) Google Scholar On the other hand, acute hyperkalemia can be fatal. Therefore, it is important to elucidate the mechanism of BP reduction and K+ excretion after K+ intake. Sodium-chloride cotransporter (NCC) is expressed in distal convoluted tubules (DCTs) and plays an important role in the regulation of BP and K+ homeostasis. Altering sodium (Na+) delivery to the connecting tubules and cortical collecting ducts, which are key sites of K+ secretion, regulates K+ excretion.4Terker A.S. Zhang C. McCormick J.A. et al.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 (288) Google Scholar The molecular mechanism of NCC regulation has been investigated through the study of pseudohypoaldosteronism type II—Gordon syndrome, which is characterized by hereditary hypertension with hyperkalemia and metabolic acidosis. NCC is phosphorylated and activated by Ste20-related proline- and alanine-rich kinase (SPAK) kinase, and SPAK is regulated by Kelch-like protein 3 (KLHL3)- with no lysine kinase 4 (WNK4) cascade.5Wakabayashi M. Mori T. Isobe K. et al.Impaired KLHL3-mediated ubiquitination of WNK4 causes human hypertension.Cell Rep. 2013; 3: 858-868Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 6Susa K. Sohara E. Rai T. et al.Impaired degradation of WNK1 and WNK4 kinases causes PHAII in mutant KLHL3 knock-in mice.Hum Mol Genet. 2014; 23: 5052-5060Crossref PubMed Scopus (65) Google Scholar In previous animal studies, a low-K+ diet caused increases in the amount and phosphorylation of NCC4Terker A.S. Zhang C. McCormick J.A. et al.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 (288) Google Scholar, 7Vallon V. Schroth J. Lang F. et al.Expression and phosphorylation of the Na+-Cl- cotransporter NCC in vivo is regulated by dietary salt, potassium, and SGK1.Am J Physiol Renal Physiol. 2009; 297: F704-F712Crossref PubMed Scopus (194) Google Scholar, 8Castañeda-Bueno M. Cervantes-Perez L.G. Rojas-Vega L. et al.Modulation of NCC activity by low and high K(+) intake: insights into the signaling pathways involved.Am J Physiol Renal Physiol. 2014; 306: F1507-F1519Crossref PubMed Scopus (76) Google Scholar, 9Terker A.S. Zhang C. Erspamer K.J. et al.Unique chloride-sensing properties of WNK4 permit the distal nephron to modulate potassium homeostasis.Kidney Int. 2016; 89: 127-134Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 10Takahashi D. Mori T. Nomura N. et al.WNK4 is the major WNK positively regulating NCC in the mouse kidney.Biosci Rep. 2014 May 9; 34Crossref PubMed Scopus (75) Google Scholar, 11Frindt G. Palmer L.G. Effects of dietary K on cell-surface expression of renal ion channels and transporters.Am J Physiol Renal Physiol. 2010; 299: F890-F897Crossref PubMed Scopus (98) Google Scholar, 12Wade J.B. Liu J. Coleman R. et al.SPAK-mediated NCC regulation in response to low-K+ diet.Am J Physiol Renal Physiol. 2015; 308: F923-F931Crossref PubMed Scopus (48) Google Scholar, 13Vitzthum H. Seniuk A. Schulte L.H. et al.Functional coupling of renal K+ and Na+ handling causes high blood pressure in Na+ replete mice.J Physiol. 2014; 592: 1139-1157Crossref PubMed Scopus (54) Google Scholar as well as elevation of BP.4Terker A.S. Zhang C. McCormick J.A. et al.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 (288) Google Scholar, 13Vitzthum H. Seniuk A. Schulte L.H. et al.Functional coupling of renal K+ and Na+ handling causes high blood pressure in Na+ replete mice.J Physiol. 2014; 592: 1139-1157Crossref PubMed Scopus (54) Google Scholar Elevation of BP with a low-K+ diet was dependent on NCC because NCC−/− mice did not show an elevation of BP with a low-K+ diet.4Terker A.S. Zhang C. McCormick J.A. et al.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 (288) Google Scholar A low-K+ diet also increased total and phosphorylated SPAK4Terker A.S. Zhang C. McCormick J.A. et al.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 (288) Google Scholar, 10Takahashi D. Mori T. Nomura N. et al.WNK4 is the major WNK positively regulating NCC in the mouse kidney.Biosci Rep. 2014 May 9; 34Crossref PubMed Scopus (75) Google Scholar, 12Wade J.B. Liu J. Coleman R. et al.SPAK-mediated NCC regulation in response to low-K+ diet.Am J Physiol Renal Physiol. 2015; 308: F923-F931Crossref PubMed Scopus (48) Google Scholar, 13Vitzthum H. Seniuk A. Schulte L.H. et al.Functional coupling of renal K+ and Na+ handling causes high blood pressure in Na+ replete mice.J Physiol. 2014; 592: 1139-1157Crossref PubMed Scopus (54) Google Scholar and WNK4.4Terker A.S. Zhang C. McCormick J.A. et al.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 (288) Google Scholar, 13Vitzthum H. Seniuk A. Schulte L.H. et al.Functional coupling of renal K+ and Na+ handling causes high blood pressure in Na+ replete mice.J Physiol. 2014; 592: 1139-1157Crossref PubMed Scopus (54) Google Scholar The WNK4−/− and SPAK−/− mice showed either no increase or only a blunted increase in phosphorylated NCC (pNCC) in response to a low-K+ diet, respectively.4Terker A.S. Zhang C. McCormick J.A. et al.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 (288) Google Scholar, 10Takahashi D. Mori T. Nomura N. et al.WNK4 is the major WNK positively regulating NCC in the mouse kidney.Biosci Rep. 2014 May 9; 34Crossref PubMed Scopus (75) Google Scholar, 12Wade J.B. Liu J. Coleman R. et al.SPAK-mediated NCC regulation in response to low-K+ diet.Am J Physiol Renal Physiol. 2015; 308: F923-F931Crossref PubMed Scopus (48) Google Scholar These results indicate that phosphorylation of NCC with a low-K+ diet is dependent on WNK4-SPAK kinases. In recent years, it has been found that WNK1 has a chloride (Cl−)-binding motif that affects WNK1 autophosphorylation.14Piala A.T. Moon T.M. Akella R. et al.Chloride sensing by WNK1 involves inhibition of autophosphorylation.Sci Signal. 2014; 7: ra41Crossref PubMed Scopus (238) Google Scholar Other WNKs also have a Cl−-sensing domain that is homologous to the WNK1 domain.9Terker A.S. Zhang C. Erspamer K.J. et al.Unique chloride-sensing properties of WNK4 permit the distal nephron to modulate potassium homeostasis.Kidney Int. 2016; 89: 127-134Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar A change in plasma K+ level affects the membrane potential of DCT cells, thereby altering their intracellular Cl− concentration. Therefore, WNKs are thought to be K+ sensors.4Terker A.S. Zhang C. McCormick J.A. et al.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 (288) Google Scholar, 9Terker A.S. Zhang C. Erspamer K.J. et al.Unique chloride-sensing properties of WNK4 permit the distal nephron to modulate potassium homeostasis.Kidney Int. 2016; 89: 127-134Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar Our previous study of COS7 cells highlighted the importance of K+ in stimulating the WNK cascade in low-K+ conditions.15Naito S. Ohta A. Sohara E. et al.Regulation of WNK1 kinase by extracellular potassium.Clin Exp Nephrol. 2011; 15: 195-202Crossref PubMed Scopus (25) Google Scholar As for K+ loading, there are various experimental protocols and results on the effect of high K+ on NCC. Dietary intake, rapid oral gavage, and i.v. administration of KCl decreased total and pNCC.7Vallon V. Schroth J. Lang F. et al.Expression and phosphorylation of the Na+-Cl- cotransporter NCC in vivo is regulated by dietary salt, potassium, and SGK1.Am J Physiol Renal Physiol. 2009; 297: F704-F712Crossref PubMed Scopus (194) Google Scholar, 9Terker A.S. Zhang C. Erspamer K.J. et al.Unique chloride-sensing properties of WNK4 permit the distal nephron to modulate potassium homeostasis.Kidney Int. 2016; 89: 127-134Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 11Frindt G. Palmer L.G. Effects of dietary K on cell-surface expression of renal ion channels and transporters.Am J Physiol Renal Physiol. 2010; 299: F890-F897Crossref PubMed Scopus (98) Google Scholar, 16Sorensen M.V. Grossmann S. Roesinger M. et al.Rapid dephosphorylation of the renal sodium chloride cotransporter in response to oral potassium intake in mice.Kidney Int. 2013; 83: 811-824Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 17Rengarajan S. Lee D.H. Oh Y.T. et al.Increasing plasma [K+] by intravenous potassium infusion reduces NCC phosphorylation and drives kaliuresis and natriuresis.Am J Physiol Renal Physiol. 2014; 306: F1059-F1068Crossref PubMed Scopus (99) Google Scholar, 18van der Lubbe N. Moes A.D. Rosenbaek L.L. et al.K+-induced natriuresis is preserved during Na+ depletion and accompanied by inhibition of the Na+-Cl- cotransporter.Am J Physiol Renal Physiol. 2013; 305: F1177-F1188Crossref PubMed Scopus (88) Google Scholar Rapid oral gavage of KHCO3 also induced dephosphorylation of NCC.16Sorensen M.V. Grossmann S. Roesinger M. et al.Rapid dephosphorylation of the renal sodium chloride cotransporter in response to oral potassium intake in mice.Kidney Int. 2013; 83: 811-824Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar However, a high-K+ diet of K+-citrate increased pNCC.8Castañeda-Bueno M. Cervantes-Perez L.G. Rojas-Vega L. et al.Modulation of NCC activity by low and high K(+) intake: insights into the signaling pathways involved.Am J Physiol Renal Physiol. 2014; 306: F1507-F1519Crossref PubMed Scopus (76) Google Scholar, 13Vitzthum H. Seniuk A. Schulte L.H. et al.Functional coupling of renal K+ and Na+ handling causes high blood pressure in Na+ replete mice.J Physiol. 2014; 592: 1139-1157Crossref PubMed Scopus (54) Google Scholar In addition to NCC, the effect of high K+ on WNK-SPAK kinase is also controversial. Rengarajan et al.17Rengarajan S. Lee D.H. Oh Y.T. et al.Increasing plasma [K+] by intravenous potassium infusion reduces NCC phosphorylation and drives kaliuresis and natriuresis.Am J Physiol Renal Physiol. 2014; 306: F1059-F1068Crossref PubMed Scopus (99) Google Scholar showed a slight decrease of phosphorylated SPAK in a high-KCl diet; however, other groups showed an increase in WNK4 and phosphorylated SPAK18van der Lubbe N. Moes A.D. Rosenbaek L.L. et al.K+-induced natriuresis is preserved during Na+ depletion and accompanied by inhibition of the Na+-Cl- cotransporter.Am J Physiol Renal Physiol. 2013; 305: F1177-F1188Crossref PubMed Scopus (88) Google Scholar as well as an increase in WNK4 mRNA level19O'Reilly M. Marshall E. Macgillivray T. et al.Dietary electrolyte-driven responses in the renal WNK kinase pathway in vivo.J Am Soc Nephrol. 2006; 17: 2402-2413Crossref PubMed Scopus (111) Google Scholar in a high-K+ diet. Other than WNK4-SPAK kinases, phosphatases are thought to regulate the phosphorylation of NCC as a counterbalance to the kinases. Indeed, in previous studies, NCC was reported to be dephosphorylated by calcinurin,20Lazelle R.A. McCully B.H. Terker A.S. et al.Renal deletion of 12 kDa FK506-binding protein attenuates tacrolimus-induced hypertension.J Am Soc Nephrol. 2016; 27: 1456-1464Crossref PubMed Scopus (37) Google Scholar protein phosphatase 1 (PP1),21Picard N. Trompf K. Yang C.L. et al.Protein phosphatase 1 inhibitor-1 deficiency reduces phosphorylation of renal NaCl cotransporter and causes arterial hypotension.J Am Soc Nephrol. 2014; 25: 511-522Crossref PubMed Scopus (51) Google Scholar and PP4.22Glover M. Mercier Zuber A. Figg N. et al.The activity of the thiazide-sensitive Na(+)-Cl(-) cotransporter is regulated by protein phosphatase PP4.Can J Physiol Pharmacol. 2010; 88: 986-995Crossref PubMed Scopus (39) Google Scholar Calcineurin is known as a calcium- and calmodulin-dependent protein serine/threonine phosphate, termed protein phosphatase 2B.23Kissinger C.R. Parge H.E. Knighton D.R. et al.Crystal structures of human calcineurin and the human FKBP12-FK506-calcineurin complex.Nature. 1995; 378: 641-644Crossref PubMed Scopus (695) Google Scholar Calcineurin inhibitors, such as tacrolimus and cyclosporine, are immunosuppressive drugs that have side effects such as hyperkalemia and hypertension. Tacrolimus and cyclosporine were reported to increase the abundance of NCC.24Hoorn E.J. Walsh S.B. McCormick J.A. et al.The calcineurin inhibitor tacrolimus activates the renal sodium chloride cotransporter to cause hypertension.Nat Med. 2011; 17: 1304-1309Crossref PubMed Scopus (250) Google Scholar, 25Melnikov S. Mayan H. Uchida S. et al.Cyclosporine metabolic side effects: association with the WNK4 system.Eur J Clin Invest. 2011; 41: 1113-1120Crossref PubMed Scopus (45) Google Scholar When the tacrolimus-binding protein (FKBP12) was specifically knocked out in the mouse kidney, the tacrolimus-induced NCC phosphorylation was not observed.20Lazelle R.A. McCully B.H. Terker A.S. et al.Renal deletion of 12 kDa FK506-binding protein attenuates tacrolimus-induced hypertension.J Am Soc Nephrol. 2016; 27: 1456-1464Crossref PubMed Scopus (37) Google Scholar Another phosphatase, PP1, was also thought to regulate NCC. Protein phosphatase inhibitor-1 (I-1), known as an endogenous inhibitor of PP1, was identified as a DCT-enriched gene product by microarray analysis of mouse DCT cells, and the I-1 knockout mouse experiment by Picard et al.21Picard N. Trompf K. Yang C.L. et al.Protein phosphatase 1 inhibitor-1 deficiency reduces phosphorylation of renal NaCl cotransporter and causes arterial hypotension.J Am Soc Nephrol. 2014; 25: 511-522Crossref PubMed Scopus (51) Google Scholar showed a decrease in pNCC and significantly lower arterial BP. These data suggested that phosphatases are involved in the normal regulation of NCC. Seeking to identify the mechanism involved in the regulation of NCC with K+ intake in vivo, we focused on the rapid decrease of NCC phosphorylation during acute K+ load in mice. Our data showed that calcineurin was activated during acute K+ load and dephosphorylated NCC. We first verified the change in NCC phosphorylation with acute oral K+ administration. KCl, K+-gluconate, or K+-citrate was fed to mice with oral gavage, and we found that all potassium compounds significantly decreased NCC phosphorylation 15 minutes after oral gavage (Figure 1A). Next, we examined the effect of changing the cation on phosphorylation of NCC. Mice were fed calcium (Ca2+) gluconate or Na+-gluconate that contained equivalent amounts of gluconate as in the K+-gluconate solution. Although K+-gluconate induced a marked reduction of NCC phosphorylation, Na+- and Ca2+-gluconate did not (Figure 1B). These results indicate that rapid dephosphorylation of NCC is specifically caused by K+, and the effect of K+ is anion independent. We also evaluated plasma potassium level after K+-gluconate oral administration and confirmed that the plasma K+ level was significantly increased (Table 1) .Table 1Blood data for the mice 15 minutes after oral gavageControl (N = 6)High K+ (N = 6)Na (mmol/l) Vehicle147 ± 2147 ± 1 Tacrolimus146 ± 2147 ± 1K (mmol/l) Vehicle4.9 ± 0.38.5 ± 0.2aP < 0.05. Tacrolimus4.8 ± 0.27.9 ± 0.2aP < 0.05.Cl (mmol/l) Vehicle111 ± 1118 ± 2 Tacrolimus114 ± 1119 ± 2pH Vehicle7.29 ± 0.017.27 ± 0.02 Tacrolimus7.22 ± 0.037.23 ± 0.01HCO3− (mmol/l) Vehicle23.7 ± 0.922.4 ± 0.9 Tacrolimus20.6 ± 0.822.9 ± 1.4Mice were pretreated with tacrolimus or vehicle. Blood was collected 15 minutes after high-potassium solution or control solution intake. There was no significant difference in serum potassium values between the vehicle-treated mice and the tacrolimus-treated mice. Data shown are mean ± SEM. Statistical significance between each groups was assessed by 1-way analysis of variance with Tukey’s test.a P < 0.05. Open table in a new tab Mice were pretreated with tacrolimus or vehicle. Blood was collected 15 minutes after high-potassium solution or control solution intake. There was no significant difference in serum potassium values between the vehicle-treated mice and the tacrolimus-treated mice. Data shown are mean ± SEM. Statistical significance between each groups was assessed by 1-way analysis of variance with Tukey’s test. We investigated WNK4, SPAK, and oxidative stress-responsive kinase-1 (OSR1) kinases, which are upstream regulators of NCC phosphorylation. There was no significant reduction of WNK4 protein expression with an acute oral K+ load (Figure 2). SPAK has several isoforms, and it is known that some isoforms have different roles in renal salt transport.26McCormick J.A. Mutig K. Nelson J.H. et al.A SPAK isoform switch modulates renal salt transport and blood pressure.Cell Metab. 2011; 14: 352-364Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar The SPAK isoforms we refer to here (∼50–60 kDa) were known to be phosphorylated for activation.5Wakabayashi M. Mori T. Isobe K. et al.Impaired KLHL3-mediated ubiquitination of WNK4 causes human hypertension.Cell Rep. 2013; 3: 858-868Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar There was no significant difference in each band of SPAK after acute K+ load. We also investigated the activity of OSR1 and found no significant difference in OSR1. We concluded that the rapid reduction of pNCC was independent of the WNK-OSR1/SPAK cascade in an acute K+ load, suggesting that activation of phosphatase might be the main mechanism of decrease in the phosphorylation of NCC. Thus, we investigated which phosphatase contributed to the dephosphorylation of NCC in an acute oral K+ load. We investigated whether PP1 and I-1 were activated by an acute oral K+ load. It is known that PP1 activity is inhibited by the phosphorylation of PP1 at Threonine-320 (T320),27Hou H. Sun L. Siddoway B.A. et al.Synaptic NMDA receptor stimulation activates PP1 by inhibiting its phosphorylation by Cdk5.J Cell Biol. 2013; 203: 521-535Crossref PubMed Scopus (42) Google Scholar, 28Wu J.Q. Guo J.Y. Tang W. et al.PP1-mediated dephosphorylation of phosphoproteins at mitotic exit is controlled by inhibitor-1 and PP1 phosphorylation.Nat Cell Biol. 2009; 11: 644-651Crossref PubMed Scopus (186) Google Scholar and I-1 inhibits the activity of PP1 by phosphorylating PP1 at T320.29Nicolaou P. Hajjar R.J. Kranias E.G. Role of protein phosphatase-1 inhibitor-1 in cardiac physiology and pathophysiology.J Mol Cell Cardiol. 2009; 47: 365-371Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar We evaluated PP1 activity by analyzing phosphorylated PP1 (pPP1) and I-1 with Western blotting using its specific antibodies. After an acute oral K+ load, there was no significant difference in PP1, pPP1, and I-1 (Figure 3A). We also investigated whether tautomycetin, a specific PP1 inhibitor, inhibits K+-induced NCC dephosphorylation. The dephosphorylation of NCC was not inhibited by tautomycetin (Figure 3B). The increase in pPP1 after tautomycetin treatment indicated the decrease in PP1 activity,27Hou H. Sun L. Siddoway B.A. et al.Synaptic NMDA receptor stimulation activates PP1 by inhibiting its phosphorylation by Cdk5.J Cell Biol. 2013; 203: 521-535Crossref PubMed Scopus (42) Google Scholar which confirmed that tautomycetin properly inhibited the PP1 activity. These data confirmed that PP1 does not play a role in the dephosphorylation of NCC after acute K+ administration. Next, we focused on another phosphatase, protein phosphatase 2B (calcineurin). We administered tacrolimus, a calcineurin inhibitor, to mice i.p. 1 hour before K+ oral gavage and found that tacrolimus dramatically inhibited K+-induced dephosphorylation of NCC (Figure 4A). We performed immunofluorescence experiments to confirm the effect of tacrolimus. After oral K+ administration, the staining of total NCC was maintained, but the staining of phosphorylated NCC was markedly reduced (Figure 4B). We confirmed the effect of tacrolimus to find the marked inhibition of the K+-induced dephosphorylation of NCC. We analyzed the plasma K+ level and found that plasma K+ was increased after K+ oral gavage in the tacrolimus-treated group in the same way that was observed in the vehicle group (Table 1). Thus, inhibition of NCC dephosphorylation by tacrolimus was not caused by differences in the plasma K+ level. Next, we investigated calmodulin, an upstream regulator of calcineurin, to confirm the involvement of calcineurin in K+-induced NCC dephosphorylation. W7 (a calmodulin inhibitor) treatment also dramatically inhibited K+-induced dephosphorylation of NCC as also seen in tacrolimus treatment (Figure 5). We also analyzed WNK4 and SPAK and found no significant difference after either tacrolimus or W7 treatment (Figures 4A and 5). These data strongly suggest that an acute K+ load activates calmodulin and calcineurin. The activated calcineurin, in turn, dephosphorylates NCC. We analyzed urinary K+ excretion to investigate the physiological contribution of calcineurin after an acute K+ load. Tacrolimus-treated mice showed significantly lower urinary K+ excretion than vehicle-treated mice at 30 and 60 minutes after high K+ intake (Figure 6). This result suggests that activation of calcineurin (which promotes dephosphorylation of NCC) plays an important role in urinary K+ excretion after a rapid K+ load. We also analyzed the plasma K+ level at 90 minutes after a K+ load. There was no significant difference between tacrolimus-treated mice and control mice. However, the plasma K+ level in tacrolimus-treated mice tended to be higher than that in control mice (the plasma K+ levels in the tacrolimus group and the vehicle group were 6.83 ± 0.50 and 6.23 ± 0.49 [mean ± SEM], respectively, P = 0.2, N = 7). In this study, we showed the involvement of calcineurin in NCC dephosphorylation after acute oral K+ administration, which regulated urinary K+ excretion in the acute phase. Calcineurin is a calcium- and calmodulin-dependent protein serine/threonine phosphatase known as protein phosphatase 2B.23Kissinger C.R. Parge H.E. Knighton D.R. et al.Crystal structures of human calcineurin and the human FKBP12-FK506-calcineurin complex.Nature. 1995; 378: 641-644Crossref PubMed Scopus (695) Google Scholar Calcineurin inhibitors, such as tacrolimus and cyclosporine, are used for immunosuppressive therapy in many diseases and conditions, including organ transplantation, autoimmune disease, and nephritis syndrome. The major side effects of calcineurin inhibitors are hyperkalemia and hypertension. A previous report found that tacrolimus increased phosphorylated NCC expression, essential in the pathophysiology of tacrolimus-induced hypertension and hyperkalemia.24Hoorn E.J. Walsh S.B. McCormick J.A. et al.The calcineurin inhibitor tacrolimus activates the renal sodium chloride cotransporter to cause hypertension.Nat Med. 2011; 17: 1304-1309Crossref PubMed Scopus (250) Google Scholar It is known that calcineurin is activated by a high K+ level in other types of cells.30Cui Z.J. Types of voltage-dependent calcium channels involved in high potassium depolarization-induced amylase secretion in the exocrine pancreatic tumour cell line AR4-2J.Cell Res. 1998; 8: 23-31Crossref Scopus (12) Google Scholar, 31Faure C. Ramos M. Girault J.A. Pyk2 cytonuclear localization: mechanisms and regulation by serine dephosphorylation.Cell Mol Life Sci. 2013; 70: 137-152Crossref PubMed Scopus (17) Google Scholar The mechanism of high K+ calcineurin activation is that high K+ induces cell depolarization, which leads to the opening of voltage-dependent Ca2+ channels.30Cui Z.J. Types of voltage-dependent calcium channels involved in high potassium depolarization-induced amylase secretion in the exocrine pancreatic tumour cell line AR4-2J.Cell Res. 1998; 8: 23-31Crossref Scopus (12) Google Scholar, 31Faure C. Ramos M. Girault J.A. Pyk2 cytonuclear localization: mechanisms and regulation by serine dephosphorylation.Cell Mol Life Sci. 2013; 70: 137-152Crossref PubMed Scopus (17) Google Scholar, 32Choi M. Scholl U.I. Yue P. et al.K+ channel mutations in adrenal aldosterone-producing adenomas and hereditary hypertension.Science. 2011; 331: 768-772Crossref PubMed Scopus (760) Google Scholar Then, an increase in intracellular Ca2+ concentration ([Ca2+]i) follows, activating calmodulin and calcineurin. In the setting of acute hyperkalemia, we hypothesize that DCT cells are also depolarized by high K+, followed by an increase in [Ca2+]i and activation of calmodulin and calcineurin (Figure 7). Although the involvement of a voltage-dependent Ca2+ channel in DCT is still unknown, it might play a role in linking the effect of K+ loading to calcineurin activation. The WNK-SPAK cascade plays an important role in the regulation of NCC activity in a low-K+ environment. Indeed, previous studies showed that the decrease in extracellular K+ reduced intracellular Cl− in the cells,33Weinstein A.M. A mathematical model of rat distal convoluted tubule. I. Cotransporter function in early DCT.Am J Physiol Renal Physiol. 2005; 289: F699-F720Crossref PubMed Scopus (64) Google Scholar, 34Weinstein A.M. A mathematical model of rat distal convoluted tubule. II. Potassium secretion along the connecting segment.Am J Physiol Renal Physiol. 2005; 289: F721-F741Crossref PubMed Scopus (53) Google Scholar and Cl− depletion stimulated WNK kinases, leading to SPAK and NCC activation.4T