Portal de Periódicos da CAPES

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

2021; Elsevier BV; Volume: 297; Issue: 2 Linguagem: Inglês

10.1016/j.jbc.2021.100915

1083-351X

Marleen L. A. Kortenoeven, Cristina Esteva‐Font, Henrik Dimke, Søren Brandt Poulsen, Sathish K. Murali, Robert A. Fenton,

Magnesium in Health and Disease

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, while patients with PHAII suffer from hyperkalemia, demonstrating 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 both NCC phosphorylation (active form) and total NCC abundance enhances Na+ delivery to downstream segments of the renal tubule to facilitate electrogenic K+ secretion and restore plasma K+ levels. Similar reductions in total and phosphorylated NCC levels are further linked with the ability of a high K+ diet to lower BP. Reduced NCC phosphorylation following high dietary K+ intake can be explained by alterations in the basolateral plasma membrane potential via the inwardly rectifying potassium channel Kir4.1/Kir5.1 (a heterotetramer of Kir4.1 and Kir5.1 channels) and modulation of the WNK-SPAK/OSR1 kinase signaling pathway (10Piala A.T. Moon T.M. Akella R. He H. Cobb M.H. Goldsmith E.J. Chloride sensing by WNK1 involves inhibition of autophosphorylation.Sci. Signal. 2014; 7: ra41Crossref PubMed Scopus (200) Google Scholar, 11Bazua-Valenti S. Chavez-Canales M. Rojas-Vega L. Gonzalez-Rodriguez X. Vazquez N. Rodriguez-Gama A. Argaiz E.R. Melo Z. Plata C. Ellison D.H. Garcia-Valdes J. Hadchouel J. Gamba G. The effect of WNK4 on the Na+-Cl- cotransporter is modulated by intracellular chloride.J. Am. Soc. Nephrol. 2015; 26: 1781-1786Crossref PubMed Scopus (96) Google Scholar, 12Cuevas C.A. Su X.T. Wang M.X. Terker A.S. Lin D.H. McCormick J.A. Yang C.L. Ellison D.H. Wang W.H. Potassium sensing by renal distal tubules requires Kir4.1.J. Am. Soc. Nephrol. 2017; 28: 1814-1825Crossref PubMed Scopus (83) Google Scholar, 13Wu P. Gao Z.X. Zhang D.D. Su X.T. Wang W.H. Lin D.H. Deletion of Kir5.1 impairs renal ability to excrete potassium during increased dietary potassium intake.J. Am. Soc. Nephrol. 2019; 30: 1425-1438Crossref PubMed Scopus (20) Google Scholar). However, such a mechanism cannot easily account for a sustained reduction in total NCC following high dietary K+ intake. Therefore, the aim of this study was to identify the mechanism of how high K+ decreases total NCC abundance. Our findings suggest that high K+ increases ubiquitin-dependent NCC degradation in a mechanism facilitated by protein phosphatase 1α (PP1α) effects on heat shock protein 70. This mechanism helps increase K+ secretion during hyperkalemia and is a novel concept for understanding how high dietary K+ can reduce BP. Aldosterone increases renal NCC expression and its phosphorylation, while total renal NCC expression and phosphorylation are lower under a high-K+ diet, even though aldosterone levels are increased (14Vallon V. Schroth J. Lang F. Kuhl D. Uchida S. 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 (177) Google Scholar, 15Frindt 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 (92) Google Scholar, 16Kim G.H. Masilamani S. Turner R. Mitchell C. Wade J.B. Knepper M.A. The thiazide-sensitive Na-Cl cotransporter is an aldosterone-induced protein.Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14552-14557Crossref PubMed Scopus (348) Google Scholar). It is thought that this reduction in NCC is triggered by an increase in plasma K+ subsequent 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). To assess the ability of long-term alterations in K+ to modulate NCC abundance independently of aldosterone, we developed an ex vivo system that utilizes renal cortical tubules isolated from mice. Incubation of such preparations in different concentrations of KCl for 24 or 48 h (choline Cl was used to balance osmolality and chloride when adjusting K+ levels) demonstrated that relative to control conditions (3.5 mM K+), NCC levels were significantly higher after incubation in 0.5 mM K+ and significantly decreased in 8 mM K+ media (Fig. 1, A and B). Similar experiments balancing altered KCl concentrations with NaCl (instead of choline Cl) showed similar results (Fig. S1A). A K+ concentration curve confirmed the effects on NCC after 24 h occurred within the physiological range, with NCC levels significantly increased in K+ concentrations lower than 3.5 mM, but decreased in K+ concentrations greater than 3.5 mM (Fig. 1C). To investigate if the effect of K+ is affected by the accompanying anion, similar tubule experiments were performed using either KCl, K-citrate, or Na-citrate. KCl and K-citrate significantly decreased the expression of NCC (Fig. S1B), indicating that the effects of K+ in this setting are independent of the anion. To rule out that the effects of high K+ to reduce NCC abundance are not due to cell death during prolonged incubation of renal tubules or cytotoxic effects of the high K+, the viability of the renal tubule suspensions was investigated. No decrease in cell number, viability, or mitochondrial metabolic activity was observed in the tubules throughout the experimental period (up to 72 h) when incubated in either control or high K+ medium (Fig 1, D and E and Fig. S2). High plasma K+ will trigger the release of aldosterone from the adrenal gland. To assess if the effects of high K+ on NCC expression were still apparent with aldosterone present, tubules were incubated in high K+ medium with or without 10 nM aldosterone for 8–48 h. At 16–48 h, high K+ significantly decreased NCC, independent of the presence of 10 nM aldosterone. In contrast, α-ENaC abundance increased significantly after 24 h aldosterone incubation, with or without high K+ (Fig. 2). Recently K+ has been shown to increase ENaC activity independently from aldosterone (17Sorensen M.V. Saha B. Jensen I.S. Wu P. Ayasse N. Gleason C.E. Svendsen S.L. Wang W.H. Pearce D. Potassium acts through mTOR to regulate its own secretion.JCI Insight. 2019; 5Google Scholar). To investigate if ENaC abundance is regulated by K+ in the cortical tubules, suspensions were incubated in low or high K+ medium for 24 or 48 h. Relative to control (3.5 mM K+), low K+ medium significantly decreased the protein expression of α-ENaC at both time points, while high K+ medium had no significant effect (Fig. S3). No differences were detected in NCC mRNA expression after incubation of cortical tubules for 24 h in low or high K+ (Fig. 3A), suggesting that the observed effect of K+ to alter NCC levels was likely independent of changes in NCC transcription and may be due to enhanced NCC degradation. Supporting such a mechanism, the effects of high K+ on NCC were absent when tubules were simultaneously incubated for 24 h with the lysosomal inhibitor chloroquine or the proteasomal inhibitor MG132 (Fig. 3, B and C). As long-term incubation with MG132 can also reduce the degradation of proteins via lysosomes (18Melikova M.S. Kondratov K.A. Kornilova E.S. Two different stages of epidermal growth factor (EGF) receptor endocytosis are sensitive to free ubiquitin depletion produced by proteasome inhibitor MG132.Cell Biol. Int. 2006; 30: 31-43PubMed Google Scholar), caution must be exercised in concluding what percentage of high K+-induced NCC degradation occurs via the lysosomal or proteasomal pathways. The posttranslational modification ubiquitylation plays an important role in proteasomal and lysosomal protein degradation, and NCC can itself be ubiquitylated (19Rosenbaek L.L. Rizzo F. Wu Q. Rojas-Vega L. Gamba G. MacAulay N. Staub O. Fenton R.A. The thiazide sensitive sodium chloride co-transporter NCC is modulated by site-specific ubiquitylation.Sci. Rep. 2017; 7: 12981Crossref PubMed Scopus (10) Google Scholar). Emphasizing a potential general role of K+ to modulate protein ubiquitylation, tubules incubated for 24 or 48 h in 0.5 mM K+ media had decreased protein ubiquitylation, whereas tubules incubated in 8 mM K+ had increased cellular ubiquitylation levels (Fig. S4A). To assess if K+ alters the quantity of ubiquitylated NCC, tubules incubated in high K+ medium with or without MG132 to block NCC degradation were immunoprecipitated using a ubiquitin antibody, and NCC and ubiquitin levels were analyzed. In line with a role of K+ to modulate protein ubiquitylation, high K+ alone led to a small increase in the total amount of ubiquitylated proteins, whereas high K+ in the presence of MG132 greatly increased the amount of ubiquitylated proteins compared with MG132 alone (Fig. 4, A and C). High K+ alone did not significantly change the amount of ubiquitylated NCC isolated (Fig. 4, A and B), probably as NCC is rapidly degraded once ubiquitylated. However, in the presence of MG132 to limit NCC degradation, high K+ significantly increased the abundance of ubiquitylated NCC compared with MG132 alone (Fig. 4, A and B). Immunoprecipitation using an NCC antibody gave similar results (not shown). To link increased ubiquitylation to the high K+-mediated reduction in total NCC levels, cortical tubules were incubated for 24 h in control or high K+ media with Pyr-41, a blocker of the ubiquitin-activating enzyme E1 (20Yang Y. Kitagaki J. Dai R.M. Tsai Y.C. Lorick K.L. Ludwig R.L. Pierre S.A. Jensen J.P. Davydov I.V. Oberoi P. Li C.C. Kenten J.H. Beutler J.A. Vousden K.H. Weissman A.M. Inhibitors of ubiquitin-activating enzyme (E1), a new class of potential cancer therapeutics.Cancer Res. 2007; 67: 9472-9481Crossref PubMed Scopus (325) Google Scholar). In total, 50 μM and 150 μM Pyr-41 prevented the decrease in NCC with high K+, with the higher dose increasing NCC levels under control and high K+ conditions (Fig. 4D). Taken together, this data demonstrates that high K+ reduces total NCC levels via an increase in ubiquitin-mediated degradation.Figure 4High K+ increases NCC ubiquitylation. A, isolated renal cortical tubules were incubated in control (3.5 mM) or high K+ medium (8.0 mM) with or without 10 μM of the proteosomal inhibitor Mg132. Lysates were subjected to immunoprecipitation using a ubiquitin antibody matrix. Immunoprecipitated samples and input samples were assessed for levels of NCC and ubiquitin. B and C, normalized band densities of NCC (B) and ubiquitin (C) relative to control (n = 6) Significant differences are indicated (∗p < 0.05). D, tubules were incubated in control (3.5 mM) or high K+ medium (8.0 mM) with or without Pyr-41, a blocker of the ubiquitin-activating E1 enzyme and after 24 h NCC protein levels were assessed. Significant differences are indicated (∗p < 0.05) (n = 8). B–D, shown are mean values ± SD. Comparisons were performed using a two-way ANOVA followed by a Tukey multiple comparison test. Coom, Coomassie blue staining; IP, immunoprecipitation.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The balance between ubiquitin-dependent degradation and protein folding is maintained by the chaperone proteins heat shock protein 70 (Hsp70) and Hsp90 (21Edkins A.L. CHIP: A co-chaperone for degradation by the proteasome.Subcell Biochem. 2015; 78: 219-242Crossref PubMed Scopus (54) Google Scholar). In our recent large-scale proteomics study, the abundance of these and other heat shock proteins was increased specifically in the mouse DCT after a high dietary K+ intake (22Kortenoeven M.L.A. Cheng L. Wu Q. Fenton R.A. An in vivo protein landscape of the mouse DCT during high dietary K(+) or low dietary Na(+) intake.Am. J. Physiol. Renal Physiol. 2021; 320: F908-F921Crossref PubMed Scopus (2) Google Scholar), suggesting they play an important role in modulating NCC levels following a high K+ diet. The role of Hsp70 and Hsp90 depends on their interaction with other proteins including the C-terminus of Hsc70 interacting protein (CHIP, encoded by Stub1) and the Hsp70-Hsp90 organizing protein HOP (Stip1). If Hsp70-bound client proteins are bound to the cochaperone CHIP, this enhances degradation of the client protein (23Muller P. Ruckova E. Halada P. Coates P.J. Hrstka R. Lane D.P. Vojtesek B. C-terminal phosphorylation of Hsp70 and Hsp90 regulates alternate binding to co-chaperones CHIP and HOP to determine cellular protein folding/degradation balances.Oncogene. 2013; 32: 3101-3110Crossref PubMed Scopus (133) Google Scholar). Previous studies in cell lines have demonstrated that NCC forms complexes with the two cytoplasmic Hsp70s, Hsp70, and heat shock cognate 70 (Hsc70), as well as with CHIP and HOP, and that a CHIP-NCC interaction promotes NCC ubiquitylation and degradation (24Donnelly B.F. Needham P.G. Snyder A.C. Roy A. Khadem S. Brodsky J.L. Subramanya A.R. Hsp70 and Hsp90 multichaperone complexes sequentially regulate thiazide-sensitive cotransporter endoplasmic reticulum-associated degradation and biogenesis.J. Biol. Chem. 2013; 288: 13124-13135Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 25Needham P.G. Mikoluk K. Dhakarwal P. Khadem S. Snyder A.C. Subramanya A.R. Brodsky J.L. The thiazide-sensitive NaCl cotransporter is targeted for chaperone-dependent endoplasmic reticulum-associated degradation.J. Biol. Chem. 2011; 286: 43611-43621Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). In agreement, in lysates from primary tubule suspensions grown in control media NCC, Hsp70 and Hsc70 could be coimmunoprecipitated (Fig. 5A), suggesting they form a complex. To investigate if heat shock proteins are involved in K+-mediated NCC downregulation, tubules were incubated for 24 h with Ver-155008, a competitive inhibitor of Hsp70/Hsc70. In total, 150 μM Ver155008 significantly reduced Hsc70 expression, which correlated with significantly increased NCC expression (Fig. 5B). In the presence of Ver-155008, the ability of high K+ to reduce NCC abundance was also completely absent. After 48 h the effects of Ver-155008 were more pronounced (Fig. S4B). Taken together, this suggests that Hsp70/Hsc70 are involved in the normal biogenesis of NCC and play an essential role in mediating high K+-induced NCC degradation. Supporting this idea, in primary tubules incubated in different concentrations of K+ for 24–48 h, Hsc70 levels increased in parallel with increasing K+ levels (Fig. 6, A and B). These K+-induced changes in Hsc70 are at least in part dependent on altered transcription, as Hsc70 (Hspa8) mRNA expression increased with increasing K+ levels (Fig. 6C).Figure 6Heat shock protein expression and phosphorylation are changed in response to high K+. A, tubules were incubated in either low K+ (0.5 mM), control (con, 3.5 mM), or high K+ (8.0 mM) medium and Hsc70 levels assessed after 24 or 48 h. The same samples were used for blotting as for generating the data for Figure 1A, hence the Coomassie gels are the same. B, summary data (mean ± SD) of normalized band densities relative to control. ∗p < 0.05 compared with control media (n = 7). Comparisons were performed using a one-way ANOVA followed by a Dunnett multiple comparison test. C, similar studies assessing Hsc70 mRNA levels using RTqPCR. Signals are normalized against 18S rRNA and expressed relative to control (mean ± SD, n = 9). ∗p < 0.05 compared with control. Comparisons were performed using a one-way ANOVA followed by a Dunnett multiple comparison test. D, tubules were incubated in either control (Con, 3.5 mM K+) or high K+ (8.0 mM K+) medium for 8, 16, or 24 h. Tubules were harvested and subjected to immunoblotting for NCC, Hsc70, phosphorylated Hsc70, or Prot20s. Summary data for NCC (E), Hsc70 (F), and phosphorylated Hsc70/total Hsc70 (G) show normalized signal intensity relative to time matched control (n = 7) and significant differences are indicated (∗p < 0.05). E–G, shown are mean values ± SD. Comparisons were performed using an unpaired Student's t test. Coom, Coomassie blue staining.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Whether interaction with Hsp70/Hsc70 results in ubiquitin-dependent degradation depends on their interaction with other proteins including CHIP and HOP. Dephosphorylated Hsp70/Hsc70 preferentially binds to CHIP, enhancing degradation of the client protein (23Muller P. Ruckova E. Halada P. Coates P.J. Hrstka R. Lane D.P. Vojtesek B. C-terminal phosphorylation of Hsp70 and Hsp90 regulates alternate binding to co-chaperones CHIP and HOP to determine cellular protein folding/degradation balances.Oncogene. 2013; 32: 3101-3110Crossref PubMed Scopus (133) Google Scholar). In isolated tubules incubated in high K+ media for 8 and 16 h, phosphorylated Hsc70/Hsp70 was significantly decreased compared with tubules grown in control media (Fig. 6, D–G). A significant reduction in NCC abundance was only apparent after 16 h, suggesting that the reduction in phosphorylated Hsc70/Hsp70 precedes the high K+-mediated NCC degradation. Protein phosphatase 1 can bind and dephosphorylate members of the Hsp70 family (23Muller P. Ruckova E. Halada P. Coates P.J. Hrstka R. Lane D.P. Vojtesek B. C-terminal phosphorylation of Hsp70 and Hsp90 regulates alternate binding to co-chaperones CHIP and HOP to determine cellular protein folding/degradation balances.Oncogene. 2013; 32: 3101-3110Crossref PubMed Scopus (133) Google Scholar, 26Polanowska-Grabowska R. Simon Jr., C.G. Falchetto R. Shabanowitz J. Hunt D.F. Gear A.R. Platelet adhesion to collagen under flow causes dissociation of a phosphoprotein complex of heat-shock proteins and protein phosphatase 1.Blood. 1997; 90: 1516-1526Crossref PubMed Google Scholar, 27Flores-Delgado G. Liu C.W. Sposto R. Berndt N. A limited screen for protein interactions reveals new roles for protein phosphatase 1 in cell cycle control and apoptosis.J. Proteome Res. 2007; 6: 1165-1175Crossref PubMed Scopus (41) Google Scholar). Theoretically this interaction could dephosphorylate Hsp70/Hsc70 leading to association of NCC with CHIP and subsequent targeting for degradation. PP1 activity has been linked to short-term modulation of NCC function via the protein phosphatase 1 inhibitor-1 (I-1) (28Picard N. Trompf K. Yang C.L. Miller R.L. Carrel M. Loffing-Cueni D. Fenton R.A. Ellison D.H. Loffing J. 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 (47) Google Scholar, 29Penton D. Moser S. Wengi A. Czogalla J. Rosenbaek L.L. Rigendinger F. Faresse N. Martins J.R. Fenton R.A. Loffing-Cueni D. Loffing J. Protein phosphatase 1 inhibitor-1 mediates the cAMP-dependent stimulation of the renal NaCl cotransporter.J. Am. Soc. Nephrol. 2019; 30: 737-750Crossref PubMed Scopus (12) Google Scholar), and the abundance of the alpha isoform of the PP1 catalytic subunit (PP1α) increases specifically in the DCT of mice fed a high K+ diet for 4 days (22Kortenoeven M.L.A. Cheng L. Wu Q. Fenton R.A. An in vivo protein landscape of the mouse DCT during high dietary K(+) or low dietary Na(+) intake.Am. J. Physiol. Renal Physiol. 2021; 320: F908-F921Crossref PubMed Scopus (2) Google Scholar). However, a role of PP1 for modulating NCC abundance has not been investigated. To investigate if K+ has a direct effect on PP1 expression, primary tubules were grown in different concentrations of K+. PP1α (Ppp1ca) mRNA expression increased after high K+ and decreased after low K+ (Fig. 7A), whereas alterations in K+ had no effect on mRNA levels of the PP1β (Ppp1cb) or PP1γ (Ppp1cg) subunits (Fig. 7A). In agreement with the changes in mRNA, PP1α protein levels were significantly lower after 24–48 h incubation in 0.5 mM K+ and increased after incubation in 8 mM K+ relative to control media (3.5 mM K+) (Fig. 7, B and C), a mirror opposite of what was observed with NCC (Fig. 1A). Experiments balancing altered KCl concentrations with NaCl (instead of choline Cl) showed similar results (Fig. S5A). Furthermore, aldosterone did not alter PP1α levels nor did it affect the high K+-mediated increase in PP1α (Fig. S5B). In lysates from primary tubule suspensions grown in control media, NCC and PP1α could also be coimmunoprecipitated (Fig. 5A), suggesting they form a complex. The interaction of NCC with PP1α and Hsc70/Hsp70 was further investigated by growing tubules in high K+ or control medium and subsequently performing coimmunoprecipitations using an antibody for total NCC. In input samples, NCC expression decreased, as well as phosphorylated Hsc70/Hsp70, in agreement with a PP1-mediated dephosphorylation of Hsc70. The coimmunoprecipitated samples show that the interaction with NCC is not changed by incubating in high K+ medium, suggesting that this complex is present independent of K+ concentration (Fig. S6). To confirm a role of PP1 activity for modulating the long-term effects of high K+ on NCC, tubules were incubated for 24 h in control or high K+ media with or without the PP1-inhibitor tautomycetin. The decrease in total NCC observed in high K+ medium was completely blocked by tautomycetin (Fig. 7, D and E), confirming that PP1 is involved in the long-term effects of K+ on NCC. In agreement with the effect of tautomycetin, the PP1/PP2A inhibitor Calyculin A increased total NCC expression and blocked the decrease in total NCC observed in high K+ medium (Fig. 7F). A role for PP2B/PP3 (calcineurin) in mediating the short-term effects of high K+ on phosphorylated NCC has been proposed (30Shoda W. Nomura N. Ando F. Mori Y. Mori T. Sohara E. Rai T. Uchida S. Calcineurin inhibitors block sodium-chloride cotransporter dephosphorylation in response to high potassium intake.Kidney Int. 2017; 91: 402-411Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). However, inhibiting PP2B/PP3 with deltamethrin (Fig. 7D) or Fk506 (tacrolimus) (Fig. 7E) did not prevent the decrease in NCC following 24 h of high K+. At high concentrations, tautomycetin and Calyculin A can inhibit PP2A (31Mitsuhashi S. Matsuura N. Ubukata M. Oikawa H. Shima H. Kikuchi K. Tautomycetin is a novel and specific inhibitor of serine/threonine protein phosphatase type 1, PP1.Biochem. Biophys. Res. Commun. 2001; 287: 328-331Crossref PubMed Scopus (91)