Volume 83, Issue 5, Pages 811-824 (May 2013) Rapid dephosphorylation of the renal sodium chloride cotransporter in response to oral potassium intake in mice  Mads V. Sorensen, Solveig Grossmann, Marian Roesinger, Nikolay Gresko, Abhijeet P. Todkar, Gery Barmettler, Urs Ziegler, Alex Odermatt, Dominique Loffing-Cueni, Johannes Loffing  Kidney International  Volume 83, Issue 5, Pages 811-824 (May 2013) DOI: 10.1038/ki.2013.14 Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 1 Plasma values as a function of time after gavage in control (Ctrl; red), KCl (blue), KHCO3 (green), and NaCl (black)-loaded mice. (a) Plasma [K+] versus time in the four experimental groups. Please note that plasma [K+] is significantly elevated already after 15min in KCl and KHCO3 loaded animals in comparison with Ctrl mice. (b) Plasma [Cl−] after gavage. Significant increase in plasma [Cl−] in the KCl group 30min post gavage. (c) Plasma [HCO3−] is increased in blood samples from the KHCO3 group compared with the Ctrl group at the time points 30 and 120min after gavage. (d) [Na+] concentration in the plasma is augmented in the NaCl group shortly after the gavage at time point 15min. (e) Plasma aldosterone (aldo) is significantly elevated in samples from KCl-loaded animals 30, 180, and 360min after gavage when compared with Ctrl. Graphs depict means ±s.e.m. *P<0.05, **P<0.01. Measurements are from three mice per group. Kidney International 2013 83, 811-824DOI: (10.1038/ki.2013.14) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 2 Urine cation concentrations normalized to creatinine concentrations from mice receiving control (Ctrl; red), KCl (blue), and KHCO3 (green) solutions through gavage. (a) Single measurements of urinary [K+]/[Crea] as a function of time after gavage in the three experimental groups. (b) Urinary [K+]/[Crea] compiled in time intervals 0; 30–60; 61–180; 181–360; and 361–480min after gavage to allow for statistical analyses. (c) Urinary [Na+]/[Crea] after gavage in the three experimental groups. (d) Urinary [Na+]/[Crea] compiled in the same time intervals as in b. Note the striking increases of [Na+]/[Crea] in the K+-loaded groups. *P<0.05 and **P<0.01 between Ctrl and experimental groups. #P<0.05 between the ‘zero’ and the time intervals within the same experimental group. Bar graphs depict means±SEM. Samples from six mice in each experimental group. Kidney International 2013 83, 811-824DOI: (10.1038/ki.2013.14) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 3 KCl rapidly dephosphorylates NaCl cotransporter (NCC). Parvalbumin promoter (PV-EGFP) (enhanced green fluorescent protein under the parvalbumin promoter) male mice received control (Ctrl) and KCl solutions through gavage and kidneys were collected after 15, 30, and 120min. Total NCC (tNCC) and phosphorylation of NCC in 50μg of membrane protein fractions of whole-kidney lysates were detected using anti-tNCC and anti-phospho NCC (pT53, pT58, pS71, and pS89) antibodies. Detection of β-actin served as loading control. Quantitative analysis of signals was performed with an infrared-based imaging system. Please note the stable tNCC abundance, but the marked reduction of NCC phosphorylation at all phospho sites following KCl loading. Graphs depict means±s.e.m.; n=3 mice in each experimental group. *P<0.05, **P<0.01. Kidney International 2013 83, 811-824DOI: (10.1038/ki.2013.14) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 4 Effect of dietary K+ intake on urinary ion excretion, plasma K+ concentration, and NaCl cotransporter (NCC). After overnight fasting, mice received either a 0.05 or 2% K+ diet. (a) Single measurements of urinary [K+]/[Crea] as a function of time after gavage in the two experimental groups. (b) Urinary [K+]/[Crea] compiled in time intervals 0; 30–60; 61–180; 181–360; and 361–480min after gavage to allow for statistical analyses. Please note the progressive rise in urinary [K+]/[Crea] in K+-loaded animals. (c) Urinary [Na+]/[Crea] after gavage in the two experimental groups. (d) Urinary [Na+]/[Crea] compiled at the same time intervals as in b. Note the more pronounced increase in [Na+]/[Crea] in the 2% K+-fed mice compared with the 0.05% K+-fed mice; n=6 mice per experimental group. *P<0.05 and **P<0.01 between control (Ctrl) and experimental groups. (e) Plasma [K+] (mmol/l) in mice 1h after refeeding 0.05 and 2% K+ diets. (f) Total NCC (tNCC) and phospho NCC (pT58) abundance in kidneys collected from mice 1h after refeeding 0.05 and 2% K+ diets; n=3 mice per experimental group. *P<0.05 between experimental groups. Kidney International 2013 83, 811-824DOI: (10.1038/ki.2013.14) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 5 KHCO3, but not NaCl, reduces NaCl cotransporter (NCC) phosphorylation. Parvalbumin promoter (PV-EGFP) (enhanced green fluorescent protein under the parvalbumin promoter) male mice received control (Ctrl), KHCO3 (a), and NaCl (b) solutions through gavage. Kidneys were collected at 15, 30, and 120min post gavage. Total NCC (tNCC) and phosphorylated NCC were detected using anti-tNCC and anti-phospho (pT53, pT58) NCC antibodies. As a loading control, β-actin was detected. Note that KHCO3, but not NaCl, induces NCC dephosphorylation. Quantitative analysis of signals was performed with an infrared-based imaging system. The same Ctrl samples were used in panel a and b. Graphs depict means±s.e.m.; n=3 mice per experimental group. *P<0.05. Kidney International 2013 83, 811-824DOI: (10.1038/ki.2013.14) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 6 Immunofluorescence and immunogold labeling of total NaCl cotransporter (tNCC) and phospho pT53 NCC in distal convoluted tubules (DCTs) 30min post control (Ctrl) and KCl gavage. (a–d) Immunofluorescence and (e–h) immunogold labeling. The DCTs of Ctrl (a) and KCl loaded (b) mice show a similar apical immunofluorescence for tNCC. In contrast, immunostaining for pT53 is clearly reduced in DCTs from KCl-loaded animals (d) compared with Ctrl animals (c). Immunogold labeling (12nm gold particles) of tNCC is found at the apical membrane (arrows) and intracellular vesicles (arrow heads), with equal density in DCT cells of both Ctrl (e) and KCl (f) mice. Immunogold labeling for pT53 is found only in the apical plasma membrane (arrows) in Ctrl (g) and KCI-load mice (h). Consistent with the immunofluorescence data, the immunogold labeling for pT53 is absent from some DCT cells and reduced in other DCT cells of KCl-loaded mice compared with Ctrl. The dotted curve in panel h highlights the cell boundary between an unstained (left) and a weakly stained DCT cell. Kidney International 2013 83, 811-824DOI: (10.1038/ki.2013.14) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 7 Long-term (6h) effects of KCl gavage on total NaCl cotransporter (tNCC), NCC phosphorylation, epithelial sodium channel (ENaC) abundance, and proteolytic cleavage. Parvalbumin promoter (PV-EGFP) (enhanced green fluorescent protein under the parvalbumin promoter) male mice received control (Ctrl) and KCl solutions through gavage. Kidneys were collected after 30 and 360min. The abundance of total and phosphorylated NCC, as well as of all subunits of ENaC, was detected using anti-total NCC, anti-phospho NCC (pT53, pT58), and anti-α/β/γ ENaC antibodies. β-Actin was detected as a loading control. Quantitative analysis of signals was performed with an infrared-based imaging system. Please note sustained NCC dephosphorylation and increased levels of proteolytic cleaved forms of α-(30kDa) and γ-ENaC (70kDa) 6h after gavage in KCl-loaded mice. Graphs depict means±s.e.m.; n=3 mice per experimental group. *P<0.05; **P<0.01. Kidney International 2013 83, 811-824DOI: (10.1038/ki.2013.14) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 8 Spot urine cation excretion in NaCl cotransporter (NCC)+/+ (red) and NCC−/− (blue) mice receiving control (Ctrl; faint colored) and KCl (strong colored) solutions through gavage. (a) Single measurements of urinary [K+]/[Crea] as a function of time after gavage in the four experimental groups. (b) Urinary [K+]/[Crea] compiled in time intervals 0; 30–60; 61–180; 181–360; and 361–480min after gavage to allow for statistical analyses. No differences between the genotypes were seen. (c) Real-time urinary [Na+]/[Crea] after gavage in the four experimental groups. (d) Urinary [Na+]/[Crea] compiled in the same time intervals as in b. Please note the significant difference between urinary [Na+]/[Crea] from KCl-loaded NCC+/+ and NCC−/− mice in the intervals from 30 to 180min post gavage. *P<0.05 and **P<0.01 between experimental group and NCC+/+ Ctrl group at same time interval. #P<0.05 between ‘zero’ and the experimental time intervals within the same experimental group. $P<0.05 between K+-loaded NCC+/+ and NCC+/+ at the same time interval. Bar graphs depict means±s.e.m. Samples from six animals in the K+-loaded groups and five animals in Ctrl groups. Kidney International 2013 83, 811-824DOI: (10.1038/ki.2013.14) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 9 K+-induced dephosphorylation of NaCl cotransporter (NCC) is independent of aldosterone. Aldosterone synthase knockout (AS−/−) and wild-type (AS+/+) male mice received control (Ctrl) and KCl solutions through gavage. Kidneys were collected after 30min. Total NCC and phosphorylated NCC were detected using anti-total NCC and anti-phospho NCC (pT53, pT58) antibodies. As a loading control, β-actin was detected. Quantitative analysis of signals was performed with an infrared-based imaging system. No changes in total NCC (tNCC) within each genotype were observed. Dephosphorylation of NCC (pT53 and pT58) in response to K+ loading was seen in both genotypes. Graphs depict means±s.e.m.; n=3 mice per experimental group. *P<0.05. Kidney International 2013 83, 811-824DOI: (10.1038/ki.2013.14) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 10 Changes in extracellular K+ concentration are not sufficient to induce NaCl cotransporter (NCC) dephosphorylation. Freshly isolated kidney tubules from parvalbumin promoter (PV-EGFP) (enhanced green fluorescent protein under the parvalbumin promoter) male mice were exposed ex vivo for 30min to normal and high K+ (5 and 10mmol/l KCl, respectively) solutions. In control experiments, dephosphorylation of proteins was inhibited by adding 50nM of the phosphatase inhibitor calyculin A (CA). Phosphorylated NCC was detected using anti-phospho (pT53 and pT58) NCC antibodies. As a loading control, β-actin was detected. Quantitative analysis of signals was performed with an infrared-based imaging system. Graphs depict means±s.e.m.; n=3 mice per experimental group. **P<0.01. Kidney International 2013 83, 811-824DOI: (10.1038/ki.2013.14) Copyright © 2013 International Society of Nephrology Terms and Conditions