Rapid dephosphorylation of the renal sodium chloride cotransporter in response to oral potassium intake in mice

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1 & 213 International Society of Nephrology basic research see commentary on page 779 Rapid dephosphorylation of the renal sodium chloride cotransporter in response to oral potassium intake in mice Mads V. Sorensen 1,4, Solveig Grossmann 1,4, Marian Roesinger 1, Nikolay Gresko 1, Abhijeet P. Todkar 1, Gery Barmettler 2, Urs Ziegler 2, Alex Odermatt 3, Dominique Loffing-Cueni 1 and Johannes Loffing 1 1 Institute of Anatomy, University of Zurich, Zurich, Switzerland; 2 Center for Microscopy and Image Analysis (ZMB), University of Zurich, Zurich, Switzerland and 3 Department of Pharmaceutical Sciences, University of Basel, Basel, Switzerland A dietary potassium load induces a rapid kaliuresis and natriuresis, which may occur even before plasma potassium and aldosterone (aldo) levels increase. Here we sought to gain insight into underlying molecular mechanisms contributing to this response. After gastric gavage of 2% potassium, the plasma potassium concentrations rose rapidly (.25 h), followed by a significant rise of plasma aldo (.5 h) in mice. Enhanced urinary potassium and sodium excretion was detectable as early as spot urines could be collected (about.5 h). The functional changes were accompanied by a rapid and sustained (.25 6 h) dephosphorylation of the NaCl cotransporter (NCC) and a late (6 h) upregulation of proteolytically activated epithelial sodium channels. The rapid effects on NCC were independent from the coadministered anion. NCC dephosphorylation was also aldoindependent, as indicated by experiments in aldo-deficient mice. The observed urinary sodium loss relates to NCC, as it was markedly diminished in NCC-deficient mice. Thus, downregulation of NCC likely explains the natriuretic effect of an acute oral potassium load in mice. This may improve renal potassium excretion by increasing the amount of intraluminal sodium that can be exchanged against potassium in the aldo-sensitive distal nephron. Kidney International (213) 83, ; doi:1.138/ki ; published online 27 February 213 KEYWORDS: aldosterone; hyperkalemia; NCC phosphorylation; renal ion transporters Correspondence: Johannes Loffing, Institute of Anatomy, University of Zurich, Zurich, Switzerland. johannes.loffing@anatom.uzh.ch 4 The first two authors contributed equally to this work. Received 18 July 212; revised 12 November 212; accepted 21 November 212; published online 27 February 213 In mammals, plasma K þ concentration must be kept within a narrow range to secure normal function of excitable cells (e.g., neurons, skeletal and cardiac myocytes). Peaks of high K þ intake occur in normal human diet (e.g., after drinking large amounts of fruit juices) and single meals might have K þ contents equaling the entire extracellular pool, resulting in a homeostatic challenge for the organism. Consequently, rapid physiological responses are needed to restore normal plasma K þ. This involves both extrarenal and renal elements. The extrarenal factors include shift of K þ from extracellular to intracellular fluid compartments by insulin and/or b-adrenergic activation of Na þ /K þ -ATPase in various tissues, including muscle and liver. 1 Nevertheless, the final control of K þ homeostasis is achieved by K þ excretion via the kidneys and, to a lesser extent, the colon. 2,3 In the kidney, regulated K þ secretion occurs in the aldosterone (aldo)-sensitive distal nephron (ASDN), which comprises the late distal convoluted tubule (DCT2), the connecting tubule, and the collecting duct. 4 In the ASDN, K þ secretion follows the pump leak model: K þ is taken up through the basolateral Na þ /K þ -ATPase and excreted across the luminal membrane through renal outer medulla and big conductance K þ channels. 2,4,5 A concomitant activation of the absorptive epithelial sodium channel (ENaC) in the apical membrane of ASDN cells increases the electrochemical driving force for K þ secretion. 2 Thus, there is a close interdependence of Na þ absorption through ENaC and K þ secretion through renal K þ channels. 2,5 Aldo is known to stimulate ENaC, the Na þ /K þ -ATPase, 4 and potentially renal outer medulla K þ channels, 2 through a transcriptional response that alters the expression of the iontransporting and regulatory proteins that control transport activity. However, the functional response to enhanced transcription is thought to have a delay of at least 1 h. 4 The kaliuretic effect of K þ intake occurs faster and often even before plasma aldo increases. 6 Moreover, previous studies in humans and animal models indicated that the early kaliuretic response is accompanied by renal Na þ loss, 7 13 which is at odds with the expected Na þ -retaining effect of aldo. The cellular and molecular mechanisms underlying this Kidney International (213) 83,

2 basic research MV Sorensen et al.: Acute homeostatic effects of potassium loading paradox natriuresis in response to an oral K þ load are unclear. On the basis of experiments on dogs, Vander 8 concluded that K þ inhibits tubular Na þ absorption. Later studies suggested that dietary K þ intake may reduce Na þ transport in the proximal tubule 14 and/or in the thick ascending limb. 15,16 In the context of the identification and functional characterization of With No Lysine (WNK) kinases as regulators of the thiazide-sensitive NaCl cotransporter (NCC) in the DCT, the hypothesis was raised that the WNK kinases may act as molecular switches, which shut off electroneutral NaCl cotransport in the DCT in response to K þ intake. 17 Support for this hypothesis comes from the observation that a high K þ diet upregulates the mrnas of the WNK isoforms, WNK4 and KS-WNK1, in mouse kidneys. 18 Moreover, studies on rats showed a diminished natriuretic response to thiazides after dietary K þ loading. 19 Similarly, Vallon et al. 2 reported a mild reduction of NCC abundance and phosphorylation in mice kept on a high dietary K þ intake compared with mice kept on a low K þ diet. However, these experiments were conducted in animals kept for days on K þ -supplemented diets and did not address the acute effects of a K þ load, as it occurs during a K þ -rich meal. Furthermore, these experiments were conducted with KCl supplementation only. It remained unclear whether the observed effects were related to the cation or the coadministered anion. In fact, previous studies highlighted that the type of both cation and anion need to be considered in feeding experiments with electrolyte-supplemented diets. 21 Moreover, there is evidence that the activity of the NCC-regulating STE2/ SPS1-related proline-/alanine-rich kinases SPAK and ORS1 is affected by changes in extracellular chloride concentrations, 22,23 which could act as a confounding factor when KCl is fed. In the present study, we show in mice that a gastric load of 2% K þ causes a rapid aldo-independent dephosphorylation of NCC at four different phosphorylation sites, which goes along with a rapid kaliuresis and natriuresis that precedes the aldo-dependent proteolytic activation of ENaC. The observed changes depend on the increased K þ intake rather than on the coadministered anion (Cl or HCO 3 ). RESULTS Effects of gavage on plasma ion and aldo levels, urinary output, and ion excretion Although control () mice showed rather stable plasma K þ,na þ,cl, and HCO 3 levels, mice receiving gavages of either KCl, KHCO 3, or NaCl solutions showed significant changes in their plasma ion levels, reflecting the given ion loads (Figure 1a d). Interestingly, the increases in plasma [K þ ] and [Na þ ] in response to the given ion solution reached maximum values after 15 3 min, whereas the plasma concentrations for [Cl ] and [HCO 3 ] concentrations peaked at later time points (3 12 min). The reason for this different kinetic of plasma ion levels is unclear. Remarkably, although peak values of plasma [K þ ] reached 1 mm and remained high for 6 h (7.3±.2 mmol/l vs. 5.±.1 mmol/l in mice at 36 min), the K þ -loaded mice did not show any signs of illness (e.g., reduced activity, paralysis, cardiac arrests). However, mice with KCl loading revealed a significant and sustained elevation of plasma aldo levels compared with mice (Figure 1e). The plasma aldo levels were not significantly elevated 15 min after gavage, but rose sharply (fourfold) after 3 min. At 12 and 36 min, plasma aldo levels still remained approximately twofold higher than in animals. Following gavage, urinary creatinine concentrations dropped (Supplementary Figure S1 online) and urination frequency increased similarly in all mice (1.25±.18,.33±.11, and.6±.4 urinations per mouse per hour in 3 h, 3 6 h, and 6 8 h, respectively), likely as a function of the gastric water load. Analysis of the spot urines of mice showed that the gastric gavage of the ion-free sucrose solution was followed by a transient (3 18 min) drop of urinary [K þ ]/[creatinine] and [Na þ ]/[creatinine]. In contrast, mice with gastric gavages of KCl and KHCO 3 showed significantly elevated urinary [K þ ]/[creatinine] during the early (3 18 min) and late ( min) urine sampling periods (Figure 2a and b). Notably, the KCl- and KHCO 3 -induced early kaliuresis was accompanied by a significant increase in urinary [Na þ ]/[creatinine] levels (Figure 2c and d), peaking within the first 12 min. Assuming that urinary creatinine excretion is constant during the sampling period of 36 min, the [electrolyte]/[creatinine] data suggest an increased urinary K þ and Na þ excretion, following the gastric K þ load. This interpretation is supported by experiments, in which urine was collected at 3 and 3 6 h intervals post gavage, and urinary ion and creatinine excretions were measured (Supplementary Figure S2 online). For the sake of simplicity, [electrolyte]/[creatinine] and urinary ion excretion are used synonymously. Gastric gavage of KCl leads to a rapid dephosphorylation of NCC To analyze the effect of oral KCl load on NCC abundance and phosphorylation, kidneys of - and KCl-loaded mice were collected 15, 3, and 12 min after gastric gavage and analyzed by western blot analysis, using antibodies recognizing total NCC (tncc) and NCC phosphorylated at amino acids T53, T58, S71, and S89 (, pt58, ps71, and ps89), respectively. mice showed stable tncc and phospho NCC protein levels following gavage (Figure 3). KCl loading did not have any effect on tncc (Figure 3), but revealed a rapid and pronounced decrease of the phosphorylation of NCC at all four investigated phospho sites (Figure 3). Similar observations were made in female parvalbumin promoter (PV-EGFP) (enhanced green fluorescent protein (EGFP) under the parvalbumin promoter), wild-type male C57/Bl6, and NMRI mice analyzed 3 min after gavage (data not shown), indicating that the rapid NCC dephosphorylation is independent of the gender, the mouse strain, or transgenic EGFP expression in the DCT. 812 Kidney International (213) 83,

3 MV Sorensen et al.: Acute homeostatic effects of potassium loading basic research Plasma K + (mmol/l) Plasma CI (mmol/l) Plasma HCO 3 (mmol/l) Plasma Na + (mmol/l) Plasma aldo (nm) KCI KHCO 3 NaCl Figure 1 Plasma values as a function of time after gavage in control (; red), KCl (blue), KHCO 3 (green), and NaCl (black)-loaded mice. (a) Plasma [K þ ] versus time in the four experimental groups. Please note that plasma [K þ ] is significantly elevated already after 15 min in KCl and KHCO 3 loaded animals in comparison with mice. (b) Plasma [Cl ] after gavage. Significant increase in plasma [Cl ] in the KCl group 3 min post gavage. (c) Plasma [HCO 3 ] is increased in blood samples from the KHCO 3 group compared with the group at the time points 3 and 12 min after gavage. (d) [Na þ ] concentration in the plasma is augmented in the NaCl group shortly after the gavage at time point 15 min. (e) Plasma aldosterone (aldo) is significantly elevated in samples from KCl-loaded animals 3, 18, and 36 min after gavage when compared with. Graphs depict means ±s.e.m. Po.5, Po.1. Measurements are from three mice per group. Feeding of a K þ -rich diet mimics the effect of K þ loading by gastric gavage Although gastric K þ loading through gavage ensures a wellcontrolled timing and dosing of the experimental stimulus, it has the disadvantage of being a forced and artificial procedure. Therefore, we analyzed whether a voluntary K þ intake through a K þ -enriched diet may provoke similar changes as the gavage. Mice were fasted overnight before they were offered ad libitum a chow containing either low (.5%) or high (2%) K þ in the morning. Although urinary [K þ ]/ [creatinine] remained low in.5% K þ -fed mice, it rose progressively in the 2% K þ -fed mice during the 8 hours urine sampling period (Figure 4a and b). As both diets contained.3% Na þ, urinary [Na þ ]/[creatinine] increased after feeding in both groups, but it reached much higher values in the 2% K þ -fed mice (Figure 4c and d), indicating that part of the enhanced Na þ excretion reflects K þ -induced natriuresis. Similar to the gavage experiments, the enhanced urinary [K þ ]/[creatinine] and [Na þ ]/[creatinine] in the K þ -loaded mice were preceded by a significant increase in plasma K þ levels (Figure 4e) and a significant downregulation of NCC phosphorylation, but not tncc abundance (Figure 4f). The delayed detection of increased urinary Na þ and K þ excretion compared with NCC Kidney International (213) 83,

4 basic research MV Sorensen et al.: Acute homeostatic effects of potassium loading 6 3 2% K + + CI # # 2% K + + HCO 3 Urinary [K + ]/[Crea] 4 2 Urinary [K + ]/[Crea] 2 1 # # # # Time intervals (min) Urinary [Na + ]/[Crea] Urinary [Na + ]/[Crea] % K + + CI # # 2% K + + HCO 3 # Time intervals (min) Figure 2 Urine cation concentrations normalized to creatinine concentrations from mice receiving control (; red), KCl (blue), and KHCO 3 (green) solutions through gavage. (a) Single measurements of urinary [K þ ]/[Crea] as a function of time after gavage in the three experimental groups. (b) Urinary [K þ ]/[Crea] compiled in time intervals ; 3 6; 61 18; ; and min after gavage to allow for statistical analyses. (c) Urinary [Na þ ]/[Crea] after gavage in the three experimental groups. (d) Urinary [Na þ ]/[Crea] compiled in the same time intervals as in b. Note the striking increases of [Na þ ]/[Crea] in the K þ -loaded groups. Po.5 and Po.1 between and experimental groups. # Po.5 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. dephosphorylation and hyperkalemia might be related, at least in part, to the lag phase between stimulated renal ion excretion and the voluntary voiding of urine in the mice. Dephosphorylation of NCC protein is dependent on the K þ rather than Cl load To investigate whether the described KCl-induced NCC dephosphorylation was a result of the K þ or Cl load or their combination, we conducted gavage experiments with KHCO 3 or NaCl solutions that contained equivalents of K þ or Cl as in the given KCl solution. In these experiments, KHCO 3, but not NaCl, mimicked the effects of KCl. Compared with, NCC phosphorylation at T53 and T58 was profoundly reduced 15, 3, and 12 min after the gavage of KHCO 3. NaCl gavage did not have any effect on abundance or phosphorylation of NCC at any of the analyzed time points (Figure 5). K þ loading reduces phosphorylation of NCC at the plasma membrane To investigate whether the gastric K þ loading changes the subcellular localization of NCC, immunohistochemical analysis was performed using antibodies against tncc and NCC phosphorylated at position T53 (Figure 6). In mice, both the antibodies showed a strong apical immunofluorescent labeling of DCT cells (Figure 6a and c). In mice killed 3 min after the gavage of KCl, the strong apical labeling for tncc was maintained (Figure 6b), but the immunostaining for was markedly reduced compared with mice (Figure 6c and d). Some of the DCT cells in K þ -loaded animals also lacked any phosphorylated NCC (Figure 6d). Immunoelectron microscopy confirmed previous studies, 24,25 which showed that tncc is found at the apical plasma membrane and in subapical vesicles (Figure 6e), while phosphorylated NCC resides almost exclusively at the plasma membrane (Figure 6g). Consistent with the immunofluorescent observations, the abundance and subcellular localization of tncc was unaffected in K þ -loaded mice (Figure 6f), whereas the labeling of phosphorylated NCC was profoundly reduced or even lacking in DCT cells in response to the gastric gavage of KCl (Figure 6h). K þ loading causes early dephosphorylation of NCC and late proteolytic cleavage of ENaC The above-described urine analyses pointed to sustained kaliuresis but only transient natriuresis within the first 3 h following an oral K þ load. To test whether the reversed 814 Kidney International (213) 83,

5 MV Sorensen et al.: Acute homeostatic effects of potassium loading basic research KCI tncc 15 min 3 min 12 min 15 min 3 min 12 min 13 tncc/β-actin 1..5 pt /β-actin pt58/β-actin ps71 ps ps71/β-actin ps89/β-actin , 15 min, 3 min, 12 min KCI, 15 min KCI, 3 min KCI, 12 min Figure 3 KCl rapidly dephosphorylates NaCl cotransporter (NCC). Parvalbumin promoter (PV-EGFP) (enhanced green fluorescent protein under the parvalbumin promoter) male mice received control () and KCl solutions through gavage and kidneys were collected after 15, 3, and 12 min. Total NCC (tncc) and phosphorylation of NCC in 5 mg of membrane protein fractions of whole-kidney lysates were detected using anti-tncc and anti-phospho NCC (, pt58, ps71, and ps89) antibodies. Detection of b-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 ¼ 3 mice in each experimental group. Po.5, Po.1. natriuresis could be related to recovery of NCC phosphorylation, we studied NCC protein abundance and phosphorylation levels in mice 6 h after gastric gavage (Figure 7). tncc abundance did not differ between and K þ -loaded mice, and phosphorylated NCC remained very low even 6 h after the KCl gavage. Thus, rephosphorylation of NCC unlikely accounted for the reversed urinary Na þ excretion. Therefore, we tested for the activation of other renal Na þ -absorptive mechanisms. The most obvious candidate for enhanced Na þ reabsorption in response to K þ loading was ENaC. Previous studies by others suggested that the appearance of proteolytically cleaved low molecular weight forms of a- and g- ENaC correlate with ENaC activity We did not see any effect of oral K þ loading on the protein abundance and electrophoretic profile of a-, b-, and g-enac protein within the first 2 h post gavage (Supplementary Figure S3 online). However, 6 h post gavage of KCl we detected, in addition to the full length a- and g-enac forms an increased amount of the proteolytically cleaved B3 and B7 kda fragments of a-enac and g-enac, respectively (Figure 7). For the b-subunit of ENaC, no differences between and K þ -loaded mice were observed at any time point. NCC-deficient mice show reduced K þ -induced natriuresis To address whether the observed early natriuresis in response to an oral K þ load was related to a functional downregulation of NCC, we used NCC-deficient mice. After K þ loading, NCC þ / þ and NCC / mice had similar [K þ ]/ [creatinine] levels in the urine (Figure 8a and b). However, although a tendency of an increased urinary Na þ excretion / was observed in NCC mice, the natriuretic effect was clearly less pronounced than in NCC þ / þ mice. Within the first 3 h after the gavage, urinary [Na þ ]/[creatinine] levels in NCC / mice reached less than 5% of the peak levels of NCC þ / þ animals (Figure 8c and d). Thus, the early renal Na þ loss after an acute oral K þ load depends on NCC. / Nevertheless, the mild natriuretic effect seen in NCC mice suggests that NCC-independent effects contribute as well. High K þ intake was also suggested to reduce Na þ Kidney International (213) 83,

6 basic research MV Sorensen et al.: Acute homeostatic effects of potassium loading.5% K + diet 2% K + diet 4 25 Urinary [K + ]/[Crea] Urinary [K + ]/[Crea] Time intervals (min) Urinary [Na + ]/[Crea] Urinary [Na + ]/[Crea] % K + diet 2% K + diet Time intervals (min) Plasma [K + ] % K + 2% K + tncc pt58 NCC.5% K + 2% K tncc/β-actin pt58/β-actin % K + 2% K + Figure 4 Effect of dietary K þ intake on urinary ion excretion, plasma K þ concentration, and NaCl cotransporter (NCC). After overnight fasting, mice received either a.5 or 2% K þ diet. (a) Single measurements of urinary [K þ ]/[Crea] as a function of time after gavage in the two experimental groups. (b) Urinary [K þ ]/[Crea] compiled in time intervals ; 3 6; 61 18; ; and min after gavage to allow for statistical analyses. Please note the progressive rise in urinary [K þ ]/[Crea] in K þ -loaded animals. (c) Urinary [Na þ ]/[Crea] after gavage in the two experimental groups. (d) Urinary [Na þ ]/[Crea] compiled at the same time intervals as in b. Note the more pronounced increase in [Na þ ]/[Crea] in the 2% K þ -fed mice compared with the.5% K þ -fed mice; n ¼ 6 mice per experimental group. Po.5 and Po.1 between control () and experimental groups. (e) Plasma [K þ ] (mmol/l) in mice 1 h after refeeding.5 and 2% K þ diets. (f) Total NCC (tncc) and phospho NCC (pt58) abundance in kidneys collected from mice 1 h after refeeding.5 and 2% K þ diets; n ¼ 3 mice per experimental group. Po.5 between experimental groups. transport in the proximal tubule 14 and/or in the thick ascending limb. 15,16 K þ -induced dephosphorylation of NCC is independent of aldo To investigate whether the K þ -induced dephosphorylation of NCC was dependent on aldo, we used mice lacking aldo synthase (AS). 3 Consistent with the idea that NCC is an aldo-induced protein, 31 AS / mice showed only about 5% of NCC protein abundance and phosphorylation than wild-type mice (Figure 9). Oral K þ loading supressed NCC phosphorylation almost completely in both genotypes, whereas tncc abundance remained stable. Owing to the different baseline values, the relative decrease in NCC phosphorylation was more profound in AS þ / þ mice ( 92% for and 91% for pt58; Po.5) than in AS / mice ( 61% for and 82% for pt58; P ¼.8 and Po.5, respectively), but the final result was similar in both genotypes (Figure 9). High levels of extracellular K þ do not directly trigger NCC dephosphorylation To address whether the increased plasma K þ concentration could act as a direct trigger on DCT cells to decrease NCC phosphorylation, we conducted ex vivo experiments with freshly isolated renal tubules incubated in buffers containing either 5 or 1 mmol/l K þ. The level of NCC phosphorylation was similar in samples incubated in 5 and 1 mmol/l K þ (Figure 1). As a positive control for alterable NCC phosphorylation, the tubule suspensions were incubated in 816 Kidney International (213) 83,

7 MV Sorensen et al.: Acute homeostatic effects of potassium loading basic research KHCO 3 15 min 3 min 12 min 15 min 3 min 12 min 2. tncc 13 tncc/β-actin /β-actin pt58 NaCI 15 min 3 min 12 min 15 min 3 min 12 min 13 pt58/β-actin 1..5., 15 min, 3 min, 12 min KHCO 3, 15 min KHCO 3, 3 min KHCO 3, 12 min tncc 13 tncc/β-actin /β-actin pt58 13 pt58/β-actin 1.., 15 min, 3 min, 12 min NaCI, 15 min NaCI, 3 min NaCI, 12 min Figure 5 KHCO 3, but not NaCl, reduces NaCl cotransporter (NCC) phosphorylation. Parvalbumin promoter (PV-EGFP) (enhanced green fluorescent protein under the parvalbumin promoter) male mice received control (), KHCO 3 (a), and NaCl (b) solutions through gavage. Kidneys were collected at 15, 3, and 12 min post gavage. Total NCC (tncc) and phosphorylated NCC were detected using anti-tncc and anti-phospho (, pt58) NCC antibodies. As a loading control, b-actin was detected. Note that KHCO 3, but not NaCl, induces NCC dephosphorylation. Quantitative analysis of signals was performed with an infrared-based imaging system. The same samples were used in panel a and b. Graphs depict means±s.e.m.; n ¼ 3 mice per experimental group. Po.5. the presence of Ser/Thr phosphatase inhibitor calyculin A. The addition of calyculin A significantly increased NCC phosphorylation independent of the extracellular K þ concentration (Figure 1). DISCUSSION Our study reveals that a single gastric K þ load given to mice leads to a prompt renal response that includes both kaliuresis and natriuresis, which goes along with an early and sustained (15 min - 6 h) dephosphorylation of NCC and a late (6 h) proteolytic activation of ENaC. The dephosphorylation of NCC strictly depends on K þ and is independent from the coadministered anion. NCC deactivation likely explains the early natriuresis and may contribute to improved K þ excretion in the ENaC-positive ASDN. Dephosphorylation of NCC is independent of altered plasma aldo and direct effects of enhanced extracellular K þ concentrations on DCT cells. The increased urinary K þ excretion and the activation of ENaC in response to gastric K þ load are expected and are in line with numerous studies that analyzed the effect of dietary K þ intake on renal K þ handling and ENaC activity in the ASDN In contrast, the early natriuresis after the Kidney International (213) 83,

8 basic research MV Sorensen et al.: Acute homeostatic effects of potassium loading tncc tncc KCI 1 μm 5 nm Figure 6 Immunofluorescence and immunogold labeling of total NaCl cotransporter (tncc) and phospho NCC in distal convoluted tubules (DCTs) 3 min post control () and KCl gavage. (a d) Immunofluorescence and (e h) immunogold labeling. The DCTs of (a) and KCl loaded (b) mice show a similar apical immunofluorescence for tncc. In contrast, immunostaining for is clearly reduced in DCTs from KCl-loaded animals (d) compared with animals (c). Immunogold labeling (12 nm gold particles) of tncc is found at the apical membrane (arrows) and intracellular vesicles (arrow heads), with equal density in DCT cells of both (e) and KCl (f) mice. Immunogold labeling for is found only in the apical plasma membrane (arrows) in (g) and KCI-load mice (h). Consistent with the immunofluorescence data, the immunogold labeling for is absent from some DCT cells and reduced in other DCT cells of KCl-loaded mice compared with. The dotted curve in panel h highlights the cell boundary between an unstained (left) and a weakly stained DCT cell. gastric K þ load is surprising, although this phenomenon has been consistently observed in sheep, rats, and humans. 6,12,35,36 Now, we link this acute natriuretic effect to a rapid dephosphorylation of mouse NCC at T53, T58, S71, and S89 (corresponding to T55, T6 S73, and S91 in humans 22 ), which is thought to inactivate the transporter. 37 Notably, the profound acute regulation of NCC shown here contrasts with the rather small or missing effects on NCC phosphorylation and abundance that have been reported to occur in rodents with chronic (days) changes in dietary K þ intake. 2,32,33 The reasons for these discrepant effects are unclear, but it is possible that the downregulation of NCC in the acute setting is counterbalanced in the long run by compensatory mechanisms that prevent chronic renal Na þ loss. The rapidity and the magnitude of the effect of K þ loading on NCC phosphorylation are striking. One could argue that the observed effect is an unphysiological response to the forced K þ loading through gavage that caused exceedingly high plasma [K þ ] values of B1 mmol/l. However, we made similar observations in mice fed a high K þ diet (2% K þ ) that is close to the one found in natural food (e.g., in soy beans, herbs, dried apricots, and dried tomatoes). With the 2% K þ diet, plasma [K þ ] rose to B7 mmol/l, which still might be in the physiological range for mice. In a recent review, Meneton et al. reported that in mice on a standard diet, plasma [K þ ] may vary between 3.5 and 8.5 mmol/l. 38 These huge variations of plasma [K þ ] might be related to the high food intake of mice related to their body weight. Adult mice usually eat g of dry food per day (own observation). With a K þ content of.8% (our standard mouse chow), this equals a daily K þ intake of 3 4 mg. Estimating that the extracellular volume is 2% of the body weight 39 and assuming an extracellular K þ concentration of 4 5 mmol/l, the total extracellular pool of K þ in a 25 g mouse is only.8 1. mg K þ. Thus, the standard daily K þ intake of a mouse exceeds the amount of K þ in the extracellular volume by a factor of 4. In humans, the factor is reported to be approximately one. 3 Thus, it is not unexpected that depending on variations in food intake, plasma [K þ ] values may vary in mice much more than in humans. The NCC protein is not only localized to the apical plasma membrane, but also resides in subapical vesicles. This raised the question whether the K þ -induced dephosphorylation of NCC might be accompanied by rapid endocytosis of NCC. However, although our immunofluorescent and immunogold studies confirmed the rapid dephosphorylation of apical NCC, the amount of tncc in the apical plasma membrane remained rather stable, at least within the analyzed first 3 min following gavage. As such, the data are consistent with the view that the NCC activity is regulated by phosphorylation/dephosphorylation in the apical membrane rather than by altered vesicular trafficking to or from the membrane. This conclusion is supported by findings in vasopressintreated rats 24 and mice deficient for the NCC-regulating kinase SPAK, 4 but contrasts with previous data that pointed to a regulation of NCC by vesicular trafficking in response to altered dietary Na þ intake, 41 angiotensin II stimulation, 42 and vasopressin treatment. 25 In principle, a reduced phosphorylation of a protein can result from reduced kinase and/or an increased phosphatase 818 Kidney International (213) 83,

9 MV Sorensen et al.: Acute homeostatic effects of potassium loading basic research 3 min 36 min KCI KCI tncc tncc/β-actin pt53/β-actin pt58 13 p T58/β-actin α-enac β-enac γ-enac α-enac (95 kda)/ β-actin γ-enac (9 kda)/ β-actin β-enac/β-actin , 3 min KCI, 3 min, 36 min KCI, 36 min α-enac (3 kda)/ β-actin γ-enac (7 kda)/ β-actin , 3 min KCI, 3 min, 36 min KCI, 36 min Figure 7 Long-term (6 h) 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 () and KCl solutions through gavage. Kidneys were collected after 3 and 36 min. The abundance of total and phosphorylated NCC, as well as of all subunits of ENaC, was detected using anti-total NCC, anti-phospho NCC (, pt58), and antia/b/g ENaC antibodies. b-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 a-(3 kda) and g-enac (7 kda) 6 h after gavage in KCl-loaded mice. Graphs depict means±s.e.m.; n ¼ 3 mice per experimental group. Po.5; Po.1. activity. The phosphorylation of NCC is controlled by a complex network of kinases that includes the WNK1, WNK4, and SPAK/OSR1 kinases. 23, Although SPAK and OSR1 are thought to control the phosphorylation of NCC at residues analyzed in this study, the WNK kinases were shown to control the activity of SPAK/OSR1. 45 In turn, phosphatases, such as protein phosphatase-4, are thought to counterbalance the kinases acting on NCC. 46 We did not analyze the impact of the gastric K þ load on this kinase and phosphatase network; however, previous studies indicated that chronic changes in dietary K þ have an impact on the expression of WNK kinases 18 or signal pathways downstream of WNK. Gene-modified mouse models with lacking or altered activity of SPAK, WNK1, WNK4, and other NCC regulators have been generated (reviewed by Hoorn et al. 44 ), which might be used to further dissect underlying mechanisms. The signals by which a gastric K þ load reduces NCC phosphorylation are unclear. Several hormones were shown Kidney International (213) 83,

10 basic research MV Sorensen et al.: Acute homeostatic effects of potassium loading NCC +/+ KCI NCC / KCI NCC +/+ NCC / Urinary [K + ]/[Crea] Urinary [K + ]/[Crea] # # # # # Time intervals (min) Urinary [NA + ]/[Crea] Urinary [NA + ]/[Crea] NCC +/+ KCI NCC / KCI NCC +/+ NCC / #,$ #,$ $ # Time intervals (min) Figure 8 Spot urine cation excretion in NaCl cotransporter (NCC) þ / þ (red) and NCC / (blue) mice receiving control (; faint colored) and KCl (strong colored) solutions through gavage. (a) Single measurements of urinary [K þ ]/[Crea] as a function of time after gavage in the four experimental groups. (b) Urinary [K þ ]/[Crea] compiled in time intervals ; 3 6; 61 18; ; and min after gavage to allow for statistical analyses. No differences between the genotypes were seen. (c) Real-time urinary [Na þ ]/[Crea] after gavage in the four experimental groups. (d) Urinary [Na þ ]/[Crea] compiled in the same time intervals as in b. Please note the significant difference between urinary [Na þ ]/[Crea] from KCl-loaded NCC þ / þ and NCC / mice in the intervals from 3 to 18 min post gavage. Po.5 and Po.1 between experimental group and NCC þ / þ group at same time interval. # Po.5 between zero and the experimental time intervals within the same experimental group. $ Po.5 between K þ -loaded NCC þ / þ and NCC þ / þ at the same time interval. Bar graphs depict means±s.e.m. Samples from six animals in the K þ -loaded groups and five animals in groups AS +/+ AS / AS +/+ AS / tncc KCI KCI tncc/β-actin /β-actin pt pt58/β-actin KCI KCI Figure 9 K þ -induced dephosphorylation of NaCl cotransporter (NCC) is independent of aldosterone. Aldosterone synthase knockout (AS / ) and wild-type (AS þ / þ ) male mice received control () and KCl solutions through gavage. Kidneys were collected after 3 min. Total NCC and phosphorylated NCC were detected using anti-total NCC and anti-phospho NCC (, pt58) antibodies. As a loading control, b-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 ( and pt58) in response to K þ loading was seen in both genotypes. Graphs depict means±s.e.m.; n ¼ 3 mice per experimental group. Po Kidney International (213) 83,

11 MV Sorensen et al.: Acute homeostatic effects of potassium loading basic research 5 mmol/l KCI 1 mmol/l KCI 5 mmol/l KCI+ 5 nm CA 1 mmol/l KCI+ 5 nm CA /β-actin pt pt58/β-actin mmol/l KCI 1 mmol/l KCI 5 mmol/l KCI + 5 nm CA 5 mmol/l KCI + 5 nm CA Figure 1 Changes in extracellular K þ 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 3 min to normal and high K þ (5 and 1 mmol/l KCl, respectively) solutions. In control experiments, dephosphorylation of proteins was inhibited by adding 5 nm of the phosphatase inhibitor calyculin A (CA). Phosphorylated NCC was detected using anti-phospho ( and pt58) NCC antibodies. As a loading control, b-actin was detected. Quantitative analysis of signals was performed with an infrared-based imaging system. Graphs depict means±s.e.m.; n ¼ 3 mice per experimental group. Po.1. to control NCC phosphorylation in the kidney, including aldo, 31,47 vasopressin, 24,25 and angiotensin II. 31 However, of these hormones, only aldo appears to be regulated by dietary K þ intake. 48 Nevertheless, aldo is unlikely the potential mediator of the K þ -induced effect on NCC, because (i) the dephosphorylation of NCC appears before the increase in plasma aldo, (ii) the NCC dephosphorylation is also seen in AS-deficient mice, and (iii) aldo is considered to increase rather than decrease NCC phosphorylation. 47 That aldoindependent factors contribute to the rapid renal control of K þ homeostasis was stressed already by Rabinowitz 49 more than 2 years ago. He suggested that dietary K þ intake may cause the release of unknown kaliuretic factors that have an impact on the kidney and explain the rapid and aldoindependent homeostatic response of the kidney to a gastric K þ load. Rabinowitz 49 and, more recently, Youn 3 also suggested that this renal response can occur even in the absence of any measurable change in plasma K þ. Our experimental protocols (i.e., gastric K þ loading by gavage or voluntary feeding) caused profound increases in plasma K þ concentrations. Therefore, we cannot exclude that NCC dephosphorylation may occur only when plasma K þ is increased. At least, our ex vivo experiments on isolated renal tubules indicated that high extracellular K þ concentrations per se are not sufficient to trigger NCC dephosphorylation through direct effects on DCT cells. In contrast to the rapid dephosphorylation of NCC, the observed proteolytic cleavage of ENaC might be aldodependent. The induction of plasma aldo peaks at 3 min and hence precedes the appearance of the low molecular weight forms of a- and g-enac. The observed time course is fully compatible with the known effect of aldo that usually includes a lag period in which a transcriptional program is activated to stimulate ENaC. 4 Thus, based on our observations, we propose a two-phase model for the renal adaptation to an acute gastric K þ load. In the first phase, an aldo-independent dephosphorylation and inactivation of NCC leads to an immediate enhanced delivery of Na þ to the downstream localized ASDN. In the ASDN, the enhanced tubular Na þ load increases the concentration gradient for Na þ reabsorption through ENaC, which improves the electrochemical driving force for K þ secretion through apical K þ channels. In phase two, an aldo-dependent activation of ENaC further improves the electrochemical driving forces for K þ secretion and also limits the renal Na þ loss. On the first glance, the similar K þ load-induced K þ secretion in NCC-wild-type and NCC-deficient mice appear to contradict our interpretation that NCC downregulation contributes to early kaliuresis. However, NCC-deficient mice have, as a consequence of their lifelong NCC deficiency, already an enhanced Na þ delivery to the ASDN and an aldodependent upregulation of ENaC. 5 Thus, these mice are well prepared to excrete K þ, which might be further improved by K þ intake induced downregulation of Na þ transport in nephron segments upstream of the DCT (e.g., in proximal tubule 14 and/or thick ascending limb 15,16 ). However, the lack of NCC and hence the possibility to switch between Na þ reabsorption in DCT and ASDN may limit the dynamic range of the kidney to independently control renal Na þ and K þ secretion. In fact, NCC-deficient mice 51 and Gitelman patients 52 have an increased risk for hypokalemia. In conclusion, our study provides several novel insights into the mechanisms of the renal control of K þ homeostasis. The observed dephosphorylation and downregulation of NCC may not only improve renal K þ secretion, but likely explains the yet not well-understood early natriuresis in response to K þ load and may also contribute to the known antihypertensive effect of K þ -rich diets. 53 Kidney International (213) 83,

12 basic research MV Sorensen et al.: Acute homeostatic effects of potassium loading MATERIALS AND METHODS Animals Unless otherwise stated, the animals used in this study are male transgenic mice overexpressing green fluorescent protein under the PV-EGFP. 54 Key experiments were also repeated in female PV-EGFP mice, male wild-type C57/Bl6, and male outbreed NMRI male mice (both from Charles River, Kisslegg, Germany). Male wild-type and knockouts of NCC (NCC þ / þ and NCC / ) 55 and aldosterone synthase (AS þ / þ and AS / ) 3 were used to address the role of NCC and aldo for the observed changes in urinary ion excretion and NCC dephosphorylation, respectively. All mice were between 8 and 16 weeks old. They were maintained at a 12/12 h light/dark cycle and had access to standard chow and water ad libitum. Twelve hours before the experiment, animals were fasted. All animal experiments were conducted according to Swiss Laws and approved by the veterinary administration of the Canton of Zurich, Switzerland. General experimental setup Gastric gavage with solutions containing 2% sucrose () or 2% sucrose with either 2% K þ (512 nm) or 1.2% Na þ (512 nm) with Cl as the anion, or 2% K þ (512 nm) with HCO 3 as anion were given between 9 and 1 AM. Sucrose was added to the gavage solution, because previous data by other suggested that a caloric intake may have a permissive role for the early kaliuresis. Each mouse received 15 ml solution per gram body weight. Sampling times were 15, 3, 12, or 36 min post gavage. At these time points, blood was collected for ion analyses and aldo measurements and kidneys were collected for semiquantitative western blot analysis. In an additional experiment, mice were fasted overnight and then refed either with a.5% K þ or a 2% K þ diet for 1 h before collecting kidney and blood samples. See Supplementary Information online for details. Aldo quantification Quantification of aldo in plasma (15 ml) was performed by liquid chromatography mass spectrometry. Samples were purified by solid-phase extraction and steroids were resolved on an Atlantis T3 column (Waters, Milford, MA). The mass spectrometry was operated in atmospheric pressure electrospray-positive ionization mode. Determination of aldo was carried out by selected multiple reaction monitoring. Precursor and product ion and retention time were m/z 361.3, 3.2, and 2.4 min, respectively. Aldo was quantified from calibration curves of the ratio of the peak area of the authentic standards. Deuterized aldo was used as internal standard. See Supplementary Information online for details. Urine analysis After gavage, subsets of mice from the different experimental groups were placed in metabolic cages. Parafilm (Pechiney Plastic Packing Company, Menasha, WI) was stretched over the upper frame of the urine/stool separation cone. Urine was collected by one of the investigators (MVS) as soon as it was delivered. When animals urinated during gavage or at the end of the experiment, this urine was collected to obtain pretreatment and endpoint (48 min) data, respectively. Na þ and K þ concentrations in the urine were measured with ion chromatography. Unfortunately, the volumes of spot urines were too small for an accurate recording of volume information. To get estimates for ion excretion, the urinary concentrations of Na þ and K þ in each sample were normalized to creatinine concentrations. Moreover, in an additional experiment, larger urine samples were collected in time intervals from to 3 h and from 3 to 6 h post gavage, which allowed measurements of urine volumes and ion concentrations, and hence the calculation of ion excretion rates (Supplementary Figure S2 online). Immunoblotting Kidneys were collected and processed for immunoblotting as described. 56 Nitrocellulose membranes were incubated with primary antibodies (Supplementary Table S1 online) detecting tncc, NCC phosphorylated at T53, T58, S73, and S89, a-enac, b- ENaC, 56 g-enac, 56 and b-actin (Sigma, Buchs, Switzerland), followed by infrared dye-conjugated secondary antibodies against rabbit and mouse IgGs (LI-COR Biosciences, Bad Homburg, Germany), respectively. Antibodies against tncc, phosphorylated NCC, and a-enac were newly generated and characterized by using kidneys from NCC / 55 and a-enac / 57 mice (Supplementary Figure S4 online). See Supplementary Information online for details. Immunohistochemistry and immunogold labeling Thirty minutes post gavage, kidneys of anesthetized mice were fixed with 3% paraformaldehyde and.1% glutaraldehyde in a cacodylate-sucrose buffer as described previously 58 Thereafter, kidneys were removed and processed for either immunofluorescence or immunogold electron microscopy. See Supplementary Information online for details. Ex vivo high K þ treatment Kidneys were collected from mice perfused with 1 ml phosphatebuffered saline followed by 1 ml of digestion buffer (KREBS buffer supplemented with 1 mg/ml hyaluronidase, 1 mg/ml collagenase type-1, and.1 mg/ml DNase I (Roche, Basel, Switzerland)). Following perfusion, kidneys were separated in the cortex and medulla, and the renal cortex was minced and further digested in digestion buffer for 1 min at 37 1C. Digested cortex was filtered using 25, 212, 1, and 4 mm sieves, and the tubules caught on the 4 mm sieve were collected in ice-cooled KREBS buffer. Tubules were incubated in KREBS buffer (normal KCl: 5 mmol/l and high KCl: 1 mmol/l) in the presence and absence of the Ser/Thr phosphatase inhibitor 5 nm calyculin A at 37 1C for 3 min. Subsequent to the incubation, the tubules were centrifuged (8g, 5 min, 4 1C). The pellets were solubilized in Laemmli buffer, and western blot analysis was performed as described in Supplementary Information online. Statistics Statistics were performed with GraphPadPrism software (San Diego, CA). The data are shown as mean values±s.e.m. Student s t-test was used to compare between two groups. One-way analysis of variance with Bonferroni s multiple comparison post test was used to compare more groups in the same experimental series. DISCLOSURE All the authors declared no competing interests. ACKNOWLEDGMENTS We gratefully acknowledge the expert technical support of Monique Carrel, Bruno Guhl, and Thierry Da Cunha. We thank Dr Gary Shull, Dr Oliver Smithies, and Dr Edith Hummler for providing the NCC-, the AS-deficient mice, and the kidneys from a-enac-deficient mice, respectively. The blood gas analyses were performed in the Zurich Integrative Rodent Physiology (ZIRP) core facility. For ion chromatography, we had expert support from Udo Schnitzbauer, 822 Kidney International (213) 83,

13 MV Sorensen et al.: Acute homeostatic effects of potassium loading basic research who is working in the group of Dr Carsten Wagner in Zurich. We thank Dr Olivier Staub for critically reading the manuscript. We also thank the two anonymous reviewers for their thoughtful comments on the manuscript. The laboratory of Johannes Loffing is supported by the Swiss National Science Foundation project grant /1 and 313_1929/1, the National Centre of Competence in Research (NCCR) Kidney.CH, and the Zurich Centre for Integrative Human Physiology. Solveig Grossmann is a postdoctoral fellow of the Marie-Curie Fellowship European Community s Seventh Framework Programme (FP7/27 213) under grant agreement number SUPPLEMENTARY MATERIAL Table S1. Antibodies used for immunohistochemical and western blot analysis. Figure S1. Urine creatinine concentrations in mice that received by gavage (red), KCl (blue), and KHCO3 (green) solutions. Figure S2. Urinary excretion of K þ,na þ, and creatinine excretion in mice after gavage of a control (, red) or a KCl (2% K þ, blue) solution. Figure S3. Short-term (15 12 min) effects of KCl gavage on ENaC protein abundance. Figure S4. Characterization by immunoblotting of novel antibodies against tncc, phospho-ncc (, pt58, ps71, and ps89), and a-enac using kidneys from adult NCC þ / þ and NCC / mice, and newborn a-enac þ / þ and / mice, respectively. Supplementary material is linked to the online version of the paper at REFERENCES 1. Clausen T. Na þ -K þ pump regulation and skeletal muscle contractility. Physiol Rev 23; 83: Wang WH, Giebisch G. Regulation of potassium (K) handling in the renal collecting duct. Pflugers Arch 29; 458: Youn JH, McDonough AA. Recent advances in understanding integrative control of potassium homeostasis. Annu Rev Physiol 29; 71: Verrey F, Hummler E, Schild L et al. Mineralocorticoid action in the aldosterone-sensitive distal nephron. In: Alpern RJ, Hebert SC (eds). 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