Regulation of distal nephron transport by intracellular chloride and potassium

Abstract

Background:

Low potassium increases the phosphorylation and activity of the sodium chloride cotransporter (NCC) in the distal convoluted tubule of the nephron, which contributes to the hypertensive effect of the modern low potassium/high sodium diet. A central mediator of potassium regulation of NCC is the chloride-sensitive WNK (With No Lysine (K)) kinase.

Summary:

Chloride directly inhibits WNKs by binding to the active site. The mechanisms underlying WNK regulation by extracellular potassium are reviewed, as well as the modulatory effect of kidney-specific (KS)-WNK1. WNK1, but not WNK1 kinase activity, is also required for the aldosterone-independent regulation of the epithelial sodium channel by potassium. Whether intracellular chloride could be involved in this process is discussed. Recent studies demonstrating direct regulation of WNKs by intracellular potassium are also reviewed, and the potential physiological relevance to renal epithelial ion transport is discussed.

Key Messages:

WNKs are sensors of the intracellular ionic milieu. In the nephron, changes in extracellular ion concentrations, resulting in changes in intracellular ion concentration, regulate WNK activity and downstream transporters and channels to maintain total body ion homeostasis.

INTRODUCTION

High blood pressure is the leading risk factor for morbidity and mortality globally, and is increasing in prevalence [1,2]. Low dietary potassium, particularly in combination with high dietary sodium, is associated with high blood pressure [3,4]. Household substitution of 25% potassium/75% sodium salt for 100% sodium salt lowers blood pressure [5]. More than 90% of individuals worldwide, however, consume more sodium and less potassium than recommended [4,6].

Potassium has natriuretic effects [7–9], implicating the kidney in the blood pressure-lowering effects of potassium. Sodium delivery to the potassium-secreting connecting tubule and collecting duct is an important determinant of renal potassium secretion, as sodium absorption through the epithelial sodium channel in this segment generates a lumen-negative transepithelial potential that facilitates potassium secretion [10]. Variation in dietary potassium and changes in blood potassium concentration affect renal sodium absorption, with high potassium inhibiting sodium reabsorption in the proximal tubule, thick ascending limb, and distal convoluted tubule (DCT) [11–13]. Since these segments are upstream of the connecting tubule and collecting duct, the effect of high potassium will be to deliver more sodium to these segments, increasing potassium secretion. In recent years, emerging evidence points to the importance of ions themselves in regulating renal epithelial ion transport. This review will consider potential roles for intracellular chloride and potassium in the regulation of renal sodium absorption.

MECHANISMS FOR POTASSIUM REGULATION OF THE SODIUM CHLORIDE COTRANSPORTER

Potassium regulates NCC phosphorylation and activity via the WNK4-SPAK/OSR1 kinase cascade

Sodium is reabsorbed through the apical sodium chloride cotransporter (NCC) in the DCT [14]. NCC activity is regulated by phosphorylation of N-terminal serine and threonine residues, with phosphorylation increasing transporter activity [15,16]. In 2009, Vallon and colleagues were the first to demonstrate that NCC phosphorylation increased in mice fed a low-potassium diet for one week, and tended to decrease in animals fed a high potassium diet [17]. Since then, the inverse relationship between dietary potassium (or extracellular potassium concentration) and NCC phosphorylation has been demonstrated by multiple groups in response to both acute potassium administration (for example, by gavage) and more chronic regimens of dietary potassium feeding [18].

SPAK (Ste20-related proline-alanine-rich kinase) and OSR1 (oxidative stress response 1) are paralogous Ste20 kinases that phosphorylate NCC, as well as related SLC12 cation-chloride cotransporters, including the sodium-potassium-2-chloride cotransporters (NKCC1 and NKCC2) and the potassium-chloride cotransporters (KCCs) [19]. SPAK and OSR1 are regulated by the WNK (With No Lysine (K)) kinases, with WNK phosphorylation of the T-loop threonine (Thr 233 in SPAK and Thr 185 in OSR1) required for SPAK/OSR1 activation [19]. Thus, the WNK-SPAK/OSR1 signaling pathway is a major regulator of NCC activity.

WNK kinases are found in plants, animals, fungi and unicellular protists [20]. There are four mammalian WNKs, WNKs 1–4. Mammalian WNKs share a highly conserved kinase domain (~80–90% identity between human WNKs) with atypical placement of the catalytic lysine [21,22]. WNKs are found in many cell types and tissues, including the kidney, vasculature, immune cells, and the nervous system, and have been implicated in a growing list of physiological and pathophysiological processes, such as development, ion transport, cell volume regulation, hypertension, inflammation, cancer, autophagy, cerebral edema, neuronal excitability, and metabolism (reviewed in [19,21,23–30]). Of the four mammalian WNKs, WNK4 is dominant in the DCT [31,32], although gain-of-function mutations in either WNK1 or WNK4 increase NCC phosphorylation and activity, and result in hypertension and hyperkalemia in humans [19,23,33].

The ability of potassium to regulate NCC phosphorylation and activity is lost in mice in which WNK4, or SPAK and OSR1, have been knocked out [34–37], indicating that WNK4-SPAK/OSR1 signaling mediates the effect of potassium on NCC.

WNKs are chloride-sensitive kinases

The existence of a chloride-sensitive kinase was hypothesized based on the observation that conditions that decrease intracellular chloride increased NKCC1 phosphorylation [38]. Delineation of the WNK-SPAK/OSR1 kinase cascade as upstream regulators of NCC and NKCCs, and demonstration that the pathway was activated by low intracellular chloride (for example, by bathing cells in hypotonic and/or low chloride bathing medium) [16,39–43], led to the hypothesis that WNKs are chloride-sensitive kinases.

Definitive proof that WNKs are directly regulated by chloride came from a study by Goldsmith and colleagues [44]. This study demonstrated that chloride increased stability of the WNK1 kinase domain and decreased its activity by decreasing autophosphorylation of Ser 382 in the activation loop. Activation loop autophosphorylation is a necessary step in the activation of WNKs, explaining chloride’s inhibitory effect. Anomalous scattering X-ray diffraction using bromide revealed a single peak corresponding to the chloride binding site. Chloride binding occurs through hydrogen bonding to backbone amide groups and hydrophobic interactions, consistent with chloride binding sites in other proteins. Mutation of amino acids in the binding site blunted the inhibitory effects of chloride on WNK1 activity [44]. Subsequently, chloride inhibition of WNK3, WNK4 and Drosophila WNK was demonstrated [45,46]. Amongst the mammalian WNKs, WNK4 is the most chloride-sensitive in vitro and in cellular studies [45,47,48].

Early studies of the effects of WNK4 on NCC co-expressed in Xenopus oocytes showed that WNK4 inhibits NCC transport activity [49,50], but, as described above, other data indicate that WNK4 is a positive regulator of NCC activity [19,23]. Chloride regulation of WNK4 provides an explanation for this discrepancy. In NCC-expressing oocytes, intracellular chloride is approximately 58 mM, a chloride concentration which inhibits WNK4 [45,47]. Inhibited WNK4 likely binds to endogenous Xenopus WNKs, resulting in a dominant-negative effect and NCC inhibition. However, when intracellular chloride was lowered to ~20–35 mM using hypotonic low chloride bathing medium, WNK4 stimulated NCC activity, as did the chloride-insensitive WNK4 mutant in isotonic bathing medium/high intracellular chloride [47]. Intracellular chloride in the DCT has been estimated at ~16–17 mM when serum potassium is 4 mM [35,51], consistent with stimulatory effects of WNK4 on NCC activity in the DCT.

Effects of potassium on WNK activity in the DCT and NCC phosphorylation and activity

Naito et al. demonstrated an inverse relationship between extracellular potassium and OSR1 phosphorylation (a readout of WNK activity) in COS7 cells [42]. These results were recapitulated in HEK (human embryonic kidney) cells. In addition, in HEK cells expressing the basolateral DCT potassium channel Kir4.1 and the Clc-kb chloride channel, intracellular chloride and extracellular potassium increased in parallel [35,52]. Based on additional experiments in HEK cells, Terker et al. proposed that decreases in extracellular potassium result in potassium efflux through the basolateral Kir4.1/Kir5.1 potassium channel in the DCT, hyperpolarizing the membrane. This drives efflux of chloride through the Clc-kb chloride channel (Clc-k2 in mice), to lower intracellular chloride and relieve inhibition of WNK4. Stimulation of WNK4-SPAK/OSR1 signaling increases NCC phosphorylation and sodium chloride reabsorption (Fig. 1). In the downstream connecting tubule and collecting duct, sodium reabsorption through the epithelial sodium channel generates the lumen-negative charge that drives potassium secretion through potassium channels [10]. Thus, decreased delivery of sodium to these segments reduces potassium secretion, conserving potassium. Consistent with this model, pharmacologic blockade of potassium and chloride channels increased intracellular chloride in microdissected DCT [53]. The importance of Kir4.1 and Clc-kb in the response of WNK4 pathway signaling to changes in potassium was further demonstrated in studies of knockout animals [54,55].

Figure 1. Model for intracellular chloride and potassium regulation of NCC in the distal convoluted tubule.

Sodium and chloride are reabsorbed through the sodium chloride cotransporter (NCC) in the distal convoluted tubule (DCT) of the nephron. Low dietary potassium and hypokalemia increase NCC phosphorylation via the WNK4-SPAK/OSR1 kinase cascade. Chloride and potassium have direct inhibitory effects on WNK4. Lowering of intracellular chloride and, possibly, potassium, via ion efflux through the basolateral ClC-Kb/Clc-K2 chloride channel (Clc-Kb in humans, Clc-K2 in mice) and the Kir4.1/5.1 potassium channel provides a mechanism by which extracellular potassium influences NCC phosphorylation and activity to modulate salt reabsorption by the DCT. KS-WNK1 (kidney-specific WNK1) may also contribute to WNK4 activation by decreasing the inhibitory effects of chloride on WNK4, and/or through the formation of “WNK bodies.” See text for additional details. Figure adapted with permission from Wolters Kluwer Health, Inc.: Rodan AR, Intracellular chloride: a regulator of transepithelial transport in the distal nephron, Curr Opin Nephrol Hypertens, 28 (4): 360–367, DOI: 10.1097/MNH.0000000000000502.

Additional support for the idea that changes in intracellular chloride regulate WNK-dependent transepithelial ion transport came from studies in the Drosophila Malpighian tubule, in which intracellular chloride concentrations, WNK activity, and transepithelial ion transport were directly correlated. The Malpighian tubule is the renal epithelium of the fly. The primary urine is generated by fluid secretion in the main segment. The principal cells secrete potassium from the hemolymph (plasma) to the tubule lumen, and the stellate cells secrete chloride [56]. A basolateral NKCC is required for principal cell potassium secretion, and is regulated by WNK and the fly SPAK/OSR1 ortholog, Fray [57,58]. Like the DCT, the principal cell has basolateral potassium and chloride conductances [59]. Malpighian tubule WNK activity was inhibited by high potassium bath, and activated by low potassium bath [46]. When the tubule was bathed in hypotonic medium, intracellular chloride decreased, WNK was activated over 30 to 60 minutes, and transepithelial potassium flux increased in a WNK-, Fray-, and NKCC-dependent manner [46,58]. Furthermore, expression of chloride-insensitive WNK increased transepithelial potassium flux when co-expressed with the scaffold protein, Mo25 [46]. Mo25 increases the activity of SPAK, OSR1 and Fray, and also appears to have Fray-independent effects in the Malpighian tubule [46,60]. Mo25 is also expressed in the DCT [61]. Whether it plays a physiological role in DCT ion transport requires further study.

Due to the technical challenges of measuring intracellular chloride, few studies have measured chloride in the DCT. Using electron microprobe analysis of freeze-dried cryosections of the renal cortex, Beck et al. demonstrated an increase in chloride concentrations in DCT epithelial cells from 9.6 mmol/kg wet weight in rats fed a control diet, to 11 mmol/kg wet weight in rats fed a high potassium diet for at least 10 days, which increased plasma potassium from 4.2 to 5.6 mmol/L [62]. Measurements of intracellular chloride in response to low potassium diet have not been made, but modeling studies predict that intracellular chloride decreases from ~17 mM to 13 mM when plasma potassium decreases from 4 mM to 2 mM [35]. This is expected to increase WNK4 activity by ~3%, based on in vitro studies [45]. Su et al. measured intracellular chloride in microdissected DCT in response to acute changes in extracellular potassium from 5 mM to 2 mM and then to 10 mM. Transient changes lasting ~2 minutes were observed [53]. Whether WNK4 activation or inhibition occurs in this timeframe in the DCT remains to be determined.

Compelling evidence for a role for chloride regulation of WNK4 in DCT physiology came from analysis of mice in which mutations were knocked in to WNK4 to render it insensitive to chloride. NCC phosphorylation and activity were increased, and did not increase further in response to low potassium diet [63]. An open question is whether the mutant WNK4 is affecting nephron structure, as expression of constitutively active SPAK in the early DCT (DCT1) resulted in hypertrophy in that segment, with a concomitant atrophy in the connecting tubule [64]. Since changes in dietary potassium also cause tubular remodeling [65], it will be interesting to determine what the relationship is between potassium, WNK-SPAK/OSR1 signaling, and nephron structure. For example, does nephron remodeling in response to low potassium occur when WNK4 or SPAK/OSR1 are knocked out?

High potassium decreases NCC phosphorylation, but whether this is due to chloride inhibition of WNK-SPAK/OSR1 signaling has been controversial. Some studies in rodents or isolated DCT have demonstrated a decrease in NCC phosphorylation in response to high dietary or extracellular potassium, without a decrease in SPAK phosphorylation, or independent of chloride [51,66–68]. Recent studies, however, demonstrated parallel decreases in SPAK and NCC phosphorylation in isolated DCT bathed in high potassium for 30 minutes [69], and in vivo in mice receiving an acute potassium load via gavage [70]. The latter study showed that, similar to previous studies, there was no change in SPAK phosphorylation on Western blots of whole kidney lysates; rather, the decrease in SPAK phosphorylation was detected by immunofluorescence only in the DCT [70]. DCT changes may not be apparent by Western blotting given that DCT mass is relatively small compared to other nephron segments, where SPAK is also expressed. Potassium gavage failed to decrease NCC phosphorylation in mice with SPAK/OSR1 double knockout in the nephron [70], and in mice expressing chloride-insensitive WNK4 [63]. Furthermore, the sustained activation of WNK4/SPAK-OSR1 signaling after 5 days of low potassium diet prevented NCC dephosphorylation in response to an acute potassium load [70]. These findings suggest a requirement to “turn off” WNK4 signaling to decrease NCC phosphorylation in the face of an acute potassium load, and indicate that this is mediated by chloride regulation of WNK4. On the other hand, feeding chloride-insensitive WNK4 mutant mice high potassium diet for four days decreased NCC phosphorylation in mutant and control mice to a similar extent. Interpretation of this experiment is complicated by the fact that the high potassium diet paradoxically decreased plasma potassium in this study [63]. Altogether, though, these results suggest that there may be differential roles of chloride regulation of WNK4 in the response to shorter- and longer-term dietary potassium loading, with current evidence indicating a greater role for chloride inhibition of WNK4 in response to acute potassium loads. Importantly, recent studies have delineated additional mechanisms for decreasing NCC phosphorylation in response to potassium, including phosphatases and degradation of WNK4 and NCC [66,68,69,71–74]. How these mechanisms interact with chloride regulation of WNK4 signaling requires further study.

KS-WNK1 may modulate WNK4 chloride sensitivity

KS-WNK1 is a kidney-specific isoform of WNK1 with an alternative promoter that results in a shortened protein lacking the N-terminal domain, and most of the kinase domain, of full-length WNK1, while maintaining an intact C-terminal domain [75,76]. KS-WNK1 protein is increased in mice fed a low potassium diet [77]. KS-WNK1 is also required for the formation of “WNK bodies,” which are condensates that form in the DCT in response to low potassium. In addition to KS-WNK1, WNK bodies contain WNK4, SPAK and OSR1 [78,79]. KS-WNK1 knockout abolishes the inverse relationship between plasma potassium concentration and NCC phosphorylation, resulting in decreased phosphorylated NCC and hypokalemia in female mice fed a low potassium diet [80]. These results suggest that KS-WNK1 may promote the activation of NCC via activation of the WNK4-SPAK/OSR1 pathway in response to low potassium. Consistent with this idea, SPAK and NCC phosphorylation, and NCC transport activity, were increased in Xenopus oocytes co-expressing KS-WNK1 [81]. Interestingly, autophosphorylation of WNK4 Ser 335 (homologous to WNK1 Ser 382) was increased when KS-WNK1 was co-expressed, even in high chloride conditions, suggesting that KS-WNK1 may decrease WNK4 chloride sensitivity [81]. This effect was dependent on the C-terminal “HQ” motif required for WNK-WNK interactions [82]. The WNK4 C-terminus was also implicated in chloride sensitivity in another study examining WNK3/WNK4 hybrids [48]. The “4a” N-terminal exon in KS-WNK1, which is unique to KS-WNK1, is required for WNK body formation, increased WNK4 autophosphorylation, and NCC stimulation [77,78,81]. Whether and how WNK body formation influences WNK4 chloride sensitivity will be interesting to determine.

Regulation of the epithelial sodium channel by extracellular potassium

The epithelial sodium channel, ENaC, plays an important role in renal potassium homeostasis by generating the lumen-negative voltage that drives potassium secretion in the connecting tubule and collecting duct [10]. Although aldosterone, which is released in response to either volume depletion or elevated potassium, is a major regulator of ENaC [83], a recent study demonstrated that increased extracellular potassium also activates ENaC independently of aldosterone [84]. The mTORC2 (mammalian target of rapamycin complex 2)-SGK1 (serum/glucocorticoid regulated kinase 1) kinase pathway is required for this effect. WNK1 is also required, but not WNK1 kinase activity. Similar to the DCT, the basolateral Kir4.1 channel was also implicated [84]. Whether intracellular chloride could play a role was not determined in this study. However, in addition to regulating WNK1 kinase activity, a recent study in pancreatic duct cells demonstrated that intracellular chloride regulates the binding of the WNK1 kinase domain to the CFTR (cystic fibrosis transmembrane regulator) chloride channel [85]. Whether intracellular chloride could be regulating kinase-independent WNK1 functions in the connecting tubule and collecting duct requires further study.

POTASSIUM REGULATION OF WNK KINASES

WNK signaling regulates transepithelial potassium secretion in the Malpighian tubule [46,58], raising the question of whether intracellular potassium itself could regulate WNK kinases. This was investigated in Malpighian tubules expressing endogenous Drosophila WNK, and in tubules in which Drosophila WNK was knocked down and replaced with wild-type or chloride-insensitive mammalian WNK3 or WNK4. Extracellular potassium was varied and measured intracellular chloride kept constant by manipulating extracellular chloride concentrations. Even with fixed intracellular chloride, raising extracellular potassium inhibited the activity of Drosophila WNK and mammalian WNK3 and WNK4 [86]. A previous study demonstrated that Drosophila WNK activity was increased at an intracellular chloride concentration of ~15 mM compared to ~27 mM [46]. The effects of high potassium were observed at both 13 mM and 26 mM intracellular chloride concentrations, indicating additive inhibitory effects of high chloride and high potassium. High potassium also inhibited chloride-insensitive WNK3 and WNK4 mutants, suggestive of independent effects of each ion as well as distinct binding sites. Low extracellular potassium stimulated wild-type and chloride-insensitive WNK4, and ouabain treatment, which is expected to lower intracellular potassium through inhibition of the Na⁺/K⁺-ATPase, stimulated Drosophila WNK [86].

Tubule potassium content (as inferred from measurement of substituted rubidium) was increased in high potassium bath [86], but potassium could also have other effects, such as changing the membrane potential. Therefore, in vitro studies were pursued to examine whether there are direct effects of potassium on WNK kinases. Like chloride [44], potassium increased the thermal stability of Drosophila WNK, WNK1, and WNK3 kinase domains in vitro [86]. Potassium also inhibited the autophosphorylation of purified Drosophila WNK and WNK3 kinase domains [86], similar to the effects of chloride on Drosophila and mammalian WNKs [44,46]. Finally, potassium inhibited WNK3 and WNK4 phosphorylation of SPAK. Importantly, WNK3 and WNK4 were most sensitive to potassium inhibition at concentrations in the physiological range of intracellular potassium, ie, 100–150 mM [86]. Thus, potassium inhibits WNKs through direct effects on the kinase.

The physiological consequences of potassium regulation of WNKs have yet to be determined. Development of potassium-insensitive WNK mutants will be helpful in this regard, and will be aided by elucidation of WNK potassium binding sites. Although intracellular potassium concentrations are typically more constant than intracellular chloride, changes in intracellular potassium have been observed in some situations, including in the nephron. For example, in rats fed a low potassium diet for 6 weeks, which decreased plasma potassium from 4.1 to 2.0 mEq/L, intracellular potassium concentration decreased in proximal tubule epithelial cells by 8 mEq/L [87]. Another study demonstrated a 10 mM decrease in intracellular potassium concentration in the distal tubule of rats fed a low potassium diet, in which plasma potassium decreased from 4.3 to 2.9 mmol/L [88]. These studies measured intracellular potassium activity using double-barreled ion-specific electrodes. Beck et al. used electron microprobe analysis of frozen kidney tissue samples to measure potassium in rats fed a normal or low potassium diet, which decreased plasma potassium from 4.4 to 2.0 mmol/L. This method revealed a decrease in proximal tubule intracellular potassium from 150 to 118 mmol/kg of wet weight, and from 152 to 120 mmol/kg of wet weight in the distal tubule [89]. Conversely, in rats fed a high potassium diet, which increased plasma potassium from 4.3 to 5.2 mmol/L, the distal tubule intracellular potassium concentration increased by 14 mM, as measured using ion-specific electrodes [88]. Beck et al. also measured intracellular potassium using electron microprobe analysis in rats fed high-potassium diet, which increased plasma potassium from 4.2 to 5.6 mmol/L, and demonstrated an 8 mmol/kg wet weight increase in the DCT, and a 10 mmol/kg wet weight increase in the connecting tubule [62]. Thus, independent groups using varying methods have demonstrated changes in renal epithelial intracellular potassium under conditions of both chronic low and high potassium diet. Genetically encoded fluorescent potassium sensors have recently been developed [90,91], and could provide additional opportunities for measurements of intracellular potassium under different conditions.

Based on in vitro studies, an 8 mM increase in intracellular potassium is expected to decrease WNK4 activity by ~4% [86], similar to the changes expected from the small changes in intracellular chloride in the DCT (see above, “Effects of potassium on WNK activity in the DCT and NCC phosphorylation and activity”). Given these relatively small changes, and that intracellular chloride and potassium are expected to increase or decrease in parallel in response to changes in extracellular potassium, additive effects of intracellular potassium and chloride on WNK activity could help amplify the effects of small changes in extracellular potassium. Further amplification may occur through the transduction of the WNK signal by the SPAK/OSR1 kinases. Alternatively, intracellular potassium regulation of WNKs could be operative during more chronic dietary potassium loading or deficiency, in which chloride regulation of WNK signaling may be less important, as discussed above (“Effects of potassium on WNK activity in the DCT and NCC phosphorylation and activity”).

CONCLUSION

In addition to regulation by potassium and chloride, WNKs are activated by increased osmotic pressure [22,48,92–94]. Thus, WNKs are sensors of multiple components of the intracellular ionic and osmotic environment. How these signals are integrated will require further study. Intracellular sodium couples apical ENaC and basolateral Na⁺/K⁺-ATPase activity in the collecting duct. p38 kinase has been implicated in this process, but the mechanisms are unknown [95,96]. Thus, elucidating the mechanisms by which ions regulate kinases is likely to be informative in understanding the physiology of multiple nephron segments.