Hypertension is the leading contributor to premature death and disability in the world. Genetic susceptibility and high dietary salt (NaCl) consumption have long been considered to be the primary culprits but growing evidence indicates that low dietary potassium consumption has an equally important role. Although the underlying mechanisms are complex and multifactorial, the potassium switch signalling mechanism in the kidney distal convoluted tubule represents a crucial pathway with implications for preventing and treating hypertension. Comprising a Kir4.1 and Kir5.1 channel potassium-sensing mechanism, a WNK kinase-induced phosphorylation cascade, and the thiazide-diuretic-targeted sodium chloride co-transporter, the potassium switch orchestrates a physiological response in the distal nephron that maintains sodium-potassium balance over wide variations in dietary potassium intake. The potassium switch is ideally adapted for the low-salt, feast-and-famine diets of hunter-gatherers. However, low potassium consumption, which is common in high-sodium modern diets, promotes potassium conservation at the expense of increasing sodium reabsorption, exacerbating salt-sensitive hypertension and its associated cardiovascular complications. Here, we discuss current understanding of the potassium switch and how its role in kidney adaptation to the modern diet can contribute to hypertension.

Abstract NKCC2, localized to the apical membrane of thick ascending limb epithelial cells, is essential for renal salt handling and systemic electrolyte homeostasis. NKCC2 undergoes extensive ubiquitylation, with the E3 protein ligase Nedd4-2 implicated as a key regulator. However, progress has been limited by challenges expressing NKCC2 in mammalian cell lines, hindering mechanistic studies of NKCC2 ubiquitylation. Therefore, the aims of this study were to develop a mammalian cell model enabling mechanistic investigations of NKCC2 ubiquitylation, including the role of Nedd4-2 and the functional consequences of site-specific modification. A tetracycline-inducible MDCKI cell line was generated expressing human NKCC2 and used to assess Nedd4-2-dependent and site-specific ubiquitylation of NKCC2 using biochemical, imaging, and functional assays. The MDCKI cell line demonstrated stable, inducible expression of full-length human NKCC2. In this cell line, mutating the ubiquitylation site at K871 increased membrane abundance and uptake activity, without altering internalization rates. Nedd4-2 co-immunoprecipitated with NKCC2, and Nedd4-2 deletion increased total, but not membrane NKCC2 abundance. In summary, ubiquitylation on NKCC2 at K871 plays a key role in controlling NKCC2 membrane localization and thus function. Although Nedd4-2 can modulate NKCC2 abundance, it is not involved in NKCC2 trafficking. We conclude that the generated cell line provides a robust model for mechanistic studies of NKCC2 and will aid studies examining posttranslational regulation of NKCC2.

Pseudohypoaldosteronism type 1 (PHA-1) is a rare genetic disease caused by aldosterone resistance, characterized by severe sodium loss, hyperkalemia, dehydration, and vomiting. The Epithelial Na+ Channel (ENaC) is a cation channel that constitutes the rate-limiting step of transepithelial Na+ transport in many tissues and regulates blood volume and pressure. Mutations in any of its subunits (α, β, or γ) have been shown to cause PHA-1B. The present investigation is a case study of a 4-month-old female born to consanguineous parents with symptoms suggestive of a form of PHA-1. The child presented with failure to thrive, accompanied by mild hyponatremia and hyperkalemia, together with a normal anion gap metabolic acidosis. Whole exome sequencing, conducted to identify genetic variants, revealed a variant of uncertain significance, the homozygous missense mutation c.1594G > A, p. Gly532Ser in the SCNN1G gene, associated with PHA-1B3. To investigate the functional impact of this mutation, in vitro electrophysiological and biochemical studies were performed with wild type αβγ and mutant αβγG532S-ENaC. This analysis showed that the γG532S mutation reduced, but did not suppress ENaC expression and activity. The functional observation explains the mild phenotype of this novel SCNN1G mutation, which contrasts with the typically severe presentation of autosomal recessive PHA-1B. In our case, the patient showed a positive clinical response to sodium chloride supplementation alone. These findings suggest that certain missense mutations in SCNN1G may result in a milder disease course, underscoring the importance of functional studies in understanding genotype–phenotype correlations in PHA-1.

A candidate gene association analysis identified TMPRSS9 as gene for potassium sensitivity in women. To validate this finding, constitutive and conditional Tmprss9 knockout mice were generated and subjected to dietary K+ deprivation and K+ loading. Interestingly, mouse renal Tmprss9 gene expression was similar in both sexes on standard diet but differed when challenged with K+-deprivation or -loading in wildtype (WT) mice. Constitutive deficiency of Tmprss9 was evidenced on a transcriptional level in knockout (KO) mice. Serum Na+ levels were lower in male and female KO mice on low K+ (LKD), while on high K+ (HKD) diet, serum K+ only increased in male KO mice. Upon all diet conditions namely standard diet (SD), LKD and HKD the protein abundances of sodium transporting proteins like the sodium-chloride symporter (NCC), alpha and gamma epithelial sodium channel (ENaC) subunits as well as their ratio of cleaved/full length protein and the sodium-hydrogen exchanger 3 (NHE3) were similar in WT and KO mice and/or showed only minor differences. We propose that in human, TMPRSS9 may function as a sex-specific modifier gene for serum K+ handling in women, whereas in mice, male rather than female Tmprss9 KO retained serum K+ on HKD.

The endothelial ENaC (EnNaC) is mainly responsible for maintaining the mechanical properties of the endothelial cell surface, the sensitivity to the shear forces of the streaming blood and thus for vascular function. The correlation between EnNaC surface expression, the dynamics of the actin cortex, the mechanical stiffness, and nitric oxide release indicates a close structure–function relationship. Mechanical flexibility of the endothelial surface has been associated with proper vascular function, while chronic stiffening leads to endothelial dysfunction and the so-called ‘stiff endothelial cell syndrome’ (SECS). With the help of atomic force microscopy (AFM)–based nanoindentation and immunofluorescence staining in vitro and ex vivo, we investigated the underlying cellular mechanisms and signalling pathways of EnNaC-dependent endothelial behaviour. We were able to show that the interaction between EnNaC and the cortical cytoskeleton is mediated by the small GTPases RhoA, Rac1, and the Arp2/3 complex. The functional inhibition of EnNaC by the drugs amiloride and benzamil led to membrane removal of the channel within minutes. Furthermore, we could observe an involvement of mineralocorticoid receptor, SGK1 and Nedd4-2 in regulation of endothelial cell stiffness. Our study contributes further insights on complex regulation of EnNaC and elucidates its interaction with the actin cytoskeleton, which could be central to its role as a key regulator of vascular function in health and disease.

We previously reported that Na+-deprived mice lacking CAP1/Prss8 in kidney tubules maintained epithelial sodium channel-mediated sodium balance albeit persistent hypoaldosteronism, hence indicating an uncoupling from aldosterone production. This further suggested an implication of the serine protease CAP1/Prss8 (prostasin) in the cross talk of the kidney with the adrenal gland that does not express prostasin. When these knockout (Ko) mice were additionally exposed to a high K+ diet, plasma K+ levels and plasma aldosterone concentrations were normalized and no longer different from those of the control mice. The mRNA transcript expression of the adrenal aldosterone synthase Cyp11b2, which was lower in Na+-deprived CAP1/Prss8 Ko animals, was in the normal range. Plasma aldosterone levels were similar to control animals, indicating that K+ rescued the hypoaldosteronism in Na+-deprived CAP1/Prss8 Ko animals. These data suggest that CAP1/Prss8 (prostasin) is implicated in the regulation of aldosterone synthesis or production and that the consequences of CAP1/Prss8 deficiency can be compensated by high dietary K+ supplementation. Prostasin may therefore present a promising regulator of aldosterone production by affecting the adrenal steroidogenic pathway.NEW & NOTEWORTHY We explore the role of the serine protease CAP1/Prss8 in aldosterone synthesis. Described previously as a candidate gene for hypertension, the mechanism by which renal serine protease deficiency is implicated in aldosterone production is still largely unknown. Our findings underscore a role of prostasin in the regulation of aldosterone synthesis. In kidney-specific CAP1/Prss8 knockout mice, K+ supplementation is predominant over Na+ and restores normal aldosterone production proposing new pathways to treat hypo- or hypertension.

Blood pressure (BP) follows a circadian pattern that rises during the active phase of the day (morning surge) and decreases during the inactive (night dipping) phase of the day. The morning surge coincides with increased circulating glucocorticoids and aldosterone, ligands for glucocorticoid receptors and mineralocorticoid receptors, respectively. Serum- and glucocorticoid-induced kinase 1 (SGK1), a clock-controlled and glucocorticoid receptor- and mineralocorticoid receptor-induced gene, plays a role in BP regulation in human and animal models. However, the role of SGK1 in BP circadian regulation has not yet been demonstrated. Using telemetry, we analyzed BP in the inducible renal tubule-specific Sgk1(Pax8/LC1) model under basal K+ diet (1% K+) and high-K+ diet (HKD; 5% K+). Our data revealed that, under basal conditions, renal SGK1 plays a minor role in BP regulation; however, after 1 wk of HKD, Sgk1(Pax8/LC1) mice exhibited significant defects in diastolic BP (DBP), including a blunted surge, a decreased amplitude, and reduced day/night differences. After prolonged HKD (7 wk), Sgk1(Pax8/LC1) mice had lower BP than control mice and exhibited reduced DBP amplitude, together with decreased DBP day/night differences and midline estimating statistic of rhythm (MESOR). Interestingly, renal SGK1 deletion increased pulse pressure, likely secondary to an increase in circulating aldosterone. Taken together, our data suggest that 1) the kidney plays a significant role in setting the BP circadian rhythm; 2) renal tubule SGK1 mediates the BP surge and, thus, the day/night BP difference; 3) long-term renal SGK1 deletion results in lower BP in mutant compared with control mice; and 4) renal SGK1 indirectly regulates pulse pressure due to compensatory alterations in aldosterone levels.

Thiazide sensitive sodium‐chloride cotransporter (NCC) is a major salt transport pathway in the apical membrane of the nephron distal convoluted tubule. While we know the importance of its function in maintaining sodium‐potassium homeostasis, the regulation of NCC is complicated and the mechanisms are not well understood. The aim of this study is to evaluate the role of mineralocorticoid receptors (MRs) in the regulation of NCC. Here, we generated DCT‐specific MRs Knockout (KO) mice, in which MRs were deleted in cells expressing NCC, taking advantage of a cre recombinase under the control of the NCC gene. Under standard and Low‐Na+ diets, these DCT‐specific MRs‐KO mice display normal Na+/K+ balance but exhibit problems with Cl‐ regulation. These KO mice presented hypochloremia under normal diet but unexpectedly, hyperchloremia after 6d on low Na+ diet. The expression of NCC and phosphorylated NCC were both decreased in KO mice compared to that in control mice, under both standard and low‐Na+ diet. This decrease in NCC protein levels is related to a decrease in transcription levels. Under standard diet, decreased NCC expression is compensated by an increase in αENaC and pendrin expression, which is not the case under low‐salt diet. Under a low‐K+ diet, KO mice exhibit hypokalemia and volume depletion with increased blood CO2 levels and Renin mRNA levels. NCC expression and its phosphorylation are both increased in control and KO groups under low‐K+ diet compared to standard diet but, the levels of NCC protein and mRNA are still decreased in the KO group compared to the control group mice. Taken together, these results demonstrate that MRs is not necessary for the complete regulation of NCC under low‐Na+ and low‐K+ diet. On the other hand, MRs appears to be indispensable to assure proper NCC expression in adult DCT renal tubules.

Background: MR (mineralocorticoid receptor) antagonists are recommended for patients with resistant hypertension even when circulating aldosterone levels are not high. Although aldosterone activates MR to increase epithelial sodium channel (ENaC) activity, glucocorticoids also activate MR but are metabolized by 11βHSD2 (11β-hydroxysteroid dehydrogenase type 2). 11βHSD2 is expressed at increasing levels from distal convoluted tubule (DCT) through collecting duct. Here, we hypothesized that MR maintains ENaC activity in the DCT2 and early connecting tubule in the absence of aldosterone. Methods: We studied AS (aldosterone synthase)-deficient (AS−/−) mice, which were backcrossed onto the same C57BL6/J strain as kidney-specific MR knockout (KS-MR−/−) mice. KS-MR−/− mice were used to compare MR expression and ENaC localization and cleavage with AS−/− mice. Results: MR was highly expressed along DCT2 through the cortical collecting duct (CCD), whereas no 11βHSD2 expression was observed along DCT2. MR signal and apical ENaC localization were clearly reduced along both DCT2 and CCD in KS-MR−/− mice but were fully preserved along DCT2 and were partially reduced along CCD in AS−/− mice. Apical ENaC localization and ENaC currents were fully preserved along DCT2 in AS−/− mice and were not increased along CCD after low salt. AS−/− mice exhibited transient Na+ wasting under low-salt diet, but administration of the MR antagonist eplerenone to AS−/− mice led to hyperkalemia and decreased body weight with higher Na+ excretion, mimicking the phenotype of MR−/− mice. Conclusions: Our results provide evidence that MR is activated in the absence of aldosterone along DCT2 and partially CCD, suggesting glucocorticoid binding to MR preserves sodium homeostasis along DCT2 in AS−/− mice.

Na+ and K+ balance is influenced by the activity of the sodium chloride cotransporter NCC in the distal convoluted tubule. NCC activity and abundance are reduced by high extracellular K+. The E3 ubiquitin ligase neural precursor cell expressed developmentally downregulated 4–2 (Nedd4-2) has been proposed as a modulator of NCC abundance. Here, we examined the functional role of Nedd4-2 on NCC regulation and whether Nedd4-2 is important for the effects of high extracellular K+ on NCC. Total and plasma membrane levels of ubiquitylated NCC were lower in NCC-expressing MDCKI cells after Nedd4-2 deletion. NCC and phosphorylated NCC (pT58-NCC) levels were higher after Nedd4-2 deletion, and NCC levels on the plasma membrane were elevated. No significant changes were seen after Nedd4-2 knockdown in the levels of SPAK and phosphorylated SPAK (pS373-SPAK), the major NCC regulatory kinase. Nedd4-2 deficiency had no effect on the internalization rate of NCC from the plasma membrane, but NCC protein half-life was increased. In ex vivo experiments with kidney tubule suspensions from Nedd4-2 knockout (KO) mice, high K+ reduced total and pT58-NCC regardless of genotype. We conclude that Nedd4-2 is involved in ubiquitylation of NCC and modulating its plasma membrane levels and degradation. However, Nedd4-2 does not appear to be important for K+ induced reductions in NCC abundance.

Kidney tubules play a pivotal role in the maintenance of body fluid homeostasis and pH regulation. Accordingly, kidney disorders are known to be strongly associated with hypertension and acid-base imbalance. In the kidney, the intercalated cells of the nephron are the main site of acid-base balance: type A are acid-secreting whereas type B are base-secreting. The chloride bicarbonate exchanger Pendrin is present in type B intercalated cells and participates actively in regulating blood pressure and NaCl balance. It is still not entirely deciphered how Pendrin activity is regulated; nevertheless, it was previously demonstrated that Pendrin is regulated by c-AMP/Protein Kinase A (PKA) signaling pathway. Consequently, in this study, our hypothesis is that the A-Kinase anchoring protein 2 (AKAP2), strongly expressed in the kidney, regulates in time and space the intracellular signal transduction. In order to investigate AKAP2 involvement in Pendrin regulation and trafficking to the apical plasma membrane, we generated an inducible and nephron-specific Akap2 mice knockout model. By confocal microscopy, fluorescent immunostaining on kidney tissue sections showed that AKAP2 is present in the tubules and most importantly colocalizes with Pendrin at the apical plasma membrane of the intercalated cells. To show that Pendrin and AKAP2 are interacting, we used the Proximity Ligation Assay (PLA) and, positively, we could reveal the association between the two proteins. The latter finding was also confirmed in vitro taking advantage of the Opossum Kidney Cell line (OKP) stably expressing Pendrin and co-transfected with AKAP2, we could co-immunoprecipitate Pendrin and AKAP2 after crosslinking on live cells. Fluorescent immunostaining in Akap2 show that Pendrin tends to be shifted from the apical membrane (seen in control mice) to intracellular compartments. In conclusion, our data suggest that AKAP2 and Pendrin are interacting, and AKAP2 may play a key role in Pendrin trafficking to the apical plasma membrane, hence in regulating its activity.

Urinary K + potassium excretion rapidly increases after a potassium‐rich meal. The early aldosterone‐induced sgk1 gene (encoding serum and glucocorticoid‐induced kinase 1), activates potassium clearance, but the role of this kinase in the early activation of K + secretion has not been clearly defined. Here, we challenged inducible renal‐tubule‐specific Sgk1 Pax8 / LC1 knockout mice with an acute high‐potassium load (HK:5%K + ) and compared the physiological and molecular responses to control mice. We observe that urinary excretion after a K + load over the first 3 h is not dependent on SGK1 but is coincident with the rapid dephosphorylation of the Na + ,Cl − ‐cotransporter (NCC) to increase distal salt delivery. Molecular analyses indicate that whereas SGK1‐mediated phosphorylation of the ubiquitin‐protein ligase NEDD4‐2 begins to increase by 3h, SGK1‐dependent proteolytic activation of ENaC only becomes detectable after 6 h of HK intake. Consistent with SGK1‐dependent ENaC activation via inhibition of NEDD4‐2‐mediated ubiquitylation, Sgk1 Pax8 / LC1 mice are unable to efficiently inhibit NEDD4‐2 or increase ENaC cleavage after 6 h of HK. Nevertheless, no defect in acute K + balance was detected in the mutant mice after 6 h of HK. Moreover, we found that Sgk1 Pax8 / LC1 mice reduce NCC phosphorylation and NCC‐mediated salt absorption to a greater extent than control mice after a K + load, promoting increased amiloride‐sensitive Na + ‐reabsorption via ENaC to maintain adequate kaliuresis. Together, these data indicate that: (a) during the early 3 h of HK intake, K + excretion is SGK1‐independent even under an extreme K + challenge, (b) shortly after, SGK1 inhibits NEDD4‐2 and activates ENaC to stimulate K + ‐secretion, (c) SGK1‐dependent phosphorylation of NCC occurs, acting more likely as a brake pedal to prevent excessive K + loss.

Fine tuning of Na+ reabsorption takes place along the aldosterone-sensitive distal nephron, which includes the collecting duct (CD), where it is mainly regulated by aldosterone. In the CD, Na+ reabsorption is mediated by the epithelial Na+ channel and Na+ pump (Na+-K+-ATPase). Paracellular ion permeability is mainly dependent on tight junction permeability. Claudin-8 is one of the main tight junction proteins expressed along the aldosterone-sensitive distal nephron. We have previously shown a coupling between transcellular Na+ reabsorption and paracellular Na+ barrier. We hypothesized that aldosterone controls the expression levels of both transcellular Na+ transporters and paracellular claudin-8 in a coordinated manner. Here, we show that aldosterone increased mRNA and protein levels as well as lateral membrane localization of claudin-8 in cultured CD principal cells. The increase in claudin-8 mRNA levels in response to aldosterone was prevented by preincubation with 17-hydroxyprogesterone, a mineralocorticoid receptor antagonist, and by inhibition of transcription with actinomycin D. We also showed that a low-salt diet, which stimulated aldosterone secretion, was associated with increased claudin-8 abundance in the mouse kidney. Reciprocally, mice subjected to a high-salt diet, which inhibits aldosterone secretion, or treated with spironolactone, a mineralocorticoid receptor antagonist, displayed decreased claudin-8 expression. Inhibition of glycogen synthase kinase-3, Lyn, and Abl signaling pathways prevented the effect of aldosterone on claudin-8 mRNA and protein abundance, suggesting that signaling of protein kinases plays a permissive role on the transcriptional activity of the mineralocorticoid receptor. This study shows that signaling via multiple protein kinases working in concert mediates aldosterone-induced claudin-8 expression in the CD.NEW & NOTEWORTHY In this study, we showed that aldosterone modulates claudin-8 expression in cultured collecting duct principal cells and in the mouse kidney. The upregulation of claudin-8 expression in response to aldosterone is dependent on at least glycogen synthase kinase-3, Lyn, and Abl signaling pathways, indicating the participation of multiple protein kinases to the effect of aldosterone.

The Epithelial Na+ Channel, ENaC, comprised of 3 subunits (αβγ, or sometimes δβγENaC), plays a critical role in regulating salt and fluid homeostasis in the body. It regulates fluid reabsorption into the blood stream from the kidney to control blood volume and pressure, fluid absorption in the lung to control alveolar fluid clearance at birth and maintenance of normal airway surface liquid throughout life, and fluid absorption in the distal colon and other epithelial tissues. Moreover, recent studies have also revealed a role for sodium movement via ENaC in nonepithelial cells/tissues, such as endothelial cells in blood vessels and neurons. Over the past 25 years, major advances have been made in our understanding of ENaC structure, function, regulation, and role in human disease. These include the recently solved three-dimensional structure of ENaC, ENaC function in various tissues, and mutations in ENaC that cause a hereditary form of hypertension (Liddle syndrome), salt-wasting hypotension (PHA1), or polymorphism in ENaC that contributes to other diseases (such as cystic fibrosis). Moreover, great strides have been made in deciphering the regulation of ENaC by hormones (e.g., the mineralocorticoid aldosterone, glucocorticoids, vasopressin), ions (e.g., Na+ ), proteins (e.g., the ubiquitin-protein ligase NEDD4-2, the kinases SGK1, AKT, AMPK, WNKs & mTORC2, and proteases), and posttranslational modifications [e.g., (de)ubiquitylation, glycosylation, phosphorylation, acetylation, palmitoylation]. Characterization of ENaC structure, function, regulation, and role in human disease, including using animal models, are described in this article, with a special emphasis on recent advances in the field. © 2021 American Physiological Society. Compr Physiol 11:1-29, 2021.

SIRT7 is a NAD+ -dependent deacetylase that controls important aspects of metabolism, cancer, and bone formation. However, the molecular targets and functions of SIRT7 in the kidney are currently unknown. In silico analysis of kidney transcripts of the BXD murine genetic reference population revealed a positive correlation between Sirt7 and Slc12a7 mRNA expression, suggesting a link between the corresponding proteins that these transcripts encode, SIRT7, and the K-Cl cotransporter KCC4, respectively. Here, we find that protein levels and activity of heterologously expressed KCC4 are significantly modulated depending on its acetylation status in Xenopus laevis oocytes. Moreover, SIRT7 interacts with KCC4 in a NAD+ -dependent manner and increases its stability and activity in HEK293 cells. Interestingly, metabolic acidosis increases SIRT7 expression in kidney, as occurs with KCC4. In contrast, total SIRT7-deficient mice present lower KCC4 expression and an exacerbated metabolic acidosis than wild-type mice during an ammonium chloride challenge. Altogether, our data suggest that SIRT7 interacts with, stabilizes and modulates KCC4 activity through deacetylation, and reveals a novel role for SIRT7 in renal physiology.

BackgroundENaC and ROMK (Kir1.1) are expressed in the apical membrane of aldosterone‐sensitive distal nephron (ASDN). Aldosterone has been shown to play an important role in the regulation of ENaC and ROMK expression in ASDN. Also, NEDD4‐2 is an E3 ubiquitin ligase expressed in the ASDN and NEDD4‐2 has been shown to play a role in mediating the effect of aldosterone on ENaC. The aims of the present study are:1) To examine whether MR plays a similar role in regulating ENaC and ROMK in the DCT2 and CCD; 2) To examine whether NEDD4‐2 regulates ROMK channels in ASDN.MethodsThe whole‐cell recording technique has been used to measure the amiloride‐sensitive Na currents (ENaC) at −60 mV and TPNQ‐sensitive K currents (ROMK) at −40 mV in the DCT2/early connecting tubule (CNT) and in the CCD of tubule‐specific MR‐KO, NEDD4‐2 KO and corresponding WT mice on a normal rodent diet (0.4% Na and 0.9% K).ResultsDeletion of MR receptor decreased ENaC currents from10.2 pA/pf to 6.3 pA/pf (at −60 mV) in the DCT2/CNT but it largely abolished ENaC currents in the CCD (from 4.9 pA/pf to 0.7 pA/pf). In contrast, the deletion of MR had no significant effect on ROMK currents in the DCT2 since TPNQ‐sensitive K currents were the same between WT and Ks‐MR‐KO mice (WT, 51.0 pA/pf; Ks‐MR‐KO, 50.0 pA/pf at −40 mV). However, the ROMK currents of the CCD were significantly smaller in MR‐KO (22.1 pA/pf) than in WT (33.8 pA/pf), suggesting that MR plays a role in regulating ROMK only in the CCD but not in the DCT2/CNT. Deletion of NEDD4‐2 increased amiloride sensitive Na currents from 10 pA/pf to 13 pA/pf in the DCT2/CNT but it robustly increased ENaC currents from 5.1 pA/pf to 15 pA/pf in the CCD, suggesting that NEDD4‐2 plays a bigger role in regulating ENaC in the CCD than in the DCT2/CNT. Deletion of NEDD4‐2 inhibited rather than stimulated ROMK channel activity in both DCT2/CNT and in the CCD. TPNQ‐sensitive K currents were decreased from 46 pA/pf (WT) to 24.6 pA/pf (NEDD4‐2 KO) in the DCT2/CNT and from 34 pA/pf (WT) to 23.6 pA/pf (NEDD4‐2) in the CCD.ConclusionMR plays a key role in the regulation of ENaC in the CCD but to a less degree in the DCT2/CNT. ROMK channel activity is modestly regulated by MR in the CCD but not in the DCT2/CNT. NEDD4‐2 plays a role in the down‐regulation of ENaC activity in the ASDN, especially in the CCD, but it may not directly regulate ROMK.Support or Funding InformationDK54983

Significance Statement The potassium channel Kir4.1 forms the Kir4.1/Kir5.1 heterotetramer in the basolateral membrane of the distal convoluted tubule (DCT) and plays an important role in regulating the thiazide-sensitive NaCl cotransporter (NCC). Deletion of the ubiquitin ligase Nedd4-2 has been shown to increase the expression of NCC and to cause salt-sensitive hypertension. The authors demonstrated that kidney-specific deletion of Nedd4-2 in mice also stimulates Kir4.1/Kir5.1 activity in the DCT and hyperpolarizes the DCT membrane. They also found that NCC activity/expression is largely inhibited in double-knockout mice deficient in both Kir4.1 and Nedd4-2 and that NCC activity/expression is higher in these double-knockout mice compared with mice lacking only Kir4.1. These findings suggest that Nedd4-2 regulates NCC expression through modulation of basolateral Kir4.1/Kir5.1 activity and through Kir4.1-independent regulation of NCC retrieval. Background The potassium channel Kir4.1 forms the Kir4.1/Kir5.1 heterotetramer in the basolateral membrane of the distal convoluted tubule (DCT) and plays an important role in the regulation of the thiazide-sensitive NaCl cotransporter (NCC). Kidney-specific deletion of the ubiquitin ligase Nedd4-2 increases expression of NCC, and coexpression of Nedd4-2 inhibits Kir4.1/Kir5.1 in vitro . Whether Nedd4-2 regulates NCC expression in part by regulating Kir4.1/Kir5.1 channel activity in the DCT is unknown. Methods We used electrophysiology studies, immunoblotting, immunostaining, and renal clearance to examine Kir4.1/Kir5.1 activity in the DCT and NCC expression/activity in wild-type mice and mice with kidney-specific knockout of Nedd4-2, Kir4.1, or both. Results Deletion of Nedd4-2 increased the activity/expression of Kir4.1 in the DCT and also, hyperpolarized the DCT membrane. Expression of phosphorylated NCC/total NCC and thiazide-induced natriuresis were significantly increased in the Nedd4-2 knockout mice, but these mice were normokalemic. Double-knockout mice lacking both Kir4.1/Kir5.1 and Nedd4-2 in the kidney exhibited increased expression of the epithelial sodium channel α -subunit, largely abolished basolateral potassium ion conductance (to a degree similar to that of kidney-specific Kir4.1 knockout mice), and depolarization of the DCT membrane. Compared with wild-type mice, the double-knockout mice displayed inhibited expression of phosphorylated NCC and total NCC and had significantly blunted thiazide-induced natriuresis as well as renal potassium wasting and hypokalemia. However, NCC expression/activity was higher in the double-knockout mice than in Kir4.1 knockout mice. Conclusions Nedd4-2 regulates Kir4.1/Kir5.1 expression/activity in the DCT and modulates NCC expression by Kir4.1-dependent and Kir4.1-independent mechanisms. Basolateral Kir4.1/Kir5.1 activity in the DCT partially accounts for the stimulation of NCC activity/expression induced by deletion of Nedd4-2.

The mineralocorticoid hormone aldosterone plays a crucial role in the control of Na+ and K+ balance, blood volume, and arterial blood pressure, by acting in the aldosterone-sensitive distal nephron (ASDN) and stimulating a complex transcriptional, translational, and cellular program. Because the complexity of the aldosterone response is still not fully appreciated, we aimed at identifying new elements in this pathway. Here, we demonstrate that the expression of the proto-oncogene PIM3 (Proviral Integration Site of Moloney Murine Leukemia Virus 3), a serine/threonine kinase belonging to the calcium/calmodulin-regulated group of kinases, is stimulated by aldosterone in vitro (mCCDcl1 cells), ex vivo (mouse kidney slices), and in vivo in mice. Characterizing a germline Pim3-/- mouse model, we found that these mice have an upregulated Renin-Angiotensin-Aldosterone System (RAAS), with high circulating aldosterone and plasma renin activity levels on both standard or Na+ -deficient diet. Surprisingly, we did not observe any obvious salt-losing phenotype in Pim3 KO mice as shown by normal blood pressure, plasma and urinary electrolytes, as well as unchanged expression levels of the major Na+ transport proteins. These observations suggest that the potential effects of the loss of the Pim3 gene are physiologically compensated. Indeed, the 2 other family members of the PIM kinase family, PIM1 and PIM2 are upregulated in the kidney of Pim3-/- mice, and may therefore be involved in such compensation. In conclusion, our data demonstrate that the PIM3 kinase is a novel aldosterone-induced protein, but its precise role in aldosterone-dependent renal homeostasis remains to be determined.

Hypomagnesemia is associated with reduced kidney function and life-threatening complications and sustains hypokalemia. The distal convoluted tubule (DCT) determines final urinary Mg2+ excretion and, via activity of the Na+-Cl- cotransporter (NCC), also plays a key role in K+ homeostasis by metering Na+ delivery to distal segments. Little is known about the mechanisms by which plasma Mg2+ concentration regulates NCC activity and how low-plasma Mg2+ concentration and K+ concentration interact to modulate NCC activity. To address this, we performed dietary manipulation studies in mice. Compared with normal diet, abundances of total NCC and phosphorylated NCC (pNCC) were lower after short-term (3 days) or long-term (14 days) dietary Mg2+ restriction. Altered NCC activation is unlikely to play a role, since we also observed lower total NCC abundance in mice lacking the two NCC-activating kinases, STE20/SPS-1-related proline/alanine-rich kinase and oxidative stress response kinase-1, after Mg2+ restriction. The E3 ubiquitin-protein ligase NEDD4-2 regulates NCC abundance during dietary NaCl loading or K+ restriction. Mg2+ restriction did not lower total NCC abundance in inducible nephron-specific neuronal precursor cell developmentally downregulated 4-2 (NEDD4-2) knockout mice. Total NCC and pNCC abundances were similar after short-term Mg2+ or combined Mg2+-K+ restriction but were dramatically lower compared with a low-K+ diet. Therefore, sustained NCC downregulation may serve a mechanism that enhances distal Na+ delivery during states of hypomagnesemia, maintaining hypokalemia. Similar results were obtained with long-term Mg2+-K+ restriction, but, surprisingly, NCC was not activated after long-term K+ restriction despite lower plasma K+ concentration, suggesting significant differences in distal tubule adaptation to acute or chronic K+ restriction.

In the kidney, the sodium chloride potassium cotransporter NKCC2 of the thick ascending limb (TAL) of the loop of Henle plays an important role in regulation of body salt and water homeostasis. Due to a lack of a suitable polarized epithelial cell line model, the majority of functional and cell biological studies of NKCC2 have been performed in isolated TAL tubules or following expression of NKCC2 in xenopus oocytes. The aim of this study was to generate a MDCKI cell line with stable and tetracycline‐inducible expression of NKCC2. Our previous studies using this approach have demonstrated that this is an excellent approach to study another member of the same protein family, the NaCl cotransporter NCC. FRT‐MDCKI cells transfected with the A‐isoform of human NKCC2 (hNKCC2‐A) had robust NKCC2 mRNA expression after 24 h of tetracycline induction. Enhanced NKCC2 protein expression was achieved by additional treatment of cells with the histone deacetylase inhibitor, valproic acid in conjunction with tetracycline. PNGase F treatment of cell lysates determined that NKCC2 was expressed as highly glycosylated monomers and dimers. NKCC2 was observed both intracellularly and at the apical pole of MDCKI‐hNKCC2‐A cells using immunofluorescence labeling and confocal microscopy. Cycloheximide chase studies determined that the mature form of NKCC2 has a half‐life of around 12 hours, whereas the immature, non‐glycosylated form of NKCC2‐A has a much shorter half‐life. NKCC2 levels in the apical membrane increased with forskolin or hypotonic low chloride stimulation, which were accompanied with significantly increased phosphorylation at Ser126 or Thr96, respectively. We conclude that the MDCKI‐hNKCC2 A cell line is an excellent model for studying NKCC2 function and regulation. Support or Funding Information DFF ‐ Danish Council for Independent Research, Medical Sciences This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .