A New Phase for WNK Kinase Signaling Complexes as Biomolecular Condensates.

The purpose of this review is to highlight transformative advances that have been made in the field of biomolecular condensates, with special emphasis on condensate material properties, physiology, and kinases, using the With-No-Lysine (WNK) kinases as a prototypical example. To convey how WNK kinases illustrate important concepts for biomolecular condensates, we start with a brief history, focus on defining features of biomolecular condensates, and delve into some examples of how condensates are implicated in cellular physiology (and pathophysiology). We then highlight how WNK kinases, through the action of "WNK droplets" that ubiquitously regulate intracellular volume and kidney-specific "WNK bodies" that are implicated in distal tubule salt reabsorption and potassium homeostasis, exemplify many of the defining features of condensates. Finally, this review addresses the controversies within this emerging field and questions to address.

Excess dietary sodium partially restores salt and water homeostasis caused by loss of the endoplasmic reticulum molecular chaperone, GRP170, in the mouse nephron.

The maintenance of fluid and electrolyte homeostasis by the kidney requires proper folding and trafficking of ion channels and transporters in kidney epithelia. Each of these processes requires a specific subset of a diverse class of proteins termed molecular chaperones. One such chaperone is GRP170, which is an Hsp70-like, endoplasmic reticulum (ER)-localized chaperone that plays roles in protein quality control and protein folding in the ER. We previously determined that loss of GRP170 in the mouse nephron leads to hypovolemia, electrolyte imbalance, and rapid weight loss. In addition, GRP170-deficient mice develop an AKI-like phenotype, typified by tubular injury, elevation of clinical kidney injury markers, and induction of the unfolded protein response (UPR). By using an inducible GRP170 knockout cellular model, we confirmed that GRP170 depletion induces the UPR, triggers an apoptotic response, and disrupts protein homeostasis. Based on these data, we hypothesized that UPR induction underlies hyponatremia and volume depletion in rodents, but that these and other phenotypes might be rectified by supplementation with high salt. To test this hypothesis, control and GRP170 tubule-specific knockout mice were provided with a diet containing 8% sodium chloride. We discovered that sodium supplementation improved electrolyte imbalance and reduced clinical kidney injury markers, but was unable to restore weight or tubule integrity. These results are consistent with UPR induction contributing to the kidney injury phenotype in the nephron-specific GR170 knockout model, and that the role of GRP170 in kidney epithelia is essential to both maintain electrolyte balance and cellular protein homeostasis.

MicroRNA-19 is regulated by aldosterone in a sex-specific manner to alter kidney sodium transport.

A key regulator of blood pressure homeostasis is the steroid hormone aldosterone, which is released as the final signaling hormone of the renin-angiotensin-aldosterone-signaling (RAAS) system. Aldosterone increases sodium (Na+) reabsorption in the kidney distal nephron to regulate blood volume. Unregulated RAAS signaling can lead to hypertension and cardiovascular disease. The serum and glucocorticoid kinase (SGK1) coordinates much of the Na+ reabsorption in the cortical collecting duct (CCD) tubular epithelial cells. We previously demonstrated that aldosterone alters the expression of microRNAs (miRs) in CCD principal cells. The aldosterone-regulated miRs can modulate Na+ transport and the cellular response to aldosterone signaling. However, the sex-specific regulation of miRs by aldosterone in the kidney distal nephron has not been explored. In this study, we report that miR-19, part of the miR-17-92 cluster, is upregulated in female mouse CCD cells in response to aldosterone activation. Mir-19 binding to the 3'-untranslated region of SGK1 was confirmed using a dual-luciferase reporter assay. Increasing miR-19 expression in CCD cells decreased SGK1 message and protein expression. Removal of this cluster using a nephron-specific, inducible knockout mouse model increased SGK1 expression in female mouse CCD cells. The miR-19-induced decrease in SGK1 protein expression reduced the response to aldosterone stimulation and may account for sex-specific differences in aldosterone signaling. By examining evolution of the miR-17-92 cluster, phylogenetic sequence analysis indicated that this cluster arose at the same time that other Na+-sparing and salt regulatory proteins, specifically SGK1, first emerged, indicating a conserved role for these miRs in kidney function of salt and water homeostasis.NEW & NOTEWORTHY Expression of the microRNA-17-92 cluster is upregulated by aldosterone in mouse cortical collecting duct principal cells, exclusively in female mice. MiR-19 in this cluster targets the serum and glucocorticoid kinase (SGK1) to downregulate both mRNA and protein expression, resulting in a decrease in sodium transport across epithelial cells of the collecting duct. The miR-17-92 cluster is evolutionarily conserved and may act as a novel feedback regulator for aldosterone signaling in females.

Molecular Crowding: Physiologic Sensing and Control.

The cytoplasm is densely packed with molecules that contribute to its nonideal behavior. Cytosolic crowding influences chemical reaction rates, intracellular water mobility, and macromolecular complex formation. Overcrowding is potentially catastrophic; to counteract this problem, cells have evolved acute and chronic homeostatic mechanisms that optimize cellular crowdedness. Here, we provide a physiology-focused overview of molecular crowding, highlighting contemporary advances in our understanding of its sensing and control. Long hypothesized as a form of crowding-induced microcompartmentation, phase separation allows cells to detect and respond to intracellular crowding through the action of biomolecular condensates, as indicated by recent studies. Growing evidence indicates that crowding is closely tied to cell size and fluid volume, homeostatic responses to physical compression and desiccation, tissue architecture, circadian rhythm, aging, transepithelial transport, and total body electrolyte and water balance. Thus, molecular crowding is a fundamental physiologic parameter that impacts diverse functions extending from molecule to organism.

Impact of sleep on chronobiology of micturition among healthy older adults.

Nocturia (waking to void) is prevalent among older adults. Disruption of the well-described circadian rhythm in urine production with higher nighttime urine output is its most common cause. In young adults, their circadian rhythm is modulated by the 24-h secretory pattern of hormones that regulate salt and water excretion, including antidiuretic hormone (ADH), renin, angiotensin, aldosterone, and atrial natriuretic peptide (ANP). The pattern of hormone secretion is less clear in older adults. We investigated the effect of sleep on the 24-h secretion of these hormones in healthy older adults. Thirteen participants aged ≥65 yr old underwent two 24-h protocols at a clinical research center 6 wk apart. The first used a habitual wake-sleep protocol, and the second used a constant routine protocol that removed the influence of sleep, posture, and diet. To assess hormonal rhythms, plasma was collected at 8:00 am, 12:00 pm, 4:00 pm, and every 30 min from 7:00 pm to 7:00 am. A mixed-effects regression model was used to compare subject-specific and mean trajectories of hormone secretion under the two conditions. ADH, aldosterone, and ANP showed a diurnal rhythm that peaked during sleep in the wake-sleep protocol. These nighttime elevations were significantly attenuated within subjects during the constant routine. We conclude that sleep has a masking effect on circadian rhythm amplitude of ADH, aldosterone, and ANP: the amplitude of each is increased in the presence of sleep and reduced in the absence of sleep. Disrupted sleep could potentially alter nighttime urine output in healthy older adults via this mechanism.NEW & NOTEWORTHY Nocturia (waking to void) is the most common cause of sleep interruption among older adults, and increased nighttime urine production is its primary etiology. We showed that in healthy older adults sleep affects the 24-h secretory rhythm of hormones that regulate salt-water balance, which potentially alters nighttime urine output. Further studies are needed to elucidate the impact of chronic insomnia on the secretory rhythms of these hormones.

WNK kinases sense molecular crowding and rescue cell volume via phase separation.

When challenged by hypertonicity, dehydrated cells must recover their volume to survive. This process requires the phosphorylation-dependent regulation of SLC12 cation chloride transporters by WNK kinases, but how these kinases are activated by cell shrinkage remains unknown. Within seconds of cell exposure to hypertonicity, WNK1 concentrates into membraneless condensates, initiating a phosphorylation-dependent signal that drives net ion influx via the SLC12 cotransporters to restore cell volume. WNK1 condensate formation is driven by its intrinsically disordered C terminus, whose evolutionarily conserved signatures are necessary for efficient phase separation and volume recovery. This disorder-encoded phase behavior occurs within physiological constraints and is activated in vivo by molecular crowding rather than changes in cell size. This allows kinase activity despite an inhibitory ionic milieu and permits cell volume recovery through condensate-mediated signal amplification. Thus, WNK kinases are physiological crowding sensors that phase separate to coordinate a cell volume rescue response.

Role of SPAK-NKCC1 signaling cascade in the choroid plexus blood-CSF barrier damage after stroke.

BACKGROUND: The mechanisms underlying dysfunction of choroid plexus (ChP) blood-cerebrospinal fluid (CSF) barrier and lymphocyte invasion in neuroinflammatory responses to stroke are not well understood. In this study, we investigated whether stroke damaged the blood-CSF barrier integrity due to dysregulation of major ChP ion transport system, Na+-K+-Cl- cotransporter 1 (NKCC1), and regulatory Ste20-related proline-alanine-rich kinase (SPAK). METHODS: Sham or ischemic stroke was induced in C57Bl/6J mice. Changes on the SPAK-NKCC1 complex and tight junction proteins (TJs) in the ChP were quantified by immunofluorescence staining and immunoblotting. Immune cell infiltration in the ChP was assessed by flow cytometry and immunostaining. Cultured ChP epithelium cells (CPECs) and cortical neurons were used to evaluate H2O2-mediated oxidative stress in stimulating the SPAK-NKCC1 complex and cellular damage. In vivo or in vitro pharmacological blockade of the ChP SPAK-NKCC1 cascade with SPAK inhibitor ZT-1a or NKCC1 inhibitor bumetanide were examined. RESULTS: Ischemic stroke stimulated activation of the CPECs apical membrane SPAK-NKCC1 complex, NF-κB, and MMP9, which was associated with loss of the blood-CSF barrier integrity and increased immune cell infiltration into the ChP. Oxidative stress directly activated the SPAK-NKCC1 pathway and resulted in apoptosis, neurodegeneration, and NKCC1-mediated ion influx. Pharmacological blockade of the SPAK-NKCC1 pathway protected the ChP barrier integrity, attenuated ChP immune cell infiltration or neuronal death. CONCLUSION: Stroke-induced pathological stimulation of the SPAK-NKCC1 cascade caused CPECs damage and disruption of TJs at the blood-CSF barrier. The ChP SPAK-NKCC1 complex emerged as a therapeutic target for attenuating ChP dysfunction and lymphocyte invasion after stroke.

The molecular chaperone GRP170 protects against ER stress and acute kidney injury in mice.

Molecular chaperones are responsible for maintaining cellular homeostasis, and one such chaperone, GRP170, is an endoplasmic reticulum (ER) resident that oversees both protein biogenesis and quality control. We previously discovered that GRP170 regulates the degradation and assembly of the epithelial sodium channel (ENaC), which reabsorbs sodium in the distal nephron and thereby regulates salt-water homeostasis and blood pressure. To define the role of GRP170 - and, more generally, molecular chaperones in kidney physiology - we developed an inducible, nephron-specific GRP170-KO mouse. Here, we show that GRP170 deficiency causes a dramatic phenotype: profound hypovolemia, hyperaldosteronemia, and dysregulation of ion homeostasis, all of which are associated with the loss of ENaC. Additionally, the GRP170-KO mouse exhibits hallmarks of acute kidney injury (AKI). We further demonstrate that the unfolded protein response (UPR) is activated in the GRP170-deficient mouse. Notably, the UPR is also activated in AKI when originating from various other etiologies, including ischemia, sepsis, glomerulonephritis, nephrotic syndrome, and transplant rejection. Our work establishes the central role of GRP170 in kidney homeostasis and directly links molecular chaperone function to kidney injury.

L-WNK1 is required for BK channel activation in intercalated cells.

Large-conductance K+ (BK) channels expressed in intercalated cells (ICs) in the aldosterone-sensitive distal nephron (ASDN) mediate flow-induced K+ secretion. In the ASDN of mice and rabbits, IC BK channel expression and activity increase with a high-K+ diet. In cell culture, the long isoform of with-no-lysine kinase 1 (L-WNK1) increases BK channel expression and activity. Apical L-WNK1 expression is selectively enhanced in ICs in the ASDN of rabbits on a high-K+ diet, suggesting that L-WNK1 contributes to BK channel regulation by dietary K+. We examined the role of IC L-WNK1 expression in enhancing BK channel activity in response to a high-K+ diet. Mice with IC-selective deletion of L-WNK1 (IC-L-WNK1-KO) and littermate control mice were placed on a high-K+ (5% K+, as KCl) diet for 10 or more days. IC-L-WNK1-KO mice exhibited reduced IC apical + subapical α-subunit expression and BK channel-dependent whole cell currents compared with controls. Six-hour urinary K+ excretion in response a saline load was similar in IC-L-WNK1-KO mice and controls. The observations that IC-L-WNK1-KO mice on a high-K+ diet have higher blood K+ concentration and reduced IC BK channel activity are consistent with impaired urinary K+ secretion, demonstrating that IC L-WNK1 has a role in the renal adaptation to a high-K+ diet.NEW & NOTEWORTHY When mice are placed on a high-K+ diet, genetic disruption of the long form of with no lysine kinase 1 (L-WNK1) in intercalated cells reduced relative apical + subapical localization of the large-conductance K+ channel, blunted large-conductance K+ channel currents in intercalated cells, and increased blood K+ concentration. These data confirm an in vivo role of L-WNK1 in intercalated cells in adaptation to a high-K+ diet.

Effects of extreme potassium stress on blood pressure and renal tubular sodium transport.

We characterized mouse blood pressure and ion transport in the setting of commonly used rodent diets that drive K+ intake to the extremes of deficiency and excess. Male 129S2/Sv mice were fed either K+-deficient, control, high-K+ basic, or high-KCl diets for 10 days. Mice maintained on a K+-deficient diet exhibited no change in blood pressure, whereas K+-loaded mice developed an ~10-mmHg blood pressure increase. Following challenge with NaCl, K+-deficient mice developed a salt-sensitive 8 mmHg increase in blood pressure, whereas blood pressure was unchanged in mice fed high-K+ diets. Notably, 10 days of K+ depletion induced diabetes insipidus and upregulation of phosphorylated NaCl cotransporter, proximal Na+ transporters, and pendrin, likely contributing to the K+-deficient NaCl sensitivity. While the anionic content with high-K+ diets had distinct effects on transporter expression along the nephron, both K+ basic and KCl diets had a similar increase in blood pressure. The blood pressure elevation on high-K+ diets correlated with increased Na+-K+-2Cl- cotransporter and γ-epithelial Na+ channel expression and increased urinary response to furosemide and amiloride. We conclude that the dietary K+ maneuvers used here did not recapitulate the inverse effects of K+ on blood pressure observed in human epidemiological studies. This may be due to the extreme degree of K+ stress, the low-Na+-to-K+ ratio, the duration of treatment, and the development of other coinciding events, such as diabetes insipidus. These factors must be taken into consideration when studying the physiological effects of dietary K+ loading and depletion.

Intercalated cell BKα subunit is required for flow-induced K+ secretion.

BK channels are expressed in intercalated cells (ICs) and principal cells (PCs) in the cortical collecting duct (CCD) of the mammalian kidney and have been proposed to be responsible for flow-induced K+ secretion (FIKS) and K+ adaptation. To examine the IC-specific role of BK channels, we generated a mouse with targeted disruption of the pore-forming BK α subunit (BKα) in ICs (IC-BKα-KO). Whole cell charybdotoxin-sensitive (ChTX-sensitive) K+ currents were readily detected in control ICs but largely absent in ICs of IC-BKα-KO mice. When placed on a high K+ (HK) diet for 13 days, blood [K+] was significantly greater in IC-BKα-KO mice versus controls in males only, although urinary K+ excretion rates following isotonic volume expansion were similar in males and females. FIKS was present in microperfused CCDs isolated from controls but was absent in IC-BKα-KO CCDs of both sexes. Also, flow-stimulated epithelial Na+ channel-mediated (ENaC-mediated) Na+ absorption was greater in CCDs from female IC-BKα-KO mice than in CCDs from males. Our results confirm a critical role of IC BK channels in FIKS. Sex contributes to the capacity for adaptation to a HK diet in IC-BKα-KO mice.

Nrf2 activation protects against lithium-induced nephrogenic diabetes insipidus.

Lithium (Li) is the mainstay pharmacotherapeutic mood stabilizer in bipolar disorder. Its efficacious use is complicated by acute and chronic renal side effects, including nephrogenic diabetes insipidus (NDI) and progression to chronic kidney disease (CKD). The nuclear factor erythroid-derived 2-related factor 2 (Nrf2) pathway senses and coordinates cellular responses to oxidative and electrophilic stress. Here, we identify that graded genetic activation of Nrf2 protects against Li-induced NDI (Li-NDI) and volume wasting via an aquaporin 2-independent mechanism. Renal Nrf2 activity is differentially expressed on functional segments of the nephron, and its activation along the distal tubule and collecting duct directly modulates ion transporter expression, mimicking paradoxical effects of diuretics in mitigating Li-NDI. In addition, Nrf2 reduces cyclooxygenase expression and vasoactive prostaglandin biosynthesis. Pharmacologic activation of Nrf2 confers protective effects, confirming this pathway as a potentially novel druggable target for the prevention of acute and chronic renal sequelae of Li therapy.

The endosomal trafficking factors CORVET and ESCRT suppress plasma membrane residence of the renal outer medullary potassium channel (ROMK).

Protein trafficking can act as the primary regulatory mechanism for ion channels with high open probabilities, such as the renal outer medullary (ROMK) channel. ROMK, also known as Kir1.1 (KCNJ1), is the major route for potassium secretion into the pro-urine and plays an indispensable role in regulating serum potassium and urinary concentrations. However, the cellular machinery that regulates ROMK trafficking has not been fully defined. To identify regulators of the cell-surface population of ROMK, we expressed a pH-insensitive version of the channel in the budding yeast Saccharomyces cerevisiae We determined that ROMK primarily resides in the endoplasmic reticulum (ER), as it does in mammalian cells, and is subject to ER-associated degradation (ERAD). However, sufficient ROMK levels on the plasma membrane rescued growth on low-potassium medium of yeast cells lacking endogenous potassium channels. Next, we aimed to identify the biological pathways most important for ROMK regulation. Therefore, we used a synthetic genetic array to identify non-essential genes that reduce the plasma membrane pool of ROMK in potassium-sensitive yeast cells. Genes identified in this screen included several members of the endosomal complexes required for transport (ESCRT) and the class-C core vacuole/endosome tethering (CORVET) complexes. Mass spectroscopy analysis confirmed that yeast cells lacking an ESCRT component accumulate higher potassium concentrations. Moreover, silencing of ESCRT and CORVET components increased ROMK levels at the plasma membrane in HEK293 cells. Our results indicate that components of the post-endocytic pathway influence the cell-surface density of ROMK and establish that components in this pathway modulate channel activity.

Potassium-regulated distal tubule WNK bodies are kidney-specific WNK1 dependent.

With-no-lysine (WNK) kinases coordinate volume and potassium homeostasis by regulating renal tubular electrolyte transport. In the distal convoluted tubule (DCT), potassium imbalance causes WNK signaling complexes to concentrate into large discrete foci, which we call "WNK bodies." Although these structures have been reported previously, the mechanisms that drive their assembly remain obscure. Here, we show that kidney-specific WNK1 (KS-WNK1), a truncated kinase-defective WNK1 isoform that is highly expressed in the DCT, is critical for WNK body formation. While morphologically distinct WNK bodies were evident in the distal tubules of mice subjected to dietary potassium loading and restriction, KS-WNK1 knockout mice were deficient in these structures under identical conditions. Combining in vivo observations in kidney with reconstitution studies in cell culture, we found that WNK bodies are dynamic membraneless foci that are distinct from conventional organelles, colocalize with the ribosomal protein L22, and cluster the WNK signaling pathway. The formation of WNK bodies requires an evolutionarily conserved cysteine-rich hydrophobic motif harbored within a unique N-terminal exon of KS-WNK1. We propose that WNK bodies are not pathological aggregates, but rather are KS-WNK1-dependent microdomains of the DCT cytosol that modulate WNK signaling during physiological shifts in potassium balance.

Dietary K+ and Cl- independently regulate basolateral conductance in principal and intercalated cells of the collecting duct.

The renal collecting duct contains two distinct cell types, principal and intercalated cells, expressing potassium Kir4.1/5.1 (KCNJ10/16) and chloride ClC-K2 (ClC-Kb in humans) channels on their basolateral membrane, respectively. Both channels are thought to play important roles in controlling systemic water-electrolyte balance and blood pressure. However, little is known about mechanisms regulating activity of Kir4.1/5.1 and ClC-K2/b. Here, we employed patch clamp analysis at the single channel and whole cell levels in freshly isolated mouse collecting ducts to investigate regulation of Kir4.1/5.1 and ClC-K2/b by dietary K+ and Cl- intake. Treatment of mice with high K+ and high Cl- diet (6% K+, 5% Cl-) for 1 week significantly increased basolateral K+-selective current, single channel Kir4.1/5.1 activity and induced hyperpolarization of basolateral membrane potential in principal cells when compared to values in mice on a regular diet (0.9% K+, 0.5% Cl-). In contrast, basolateral Cl--selective current and single channel ClC-K2/b activity was markedly decreased in intercalated cells under this condition. Substitution of dietary K+ to Na+ in the presence of high Cl- exerted a similar inhibiting action of ClC-K2/b suggesting that the channel is sensitive to variations in dietary Cl- per se. Cl--sensitive with-no-lysine kinase (WNK) cascade has been recently proposed to orchestrate electrolyte transport in the distal tubule during variations of dietary K+. However, co-expression of WNK1 or its major downstream effector Ste20-related proline-alanine-rich kinase (SPAK) had no effect on ClC-Kb over-expressed in Chinese hamster ovary (CHO) cells. Treatment of mice with high K+ diet without concomitant elevations in dietary Cl- (6% K+, 0.5% Cl-) elicited a comparable increase in basolateral K+-selective current, single channel Kir4.1/5.1 activity in principal cells, but had no significant effect on ClC-K2/b activity in intercalated cells. Furthermore, stimulation of aldosterone signaling by Deoxycorticosterone acetate (DOCA) recapitulated the stimulatory actions of high K+ intake on Kir4.1/5.1 channels in principal cells but was ineffective to alter ClC-K2/b activity and basolateral Cl- conductance in intercalated cells. In summary, we report that variations of dietary K+ and Cl- independently regulate basolateral potassium and chloride conductance in principal and intercalated cells. We propose that such discrete mechanism might contribute to fine-tuning of urinary excretion of electrolytes depending on dietary intake.

The renal response to potassium stress: integrating past with present.

PURPOSE OF REVIEW: The current review combines past findings with recent advances in our understanding of the homeostatic response to potassium imbalance. RECENT FINDINGS: Following the ingestion of a dietary potassium load, a combination of extrarenal and renal mechanisms act to maintain extracellular K+ within a tight window. Through hormonal regulation and direct K+ sensing, the nephron is ideally suited to respond to wide shifts in external K+ balance. Current evidence indicates that dietary K+ loading triggers a coordinated kaliuretic response that appears to involve voltage-dependent changes in sodium transport across multiple nephron segments, including the proximal tubule, medullary loop of Henle, and distal tubule. Inhibition of sodium transport in these segments would accomplish the final goal of enhancing distal NaCl delivery, luminal flow, and K+ secretion in the aldosterone sensitive distal nephron (ASDN). SUMMARY: Ongoing research seeks to define the relationship between potassium and volume homeostasis by elucidating pathways that couple renal K+ sensing and tubular function during the potassium stress response.

Endoplasmic reticulum-associated degradation of the renal potassium channel, ROMK, leads to type II Bartter syndrome.

Type II Bartter syndrome is caused by mutations in the renal outer medullary potassium (ROMK) channel, but the molecular mechanisms underlying this disease are poorly defined. To rapidly screen for ROMK function, we developed a yeast expression system and discovered that yeast cells lacking endogenous potassium channels could be rescued by WT ROMK but not by ROMK proteins containing any one of four Bartter mutations. We also found that the mutant proteins were significantly less stable than WT ROMK. However, their degradation was slowed in the presence of a proteasome inhibitor or when yeast cells contained mutations in the CDC48 or SSA1 gene, which is required for endoplasmic reticulum (ER)-associated degradation (ERAD). Consistent with these data, sucrose gradient centrifugation and indirect immunofluorescence microscopy indicated that most ROMK protein was ER-localized. To translate these findings to a more relevant cell type, we measured the stabilities of WT ROMK and the ROMK Bartter mutants in HEK293 cells. As in yeast, the Bartter mutant proteins were less stable than the WT protein, and their degradation was slowed in the presence of a proteasome inhibitor. Finally, we discovered that low-temperature incubation increased the steady-state levels of a Bartter mutant, suggesting that the disease-causing mutation traps the protein in a folding-deficient conformation. These findings indicate that the underlying pathology for at least a subset of patients with type II Bartter syndrome is linked to the ERAD pathway and that future therapeutic strategies should focus on correcting deficiencies in ROMK folding.

Renal Tubular Ubiquitin-Protein Ligase NEDD4-2 Is Required for Renal Adaptation during Long-Term Potassium Depletion.

Adaptation of the organism to potassium (K+) deficiency requires precise coordination among organs involved in K+ homeostasis, including muscle, liver, and kidney. How the latter performs functional and molecular changes to ensure K+ retention is not well understood. Here, we investigated the role of ubiquitin-protein ligase NEDD4-2, which negatively regulates the epithelial sodium channel (ENaC), Na+/Cl- cotransporter (NCC), and with no-lysine-kinase 1 (WNK1). After dietary K+ restriction for 2 weeks, compared with control littermates, inducible renal tubular NEDD4-2 knockout (Nedd4LPax8/LC1 ) mice exhibited severe hypokalemia and urinary K+ wasting. Notably, expression of the ROMK K+ channel did not change in the distal convoluted tubule and decreased slightly in the cortical/medullary collecting duct, whereas BK channel abundance increased in principal cells of the connecting tubule/collecting ducts. However, K+ restriction also enhanced ENaC expression in Nedd4LPax8/LC1 mice, and treatment with the ENaC inhibitor, benzamil, reversed excessive K+ wasting. Moreover, K+ restriction increased WNK1 and WNK4 expression and enhanced SPAK-mediated NCC phosphorylation in Nedd4LPax8/LC1 mice, with no change in total NCC. We propose a mechanism in which NEDD4-2 deficiency exacerbates hypokalemia during dietary K+ restriction primarily through direct upregulation of ENaC, whereas increased BK channel expression has a less significant role. These changes outweigh the compensatory antikaliuretic effects of diminished ROMK expression, increased NCC phosphorylation, and enhanced WNK pathway activity in the distal convoluted tubule. Thus, NEDD4-2 has a crucial role in K+ conservation through direct and indirect effects on ENaC, distal nephron K+ channels, and WNK signaling.

WNK-Cab39-NKCC1 signaling increases the susceptibility to ischemic brain damage in hypertensive rats.

With-no-lysine kinase (WNK) and Na+-K+-2Cl- cotransporter 1 (NKCC1) are involved in the pathogenesis of hypertension. In this study, we investigated the WNK-NKCC1 signaling pathway in spontaneously hypertensive rats (SHR) and their associated susceptibility to stroke injury. Basal NKCC1 protein levels were higher in SHR than in normotensive Wistar Kyoto (WKY) rat brains. After inducing ischemic stroke, adult male WKY and SHR received either saline or NKCC1 inhibitor bumetanide (10 mg/kg/day, i.p.) starting at 3-h post-reperfusion. NKCC1 inhibition blunted the extent of ischemic infarction in SHR and improved their neurobehavioral functions. Interestingly, ischemia led to increased NKCC1 phosphorylation in SHR but not in WKY rats. Pronounced elevation of WNK1, WNK2 and WNK4 protein and downregulation of WNK3 were detected in ischemic SHR, but not in ischemic WKY rats. Upregulation of WNK-NKCC1 complex in ischemic SHR brain was associated with increased Ca2+-binding protein 39 (Cab39), without increases in Ste20-related proline alanine-rich kinase or oxidative stress-responsive kinase-1. Moreover, subacute middle cerebral artery stroke human brain autopsy exhibited increased expression of NKCC1 protein. We conclude that augmented WNK-Cab39-NKCC1 signaling in SHR is associated with an increased susceptibility to ischemic brain damage and may serve as a novel target for anti-hypertensive and anti-ischemic stroke therapy.