Trimethoprim inhibits renal H+-K+-ATPase in states of K+ depletion.

There is growing consensus that under physiological conditions, collecting duct H+ secretion is independent of epithelial Na+ channel (ENaC) activity. We have recently shown that the direct ENaC inhibitor benzamil acutely impairs H+ excretion by blocking renal H+-K+-ATPase. However, the question remains whether inhibition of ENaC per se causes alterations in renal H+ excretion. To revisit this question, we studied the effect of the antibiotic trimethoprim (TMP), which is well known to cause K+ retention by direct ENaC inhibition. The acute effect of TMP (5 µg/g body wt) was assessed in bladder-catheterized mice, allowing real-time measurement of urinary pH, electrolyte, and acid excretion. Dietary K+ depletion was used to increase renal H+-K+-ATPase activity. In addition, the effect of TMP was investigated in vitro using pig gastric H+-K+-ATPase-enriched membrane vesicles. TMP acutely increased natriuresis and decreased kaliuresis, confirming its ENaC-inhibiting property. Under control diet conditions, TMP had no effect on urinary pH or acid excretion. Interestingly, K+ depletion unmasked an acute urine alkalizing effect of TMP. This finding was corroborated by in vitro experiments showing that TMP inhibits H+-K+-ATPase activity, albeit at much higher concentrations than benzamil. In conclusion, under control diet conditions, TMP inhibited ENaC function without changing urinary H+ excretion. This finding further supports the hypothesis that the inhibition of ENaC per se does not impair H+ excretion in the collecting duct. Moreover, TMP-induced urinary alkalization in animals fed a low-K+ diet highlights the importance of renal H+-K+-ATPase-mediated H+ secretion in states of K+ depletion.NEW & NOTEWORTHY The antibiotic trimethoprim (TMP) often mediates K+ retention and metabolic acidosis. We suggest a revision of the underlying mechanism that causes metabolic acidosis. Our results indicate that TMP-induced metabolic acidosis is secondary to epithelial Na+ channel-dependent K+ retention. Under control dietary conditions, TMP does not per se inhibit collecting duct H+ secretion. These findings add further argument against a physiologically relevant voltage-dependent mechanism of collecting duct H+ excretion.

Capsazepine activates amiloride-insensitive ENaC-like channels in human leukemia cells.

Sodium influx carried out by ion channels is one of the main regulators of water-salt and volume balance in cells of blood origin. Previously, we described amiloride-insensitive ENaC-like channels in human myeloid leukemia K562 cells; the intracellular regulatory mechanisms of the channels are associated with actin cytoskeleton dynamics. Recently, an extracellular mechanism of ENaC-like channels activation in K562 cells by the action of serine protease trypsin has been revealed. The other extracellular pathways that modulate ENaC (epithelial Na+ channel) activity and sodium permeability in transformed blood cells are not yet fully investigated. Here, we study the action of capsazepine (CPZ), as δ-ENaC activator, on single channel activity in K562 cells in whole-cell patch clamp experiments. Addition of CPZ (2 µM) to the extracellular solution caused an activation of sodium channels with typical features; unitary conductance was 15.1 ± 0.8 pS. Amiloride derivative benzamil (50 µM) did not inhibit their activity. Unitary currents and conductance of CPZ-activated channels were higher in Na+-containing extracellular solution than in Li+, that is one of the main fingerprints of δ-ENaC. The results of RT-PCR analysis and immunofluorescence staining also confirmed the expression of δ-hENaC (as well as α-, ß-, γ-ENaC) at the mRNA and protein level. These findings allow us to speculate that CPZ activates amiloride-insensitive ENaC-like channels that contain δ-ENaC in К562 cells. Our data reveal a novel extracellular mechanism for ENaC-like activation in human leukemia cells.

Characteristics of sodium and water retention in rats with nephrotic syndrome induced by puromycin aminonucleoside.

INTRODUCTION: Nephrotic syndrome (NS) is characterized by renal sodium and water retention. The mechanisms are not fully elucidated. METHODS: The NS rat model was established by single intraperitoneal injection of 100 mg/kg puromycin aminonucleoside (PAN). The plasma electrolyte level and urinary sodium excretion were monitored dynamically. The changes of some sodium transporters, including epithelial Na+ channel (ENaC), Na+/H+ exchanger 3 (NHE3), Na+-K+-2Cl- cotransporter 2 (NKCC2) and Na+-Cl- cotransporter (NCC) in renal cortex at different time points and the level of peripheral circulation factors were detected. RESULTS: The urinary sodium excretion of the model group increased significantly on the first day, then decreased compared with the control group, and there was no significant difference between the model group and the control group on the 12th day. The changes of peripheral circulation factors were not obvious. Some sodium transporters in renal cortex increased in varying degrees, while NKCC2 decreased significantly compared with the control group. CONCLUSIONS: The occurrence of NS edema may not be related to the angiotensin system. The decrease of urinary sodium excretion is independent of the development of albuminuria. During the 18 days of observation, it can be divided into three stages: sodium retention, sodium compensation, and simple water retention. The mechanism is related to the increased expression of α-ENaC, γ-ENaC, NHE3 and NCC in a certain period of time, the compensatory decrease of NKCC2 expression and the continuous increase of aquaporin 2 (AQP2) expression.

Amiloride evokes significant natriuresis and weight loss in kidney transplant recipients with and without albuminuria.

Albuminuria in kidney transplant recipients (KTRs) is associated with hypertension and aberrant glomerular filtration of serine proteases that may proteolytically activate the epithelial Na+ channel (ENaC). The present nonrandomized, pharmacodynamic intervention study aimed to investigate if inhibition of ENaC increases Na+ excretion and reduces extracellular volume in KTRs dependent on the presence of albuminuria. KTRs with and without albuminuria (albumin-to-creatinine ratio > 300 mg/g, n = 7, and <30 mg/g, n = 7, respectively) were included and ingested a diet with fixed Na+ content (150 mmol/day) for 5 days. On the last day, amiloride at 10 mg was administered twice. Body weight, 24-h urine electrolyte excretion, body water content, and ambulatory blood pressure as well as plasma renin, angiotensin II, and aldosterone concentrations were determined before and after amiloride. Amiloride led to a significant decrease in body weight, increase in 24-h urinary Na+ excretion, and decrease in 24-h urinary K+ excretion in both groups. Urine output increased in the nonalbuminuric group only. There was no change in plasma renin, aldosterone, and angiotensin II concentrations after amiloride, whereas a significant decrease in nocturnal systolic blood pressure and increase in 24-h urine aldosterone excretion was observed in albuminuric KTRs only. There was a significant correlation between 24-h urinary albumin excretion and amiloride-induced 24-h urinary Na+ excretion. In conclusion, ENaC activity contributes to Na+ and water retention in KTRs with and without albuminuria. ENaC is a relevant pharmacological target in KTRs; however, larger and long-term studies are needed to evaluate whether the magnitude of this effect depends on the presence of albuminuria.NEW & NOTEWORTHY Amiloride has a significant natriuretic effect in kidney transplant recipients (KTRs) that relates to urinary albumin excretion. The epithelial Na+ channel may be a relevant direct pharmacological target to counter Na+ retention and hypertension in KTRs. Epithelial Na+ channel blockers should be further investigated as a mean to mitigate Na+ and water retention and to potentially obtain optimal blood pressure control in KTRs.

Involvement of miR-495 overexpression in the pathogenesis of bronchopulmonary dysplasia in preterm infants via the targeting of NEDD4L-ENaC pathway.

Background: Bronchopulmonary dysplasia (BPD) is a severe pulmonary complication causing morbidity and mortality in preterm infants. A key histopathological feature of BPD is late lung growth retardation, in which the process of alveolarization is hindered and the mechanism of which is unclear. Emerging evidence indicates that microRNAs (miRNAs) promote the development of BPD via the inhibition of their target genes. MiR-495 has been reported to be involved in various lung diseases. However, the physiological function of miR-495 in BPD has not yet been fully understood. Methods: Differentially expressed miRNAs in peripheral blood of patients with BPD were compared with those of normal controls. A dual-luciferase reporter assay was performed to identify the target genes of miR-495. A BPD neonatal rat model was established by injecting lipopolysaccharide (LPS) in the amniotic sac of pregnant rats. The morphology of the lungs was observed using hematoxylin and eosin (HE) staining. The expression of miR-495, neural precursor cell expressed developmentally down-regulated 4-like (NEDD4L), and epithelial Na+ channel (ENaC) was tested using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blot analysis, and immunofluorescent (IF) staining. Results: The expression of miR-495 was significantly increased in the peripheral blood samples of premature infants with BPD and verified using qRT-PCR. NEDD4L was proven to be the target gene of miR-495. Additionally, miR-495 expression was also increased in the lungs of rat pups with BPD at postnatal day (P) 3 compared with the control group. qRT-PCR and Western blot results showed that NEDD4L expression was decreased while ENaC expression was increased at the transcriptional and translational levels. IF staining results showed that NEDD4L level was decreased while ENaC level was increased in the LPS-induced BPD rat model, which was consistent with abnormal changes in alveolar structure. Conclusions: The aberrant overexpression of miR-495 may contribute to the development of BPD by targeting NEDD4L-ENaC pathway, implying an imbalance in lung fluid clearance.

Rare Variants in Genes Encoding Subunits of the Epithelial Na+ Channel Are Associated With Blood Pressure and Kidney Function.

BACKGROUND: The epithelial Na+ channel (ENaC) is intrinsically linked to fluid volume homeostasis and blood pressure. Specific rare mutations in SCNN1A, SCNN1B, and SCNN1G, genes encoding the α, ß, and γ subunits of ENaC, respectively, are associated with extreme blood pressure phenotypes. No associations between blood pressure and SCNN1D, which encodes the δ subunit of ENaC, have been reported. A small number of sequence variants in ENaC subunits have been reported to affect functional transport in vitro or blood pressure. The effects of the vast majority of rare and low-frequency ENaC variants on blood pressure are not known. METHODS: We explored the association of low frequency and rare variants in the genes encoding ENaC subunits, with systolic blood pressure, diastolic blood pressure, mean arterial pressure, and pulse pressure. Using whole-genome sequencing data from 14 studies participating in the Trans-Omics in Precision Medicine Whole-Genome Sequencing Program, and sequence kernel association tests. RESULTS: We found that variants in SCNN1A and SCNN1B were associated with diastolic blood pressure and mean arterial pressure (P<0.00625). Although SCNN1D is poorly expressed in human kidney tissue, SCNN1D variants were associated with systolic blood pressure, diastolic blood pressure, mean arterial pressure, and pulse pressure (P<0.00625). ENaC variants in 2 of the 4 subunits (SCNN1B and SCNN1D) were also associated with estimated glomerular filtration rate (P<0.00625), but not with stroke. CONCLUSIONS: Our results suggest that variants in extrarenal ENaCs, in addition to ENaCs expressed in kidneys, influence blood pressure and kidney function.

Hypertension and human immunodeficiency virus: A paradigm for epithelial sodium channels?

Hypertension is a risk factor for end organ damage and death and is more common in persons with HIV compared to the general population. Several mechanisms have been studied in the pathogenesis of hypertension. Current evidence suggests that the epithelial sodium channel (ENaC) plays a key role in regulating blood pressure through the transport of sodium and water across membranes in the kidney tubules, resulting in retention of sodium and water and an altered fluid balance. However, there is scarcity of information that elucidates the role of ENaC in HIV as it relates to increasing the risk for development or pathogenesis of hypertension. This review summarized the evidence to date implicating a potential role for altered ENaC activity in contributing to hypertension in patients with HIV.

Functional characterization of ion channels expressed in kidney organoids derived from human induced pluripotent stem cells.

Kidney organoids derived from human or rodent pluripotent stem cells have glomerular structures and differentiated/polarized nephron segments. Although there is an increasing understanding of the patterns of expression of transcripts and proteins within kidney organoids, there is a paucity of data regarding functional protein expression, in particular on transporters that mediate the vectorial transport of solutes. Using cells derived from kidney organoids, we examined the functional expression of key ion channels that are expressed in distal nephron segments: the large-conductance Ca2+-activated K+ (BKCa) channel, the renal outer medullary K+ (ROMK, Kir1.1) channel, and the epithelial Na+ channel (ENaC). RNA-sequencing analyses showed that genes encoding the pore-forming subunits of these transporters, and for BKCa channels, key accessory subunits, are expressed in kidney organoids. Expression and localization of selected ion channels was confirmed by immunofluorescence microscopy and immunoblot analysis. Electrophysiological analysis showed that BKCa and ROMK channels are expressed in different cell populations. These two cell populations also expressed other unidentified Ba2+-sensitive K+ channels. BKCa expression was confirmed at a single channel level, based on its high conductance and voltage dependence of activation. We also found a population of cells expressing amiloride-sensitive ENaC currents. In summary, our results show that human kidney organoids functionally produce key distal nephron K+ and Na+ channels.NEW & NOTEWORTHY Our results show that human kidney organoids express key K+ and Na+ channels that are expressed on the apical membranes of cells in the aldosterone-sensitive distal nephron, including the large-conductance Ca2+-activated K+ channel, renal outer medullary K+ channel, and epithelial Na+ channel.

Mambalgin-2 Inhibits Lung Adenocarcinoma Growth and Migration by Selective Interaction With ASIC1/α-ENaC/γ-ENaC Heterotrimer.

Lung cancer is one of the most common cancer types in the world. Despite existing treatment strategies, overall patient survival remains low and new targeted therapies are required. Acidification of the tumor microenvironment drives the growth and metastasis of many cancers. Acid sensors such as acid-sensing ion channels (ASICs) may become promising targets for lung cancer therapy. Previously, we showed that inhibition of the ASIC1 channels by a recombinant analogue of mambalgin-2 from Dendroaspis polylepis controls oncogenic processes in leukemia, glioma, and melanoma cells. Here, we studied the effects and molecular targets of mambalgin-2 in lung adenocarcinoma A549 and Lewis cells, lung transformed WI-38 fibroblasts, and lung normal HLF fibroblasts. We found that mambalgin-2 inhibits the growth and migration of A549, metastatic Lewis P29 cells, and WI-38 cells, but not of normal fibroblasts. A549, Lewis, and WI-38 cells expressed different ASIC and ENaC subunits, while normal fibroblasts did not at all. Mambalgin-2 induced G2/M cell cycle arrest and apoptosis in lung adenocarcinoma cells. In line, acidification-evoked inward currents were observed only in A549 and WI-38 cells. Gene knockdown showed that the anti-proliferative and anti-migratory activity of mambalgin-2 is dependent on the expression of ASIC1a, α-ENaC, and γ-ENaC. Using affinity extraction and immunoprecipitation, mambalgin-2 targeting of ASIC1a/α-ENaC/γ-ENaC heteromeric channels in A549 cells was shown. Electrophysiology studies in Xenopus oocytes revealed that mambalgin-2 inhibits the ASIC1a/α-ENaC/γ-ENaC channels with higher efficacy than the ASIC1a channels, pointing on the heteromeric channels as a primary target of the toxin in cancer cells. Finally, bioinformatics analysis showed that the increased expression of ASIC1 and γ-ENaC correlates with a worse survival prognosis for patients with lung adenocarcinoma. Thus, the ASIC1a/α-ENaC/γ-ENaC heterotrimer can be considered a marker of cell oncogenicity and its targeting is promising for the design of new selective cancer therapeutics.

Chronic activation of vasopressin-2 receptors induces hypertension in Liddle mice by promoting Na+ and water retention.

The renin-angiotensin-aldosterone and arginine vasopressin-V2 receptor-aquaporin-2 (AQP2) systems converge on the epithelial Na+ channel (ENaC) to regulate blood pressure and plasma tonicity. Although it is established that V2 receptors initiate renal water reabsorption through AQP2, whether V2 receptors can also induce renal Na+ retention through ENaC and raise blood pressure remains an open question. We hypothesized that a specific increase in V2 receptor-mediated ENaC activity can lead to high blood pressure. Our approach was to test effects of chronic activation of V2 receptors in Liddle mice, a genetic mouse model of high ENaC activity, and compare differences in ENaC activity, urine Na+ excretion, and blood pressure with control mice. We found that ENaC activity was elevated in Liddle mice and could not be stimulated further by administration of desmopressin (dDAVP), a V2 receptor-specific agonist. In contrast, Liddle mice showed higher levels of expression of AQP2 and aquaporin-3, but they could still respond to dDAVP infusion by increasing phospho-AQP2 expression. With dDAVP infusion, Liddle mice excreted smaller urine volume and less urine Na+ and developed higher blood pressure compared with control mice; this hypertension was attenuated with administration of the ENaC inhibitor benzamil. We conclude that V2 receptors contribute to hypertension in the Liddle mouse model by promoting primary Na+ and concomitant water retention.NEW & NOTEWORTHY Liddle syndrome is a classic model for hypertension from high epithelial Na+ channel (ENaC) activity. In the Liddle mouse model, vasopressin-2 receptors stimulate both ENaC and aquaporin-2, which increases Na+ and water retention to such an extent that hypertension ensues. Liddle mice will preserve plasma tonicity at the expense of a higher blood pressure; these data highlight the inherent limitation in which the kidney must use ENaC as a pathway to regulate both plasma tonicity and blood pressure.

COVID-19 and Liquid Homeostasis in the Lung-A Perspective through the Epithelial Sodium Channel (ENaC) Lens.

Infections with a new corona virus in 2019 lead to the definition of a new disease known as Corona Virus Disease 2019 (COVID-19). The sever cases of COVID-19 and the main cause of death due to virus infection are attributed to respiratory distress. This is associated with the formation of pulmonary oedema that impairs blood oxygenation and hypoxemia as main symptoms of respiratory distress. An important player for the maintenance of a defined liquid environment in lungs needed for normal lung function is the epithelial sodium channel (ENaC). The present article reviews the implications of SARS-CoV-2 infections from the perspective of impaired function of ENaC. The rationale for this perspective is derived from the recognition that viral spike protein and ENaC share a common proteolytic cleavage site. This cleavage site is utilized by the protease furin, that is essential for ENaC activity. Furin cleavage of spike 'activates' the virus protein to enable binding to host cell membrane receptors and initiate cell infection. Based on the importance of proteolytic cleavage for ENaC function and activation of spike, it seems feasible to assume that virus infections are associated with impaired ENaC activity. This is further supported by symptoms of COVID-19 that are reminiscent of impaired ENaC function in the respiratory tract.

Metformin suppresses epithelial sodium channel hyperactivation and its associated phenotypes in a mouse model of obstructive lung diseases.

Chronic obstructive pulmonary disease (COPD) is one of the leading causes of death in the world, and has no radical treatment. Inhibition of amiloride-sensitive epithelial sodium ion channel (ENaC) has now been considered as a potential therapeutic target against COPD. One possible modulator of ENaC is AMP-activated protein kinase (AMPK), a key molecule that controls a wide variety of cellular signals; however, little is known about whether metformin, a clinically available AMPK activator, has a protective role against ENaC-associated chronic pulmonary phenotypes, such as emphysema and pulmonary dysfunction. We first used ENaC-overexpressing human bronchial epithelial cells (ß/γENaC-16HBE14o-) and identified that Metformin significantly reduced ENaC activity. Consistently, in vivo treatment of ENaC-overexpressing COPD mouse model (C57BL/6-ßENaC-Tg mice) showed improvement of emphysema and pulmonary dysfunction, without any detrimental effect on non-pulmonary parameters (blood glucose level etc.). Bronchoalveolar lavage fluid (BALF) and lung tissue analyses revealed significant suppression in the infiltration of neutrophils as well as the expression of inflammatory markers (KC), neutrophil gelatinase (MMP9) and macrophage elastase (MMP12) in metformin-treated C57BL/6-ßENaC-Tg mice. Overall, the present study demonstrates that metformin directly inhibits ENaC activity in vitro and provides the first evidence of therapeutical benefit of Metformin for COPD with higher ENaC activity.

SGLT2 inhibition effect on salt-induced hypertension, RAAS, and Na+ transport in Dahl SS rats.

Na+-glucose cotransporter-2 (SGLT2) inhibitors are the new mainstay of treatment for diabetes mellitus and cardiovascular diseases. Despite the remarkable benefits, the molecular mechanisms mediating the effects of SGLT2 inhibitors on water and electrolyte balance are incompletely understood. The goal of this study was to determine whether SGLT2 inhibition alters blood pressure and kidney function via affecting the renin-angiotensin-aldosterone system (RAAS) and Na+ channels/transporters along the nephron in Dahl salt-sensitive rats, a model of salt-induced hypertension. Administration of dapagliflozin (Dapa) at 2 mg/kg/day via drinking water for 3 wk blunted the development of salt-induced hypertension as evidenced by lower blood pressure and a left shift of the pressure natriuresis curve. Urinary flow rate, glucose excretion, and Na+- and Cl--to-creatinine ratios increased in Dapa-treated compared with vehicle-treated rats. To define the contribution of the RAAS, we measured various hormones. Despite apparent effects on Na+- and Cl--to-creatinine ratios, Dapa treatment did not affect RAAS metabolites. Subsequently, we assessed the effects of Dapa on renal Na+ channels and transporters using RT-PCR, Western blot analysis, and patch clamp. Neither mRNA nor protein expression levels of renal transporters (SGLT2, Na+/H+ exchanger isoform 3, Na+-K+-2Cl- cotransporter 2, Na+-Cl- cotransporter, and α-, ß-, and γ-epithelial Na+ channel subunits) changed significantly between groups. Furthermore, electrophysiological experiments did not reveal any difference in Dapa treatment on the conductance and activity of epithelial Na+ channels. Our data suggest that SGLT2 inhibition in a nondiabetic model of salt-sensitive hypertension blunts the development of salt-induced hypertension by causing glucosuria and natriuresis without changes in the RAAS or the expression or activity of the main Na+ channels and transporters.NEW & NOTEWORTHY The present study indicates that Na+-glucose cotransporter-2 (SGLT2) inhibition in a nondiabetic model of salt-sensitive hypertension blunts the development and magnitude of salt-induced hypertension. Chronic inhibition of SGLT2 increases glucose and Na+ excretion without secondary effects on the expression and function of other Na+ transporters and channels along the nephron and hormone levels in the renin-angiotensin-aldosterone system. These data provide novel insights into the effects of SGLT2 inhibitors and their potential use in hypertension.

ßENaC and ASIC2 associate in VSMCs to mediate pressure-induced constriction in the renal afferent arteriole.

In independent studies, our laboratory has shown the importance of the degenerin proteins ß-epithelial Na+ channel (ßENaC) and acid-sensing ion channel 2 (ASIC2) in pressure-induced constriction (PIC) in renal interlobar arteries. Most, but not all, of the PIC response is abolished in mice lacking normal levels of ßENaC or in ASIC2-null mice, indicating that the functions of ßENaC and ASIC2 cannot fully compensate for the loss of the other. Degenerin proteins are known to associate and form heteromeric channels in expression systems, but whether they interact biochemically and functionally in vascular smooth muscle cells is unknown. We hypothesized that ßENaC and ASIC2 interact to mediate PIC responses in renal vessels. To address this possibility, we 1) used biochemical approaches to show that ßENaC associates into high-molecular-weight complexes and immunoprecipitants with ASIC2 in vascular smooth muscle cells and then 2) examined PIC in renal afferent arterioles in mice lacking normal levels of ßENaC (ßENaCm/m) or/and ASIC2 (ASIC2-/-) using the isolated afferent arteriole-attached glomerulus preparation. We found that the sensitivity of the PIC response (slope of the relationship between intraluminal pressure and percent myogenic tone) decreased to 26%, 27%, and -8% of wild-type controls in ASIC2-/-, ßENaCm/m, and ASIC2-/-/ßENaCm/m groups, respectively, suggesting that the PIC response was totally abolished in mice deficient in both ASIC2 and ßENaC. Surprisingly, we found that resting internal diameters were 20-30% lower (60 mmHg, Ca2+ free) in ASIC2-/-/ßENaCm/m (11.3 ± 0.5 µm) mice compared with control (14.4 ± 0.6 µm, P = 0.0007, independent two-tailed t test) or singly modified (15.7 ± 1.0 to 16.3 ± 1.1 µm) mice, suggesting compensatory vasoconstriction or remodeling. We then examined mean arterial blood pressure (MAP) using radiotelemetry and glomerular injury using histological examination of renal sections. We found that 24-h MAP was mildly elevated (+8 mmHg) in ASIC2-/-/ßENaCm/m mice versus wild-type controls and the glomerular injury score was modestly increased by 38%. These findings demonstrate that myogenic constriction in afferent arterioles is dependent on normal expression of ßENaC and ASIC2 and that mice lacking normal levels of ASIC2 and ßENaC have mild renal injury and increased MAP.NEW & NOTEWORTHY Transmission of systemic blood pressure to delicate renal microvessels is a primary determinant of vascular injury in chronic kidney disease progression to end-stage renal disease. Here, we identified two degenerin family members, with an evolutionary link to mechanosensing, that interact biochemically and functionally to regulate systemic blood pressure and renal injury. Thus, degenerin proteins may serve as a target for the development of therapies to prevent or delay renal disease progression.

Pendrin-null mice develop severe hypokalemia following dietary Na+ and K+ restriction: role of ENaC.

Pendrin is an intercalated cell Cl-/[Formula: see text] exchanger thought to participate in K+-sparing NaCl absorption. However, its role in K+ homeostasis has not been clearly defined. We hypothesized that pendrin-null mice will develop hypokalemia with dietary K+ restriction. We further hypothesized that pendrin knockout (KO) mice mitigate urinary K+ loss by downregulating the epithelial Na+ channel (ENaC). Thus, we examined the role of ENaC in Na+ and K+ balance in pendrin KO and wild-type mice following dietary K+ restriction. To do so, we examined the relationship between Na+ and K+ balance and ENaC subunit abundance in K+-restricted pendrin-null and wild-type mice that were NaCl restricted or replete. Following a NaCl-replete, K+-restricted diet, K+ balance and serum K+ were similar in both groups. However, following a Na+, K+, and Cl--deficient diet, pendrin KO mice developed hypokalemia from increased K+ excretion. The fall in serum K+ observed in K+-restricted pendrin KO mice was enhanced with ENaC stimulation but eliminated with ENaC inhibition. The fall in serum K+ observed in K+-restricted pendrin KO mice was enhanced with ENaC stimulation but eliminated with ENaC inhibition. However, reducing ENaC activity also reduced blood pressure and increased apparent intravascular volume contraction, since KO mice had lower serum Na+, higher blood urea nitrogen and hemoglobin, greater weight loss, greater metabolic alkalosis, and greater NaCl excretion. We conclude that dietary Na+ and K+ restriction induces hypokalemia in pendrin KO mice. Pendrin-null mice limit renal K+ loss by downregulating ENaC. However, this ENaC downregulation occurs at the expense of intravascular volume.NEW & NOTEWORTHY Pendrin is an apical Cl-/[Formula: see text] exchanger that provides renal K+-sparing NaCl absorption. The pendrin-null kidney has an inability to fully conserve K+ and limits renal K+ loss by downregulating the epithelial Na+ channel (ENaC). However, with Na+ restriction, the need to reduce ENaC for K+ balance conflicts with the need to stimulate ENaC for intravascular volume. Therefore, NaCl restriction stimulates ENaC less in pendrin-null mice than in wild-type mice, which mitigates their kaliuresis and hypokalemia but exacerbates volume contraction.

Activation by cleavage of the epithelial Na+ channel α and γ subunits independently coevolved with the vertebrate terrestrial migration.

Vertebrates evolved mechanisms for sodium conservation and gas exchange in conjunction with migration from aquatic to terrestrial habitats. Epithelial Na+ channel (ENaC) function is critical to systems responsible for extracellular fluid homeostasis and gas exchange. ENaC is activated by cleavage at multiple specific extracellular polybasic sites, releasing inhibitory tracts from the channel's α and γ subunits. We found that proximal and distal polybasic tracts in ENaC subunits coevolved, consistent with the dual cleavage requirement for activation observed in mammals. Polybasic tract pairs evolved with the terrestrial migration and the appearance of lungs, coincident with the ENaC activator aldosterone, and appeared independently in the α and γ subunits. In summary, sites within ENaC for protease activation developed in vertebrates when renal Na+ conservation and alveolar gas exchange were required for terrestrial survival.

ROMK channels are inhibited in the aldosterone-sensitive distal nephron of renal tubule Nedd4-2-deficient mice.

We used whole cell recording to examine the renal outer medullary K+ channel (ROMK or Kir1.1) and epithelial Na+ channel (ENaC) in the late distal convoluted tubule (DCT2)/initial connecting tubule (iCNT) and in the cortical collecting duct (CCD) of kidney tubule-specific neural precursor cell-expressed developmentally downregulated protein 4-2 (Nedd4-2) knockout mice (Ks-Nedd4-2 KO) and floxed neural precursor cell-expressed developmentally downregulated 4-like (Nedd4l) mice (control). Tertiapin Q (TPNQ)-sensitive K+ currents (ROMK) were smaller in both the DCT2/iCNT and CCD of Ks-Nedd4-2 KO mice on a normal diet than in control mice. Neither high dietary salt intake nor low dietary salt intake had a significant effect on ROMK activity in the DCT2/iCNT and CCD of control and Ks-Nedd4-2 KO mice. In contrast, high dietary K+ intake (HK) increased, whereas low dietary K+ intake (LK) decreased TPNQ-sensitive K+ currents in floxed Nedd4l mice. However, the effects of dietary K+ intake on ROMK channel activity were absent in Ks-Nedd4-2 KO mice since neither HK nor LK significantly affected TPNQ-sensitive K+ currents in the DCT2/iCNT and CCD. Moreover, TPNQ-sensitive K+ currents in the DCT2/iCNT and CCD of Ks-Nedd4-2 KO mice on HK were similar to those of control mice on LK. Amiloride-sensitive Na+ currents in the DCT2/iCNT and CCD were significantly higher in Ks-Nedd4-2 KO mice than in floxed Nedd4l mice on a normal K+ diet. HK increased ENaC activity of the DCT2/iCNT only in control mice, but HK stimulated ENaC of the CCD in both control and Ks-Nedd4-2 KO mice. Moreover, the HK-induced increase in amiloride-sensitive Na+ currents was larger in Ks-Nedd4-2 KO mice than in control mice. Deletion of Nedd4-2 increased with no lysine kinase 1 expression and abolished HK-induced inhibition of with no lysine kinase 1. We conclude that deletion of Nedd4-2 increases ENaC activity but decreases ROMK activity in the aldosterone-sensitive distal nephron and that HK fails to stimulate ROMK, but robustly increases ENaC activity in the CCD of Nedd4-2-deficient mice.NEW & NOTEWORTHY We demonstrate that renal outer medullary K+ (ROMK) channel activity is inhibited in the late distal convoluted tubule/initial connecting tubule and cortical collecting duct of neural precursor cell-expressed developmentally downregulated protein 4-2 (Nedd4-2)-deficient mice. Also, deletion of Nedd4-2 abolishes the stimulatory effect of dietary K+ intake on ROMK. The lack of high K+-induced stimulation of ROMK is associated with the absence of high K+-induced inhibition of with no lysine kinase 1.

High baseline ROMK activity in the mouse late distal convoluted and early connecting tubule probably contributes to aldosterone-independent K+ secretion.

The renal outer medullary K+ channel (ROMK) is colocalized with the epithelial Na+ channel (ENaC) in the late distal convoluted tubule (DCT2), connecting tubule (CNT), and cortical collecting duct (CCD). ENaC-mediated Na+ absorption generates the electrical driving force for ROMK-mediated tubular K+ secretion, which is critically important for maintaining renal K+ homeostasis. ENaC activity is aldosterone dependent in the late CNT and early CCD (CNT/CCD) but aldosterone independent in the DCT2 and early CNT (DCT2/CNT). This suggests that under baseline conditions with low plasma aldosterone, ROMK-mediated K+ secretion mainly occurs in the DCT2/CNT. Therefore, we hypothesized that baseline ROMK activity is higher in the DCT2/CNT than in the CNT/CCD. To test this hypothesis, patch-clamp experiments were performed in the DCT2/CNT and CNT/CCD microdissected from mice maintained on a standard diet. In single-channel recordings from outside-out patches, we detected typical ROMK channel activity in both the DCT2/CNT and CNT/CCD and confirmed that ROMK is the predominant K+ channel in the apical membrane. Amiloride-sensitive and tertiapin-sensitive whole-cell currents were determined to assess ENaC and ROMK activity, respectively. As expected, baseline amiloride-sensitive current was high in the DCT2/CNT (∼370 pA) but low in the CNT/CCD (∼60 pA). Importantly, tertiapin-sensitive current was significantly higher in the DCT2/CNT than in the CNT/CCD (∼810 vs. ∼350 pA). We conclude that high ROMK activity in the DCT2/CNT is critical for aldosterone-independent renal K+ secretion under baseline conditions. A low-K+ diet significantly reduced ENaC but not ROMK activity in the DCT2/CNT. This suggests that modifying ENaC activity in the DCT2/CNT plays a key regulatory role in adjusting renal K+ excretion to dietary K+ intake.NEW & NOTEWORTHY ROMK-mediated renal K+ secretion is essential for maintaining K+ balance and requires a lumen negative transepithelial potential critically dependent on ENaC activity. Using microdissected distal mouse tubules, we demonstrated that baseline apical ROMK activity is high in the DCT2/CNT. Aldosterone-independent baseline ENaC activity is also high in the DCT2/CNT and downregulated by a low-K+ diet, which highlights the important role of the DCT2/CNT in regulating K+ secretion in an aldosterone-independent manner.

Transgenic Mice Overexpressing Human Alpha-1 Antitrypsin Exhibit Low Blood Pressure and Altered Epithelial Transport Mechanisms in the Inactive and Active Cycles.

Human alpha-1 antitrypsin (hAAT) is a versatile protease inhibitor, but little is known about its targets in the aldosterone-sensitive distal nephron and its role in electrolyte balance and blood pressure control. We analyzed urinary electrolytes, osmolality, and blood pressure from hAAT transgenic (hAAT-Tg) mice and C57B/6 wild-type control mice maintained on either a normal salt or high salt diet. Urinary sodium, potassium, and chloride concentrations as well as urinary osmolality were lower in hAAT-Tg mice maintained on a high salt diet during both the active and inactive cycles. hAAT-Tg mice showed a lower systolic blood pressure compared to C57B6 mice when maintained on a normal salt diet but this was not observed when they were maintained on a high salt diet. Cathepsin B protein activity was less in hAAT-Tg mice compared to wild-type controls. Protein expression of the alpha subunit of the sodium epithelial channel (ENaC) alpha was also reduced in the hAAT-Tg mice. Natriuretic peptide receptor C (NPRC) protein expression in membrane fractions of the kidney cortex was reduced while circulating levels of atrial natriuretic peptide (ANP) were greater in hAAT-Tg mice compared to wild-type controls. This study characterizes the electrolyte and blood pressure phenotype of hAAT-Tg mice during the inactive and active cycles and investigates the mechanism by which ENaC activation is inhibited in part by a mechanism involving decreased cathepsin B activity and increased ANP levels in the systemic circulation.

Salt sensitivity of volume and blood pressure in a mouse with globally reduced ENaC γ-subunit expression.

The epithelial Na+ channel (ENaC) promotes the absorption of Na+ in the aldosterone-sensitive distal nephron, colon, and respiratory epithelia. Deletion of genes encoding subunits of ENaC results in early postnatal mortality. Here, we present the initial characterization of a mouse with dramatically suppressed expression of the ENaC γ-subunit. We used this hypomorphic (γmt) allele to explore the importance of this subunit in homeostasis of electrolytes and body fluid volume. At baseline, γ-subunit expression in γmt/mt mice was markedly suppressed in the kidney and lung, whereas electrolytes resembled those of littermate controls. Aldosterone levels in γmt/mt mice exceeded those seen in littermate controls. Quantitative magnetic resonance measurement of body composition revealed similar baseline body water, lean tissue mass, and fat tissue mass in γmt/mt mice and controls. γmt/mt mice exhibited a more rapid decline in body water and lean tissue mass in response to a low-Na+ diet than the controls. Replacement of drinking water with 2% saline selectively and transiently increased body water and lean tissue mass in γmt/mt mice relative to the controls. Lower blood pressures were variably observed in γmt/mt mice on a high-salt diet compared with the controls. γmt/mt also exhibited reduced diurnal blood pressure variation, a "nondipping" phenotype, on a high-Na+ diet. Although ENaC in the renal tubules and colon works to prevent extracellular fluid volume depletion, our observations suggest that ENaC in other tissues may participate in regulating extracellular fluid volume and blood pressure.NEW & NOTEWORTHY A mouse with globally suppressed expression of the epithelial Na+ channel γ-subunit showed enhanced sensitivity to dietary salt, including a transient increase in total body fluid, reduced blood pressure, and reduced diurnal blood pressure variation when given a dietary NaCl challenge. These results point to a role for the epithelial Na+ channel in regulating body fluid and blood pressure beyond classical transepithelial Na+ transport mechanisms.