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.

Histone deacetylase inhibitors (HDACi) increase expression of KCa2.3 (SK3) in primary microvascular endothelial cells.

The small conductance calcium-activated potassium channel (KCa2.3) has long been recognized for its role in mediating vasorelaxation through the endothelium-derived hyperpolarization (EDH) response. Histone deacetylases (HDACs) have been implicated as potential modulators of blood pressure and histone deacetylase inhibitors (HDACi) are being explored as therapeutics for hypertension. Herein, we show that HDACi increase KCa2.3 expression when heterologously expressed in HEK cells and endogenously expressed in primary cultures of human umbilical vein endothelial cells (HUVECs) and human intestinal microvascular endothelial cells (HIMECs). When primary endothelial cells were exposed to HDACi, KCa2.3 transcripts, subunits, and functional current are increased. Quantitative RT-PCR (qPCR) demonstrated increased KCa2.3 mRNA following HDACi, confirming transcriptional regulation of KCa2.3 by HDACs. By using pharmacological agents selective for different classes of HDACs, we discriminated between cytoplasmic and epigenetic modulation of KCa2.3. Biochemical analysis revealed an association between the cytoplasmic HDAC6 and KCa2.3 in immunoprecipitation studies. Specifically inhibiting HDAC6 increases expression of KCa2.3. In addition to increasing the expression of KCa2.3, we show that nonspecific inhibition of HDACs causes an increase in the expression of the molecular chaperone Hsp70 in endothelial cells. When Hsp70 is inhibited in the presence of HDACi, the magnitude of the increase in KCa2.3 expression is diminished. Finally, we show a slower rate of endocytosis of KCa2.3 as a result of exposure of primary endothelial cells to HDACi. These data provide the first demonstrated approach to increase KCa2.3 channel number in endothelial cells and may partially account for the mechanism by which HDACi induce vasorelaxation.

Loss of miR-17~92 results in dysregulation of Cftr in nephron progenitors.

We have previously demonstrated that loss of miR-17~92 in nephron progenitors in a mouse model results in renal hypodysplasia and chronic kidney disease. Clinically, decreased congenital nephron endowment because of renal hypodysplasia is associated with an increased risk of hypertension and chronic kidney disease, and this is at least partly dependent on the self-renewal of nephron progenitors. Here, we present evidence for a novel molecular mechanism regulating the self-renewal of nephron progenitors and congenital nephron endowment by the highly conserved miR-17~92 cluster. Whole transcriptome sequencing revealed that nephron progenitors lacking this cluster demonstrated increased Cftr expression. We showed that one member of the cluster, miR-19b, is sufficient to repress Cftr expression in vitro and that perturbation of Cftr activity in nephron progenitors results in impaired proliferation. Together, these data suggest that miR-19b regulates Cftr expression in nephron progenitors, with this interaction playing a role in appropriate nephron progenitor self-renewal during kidney development to generate normal nephron endowment.

Ankyrin G Expression Regulates Apical Delivery of the Epithelial Sodium Channel (ENaC).

The epithelial sodium channel (ENaC) is the limiting entry point for Na+ reabsorption in the distal kidney nephron and is regulated by numerous hormones, including the mineralocorticoid hormone aldosterone. Previously we identified ankyrin G (AnkG), a cytoskeletal protein involved in vesicular transport, as a novel aldosterone-induced protein that can alter Na+ transport in mouse cortical collecting duct cells. However, the mechanisms underlying AnkG regulation of Na+ transport were unknown. Here we report that AnkG expression directly regulates Na+ transport by altering ENaC activity in the apical membrane. Increasing AnkG expression increased ENaC activity while depleting AnkG reduced ENaC-mediated Na+ transport. These changes were due to a change in ENaC directly rather than through alterations to the Na+ driving force created by Na+/K+-ATPase. Using a constitutively open mutant of ENaC, we demonstrate that the augmentation of Na+ transport is caused predominantly by increasing the number of ENaCs at the surface. To determine the mechanism of AnkG action on ENaC surface number, changes in rates of internalization, recycling, and membrane delivery were investigated. AnkG did not alter ENaC delivery to the membrane from biosynthetic pathways or removal by endocytosis. However, AnkG did alter ENaC insertion from constitutive recycling pathways. These findings provide a mechanism to account for the role of AnkG in the regulation of Na+ transport in the distal kidney nephron.

Specific Palmitoyltransferases Associate with and Activate the Epithelial Sodium Channel.

The epithelial sodium channel (ENaC) has an important role in regulating extracellular fluid volume and blood pressure, as well as airway surface liquid volume and mucociliary clearance. ENaC is a trimer of three homologous subunits (α, ß, and γ). We previously reported that cytoplasmic residues on the ß (ßCys-43 and ßCys-557) and γ (γCys-33 and γCys-41) subunits are palmitoylated. Mutation of Cys that blocked ENaC palmitoylation also reduced channel open probability. Furthermore, γ subunit palmitoylation had a dominant role over ß subunit palmitoylation in regulating ENaC. To determine which palmitoyltransferases (termed DHHCs) regulate the channel, mouse ENaCs were co-expressed in Xenopus oocytes with each of the 23 mouse DHHCs. ENaC activity was significantly increased by DHHCs 1, 2, 3, 7, and 14. ENaC activation by DHHCs was lost when γ subunit palmitoylation sites were mutated, whereas DHHCs 1, 2, and 14 still activated ENaC lacking ß subunit palmitoylation sites. ß subunit palmitoylation was increased by ENaC co-expression with DHHC 7. Both wild type ENaC and channels lacking ß and γ palmitoylation sites co-immunoprecipitated with the five activating DHHCs, suggesting that ENaC forms a complex with multiple DHHCs. RT-PCR revealed that transcripts for the five activating DHHCs were present in cultured mCCDcl1 cells, and DHHC 3 was expressed in aquaporin 2-positive principal cells of mouse aldosterone-sensitive distal nephron where ENaC is localized. Treatment of polarized mCCDcl1 cells with a general inhibitor of palmitoylation reduced ENaC-mediated Na+ currents within minutes. Our results indicate that specific DHHCs have a role in regulating ENaC.

Role of microRNAs in aldosterone signaling.

PURPOSE OF REVIEW: The review describes studies investigating the role of microRNAs in the signaling pathway of the mineralocorticoid hormone, aldosterone. RECENT FINDINGS: Emerging evidence indicates that aldosterone alters the expression of microRNAs in target tissues thereby modulating the expression of key regulatory proteins. SUMMARY: The mineralocorticoid hormone aldosterone is released by the adrenal glands in a homeostatic mechanism to regulate blood volume. The long-term renal action of aldosterone is to increase the retrieval of sodium from filtered plasma to restore blood pressure. Emerging evidence indicates aldosterone may alter noncoding RNAs (ncRNAs) to integrate this hormonal response in target tissue. Expression of the best characterized small ncRNAs, microRNAs, is regulated by aldosterone stimulation. MicroRNAs modulate protein expression at all steps in the renin-angiotensin-aldosterone-signaling (RAAS) system. In addition to acting as a rheostat to fine-tune protein levels in aldosterone-responsive cells, there is evidence that microRNAs down-regulate components of the signaling cascade as a feedback mechanism. The role of microRNAs is, therefore, as signal integrator, and damper in aldosterone signaling, which has implications in understating the RAAS system from both a physiological and pathophysiological perspective. Recent evidence for microRNA's role in RAAS signaling will be discussed.

A MicroRNA Cluster miR-23-24-27 Is Upregulated by Aldosterone in the Distal Kidney Nephron Where it Alters Sodium Transport.

The epithelial sodium channel (ENaC) is expressed in the epithelial cells of the distal convoluted tubules, connecting tubules, and cortical collecting duct (CCD) in the kidney nephron. Under the regulation of the steroid hormone aldosterone, ENaC is a major determinant of sodium (Na+ ) and water balance. The ability of aldosterone to regulate microRNAs (miRs) in the kidney has recently been realized, but the role of miRs in Na+ regulation has not been well established. Here we demonstrate that expression of a miR cluster mmu-miR-23-24-27, is upregulated in the CCD by aldosterone stimulation both in vitro and in vivo. Increasing the expression of these miRs increased Na+ transport in the absence of aldosterone stimulation. Potential miR targets were evaluated and miR-27a/b was verified to bind to the 3'-untranslated region of intersectin-2, a multi-domain protein expressed in the distal kidney nephron and involved in the regulation of membrane trafficking. Expression of Itsn2 mRNA and protein was decreased after aldosterone stimulation. Depletion of Itsn2 expression, mimicking aldosterone regulation, increased ENaC-mediated Na+ transport, while Itsn2 overexpression reduced ENaC's function. These findings reinforce a role for miRs in aldosterone regulation of Na+ transport, and implicate miR-27 in aldosterone's action via a novel target. J. Cell. Physiol. 232: 1306-1317, 2017. © 2016 Wiley Periodicals, Inc.

MicroRNAs and the regulation of aldosterone signaling in the kidney.

The role of small noncoding RNAs, termed microRNAs (miRs), in development and disease has been recognized for many years. The number of miRs and regulated targets that reinforce a role for miRs in human disease and disease progression is ever-increasing. However, less is known about the involvement of miRs in steady-state, nondisease homeostatic pathways. In the kidney, much of the regulated ion transport is under the control of hormonal signaling. Evidence is emerging that miRs are involved in the hormonal regulation of kidney function and, particularly, in ion transport. In this short review, the production and intra- and extracellular signaling of miRs and the involvement of miRs in kidney disease are discussed. The discussion also focuses on the role of these small biological molecules in the homeostatic control of ion transport in the kidney. MiR regulation of and by corticosteroid hormones, in particular the mineralocorticoid hormone aldosterone, is considered. While information about the role of aldosterone-regulated miRs in the kidney is limited, an increase in the research in this area will undoubtedly highlight the involvement of miRs as central mediators of hormonal signaling in normal physiology.

Aldosterone regulates microRNAs in the cortical collecting duct to alter sodium transport.

A role for microRNAs (miRs) in the physiologic regulation of sodium transport in the kidney has not been established. In this study, we investigated the potential of aldosterone to alter miR expression in mouse cortical collecting duct (mCCD) epithelial cells. Microarray studies demonstrated the regulation of miR expression by aldosterone in both cultured mCCD and isolated primary distal nephron principal cells. Aldosterone regulation of the most significantly downregulated miRs, mmu-miR-335-3p, mmu-miR-290-5p, and mmu-miR-1983 was confirmed by quantitative RT-PCR. Reducing the expression of these miRs separately or in combination increased epithelial sodium channel (ENaC)-mediated sodium transport in mCCD cells, without mineralocorticoid supplementation. Artificially increasing the expression of these miRs by transfection with plasmid precursors or miR mimic constructs blunted aldosterone stimulation of ENaC transport. Using a newly developed computational approach, termed ComiR, we predicted potential gene targets for the aldosterone-regulated miRs and confirmed ankyrin 3 (Ank3) as a novel aldosterone and miR-regulated protein. A dual-luciferase assay demonstrated direct binding of the miRs with the Ank3-3' untranslated region. Overexpression of Ank3 increased and depletion of Ank3 decreased ENaC-mediated sodium transport in mCCD cells. These findings implicate miRs as intermediaries in aldosterone signaling in principal cells of the distal kidney nephron.

Potentiation of BKCa channels by cystic fibrosis transmembrane conductance regulator (CFTR) correctors VX-445 and VX-121.

Cystic fibrosis (CF) results from mutations in the CFTR anion channel, ultimately leading to diminished transepithelial anion secretion and mucociliary clearance. CFTR correctors are therapeutics that restore the folding/trafficking of mutated CFTR to the plasma membrane. The BKCa potassium channel is also critical for maintaining lung ASL volume. Here, we show the CFTR corrector, VX-445 (Elexacaftor), a component of Trikafta, induces K+ secretion across WT and F508del CFTR primary human bronchial epithelial cells (HBEs), which was entirely inhibited by the BKCa antagonist paxilline. Similar results were observed with VX-121 - a corrector under clinical evaluation. Whole-cell patch-clamp recordings confirmed potentiated channel activity from CFTR correctors on the BKCa α-subunit, and excised patch-clamp recordings demonstrated a significant increase in open probability. In mesenteric artery, VX-445 induced a paxilline-sensitive vasorelaxation of preconstricted arteries. VX-445 also reduced action potential firing frequency in primary hippocampal and cortical neurons. VX-445 effects were observed at low micomolar concentrations (1-10 µM) - within the range reported in plasma and tissues from CF patients. We raise the possibilities that CFTR correctors gain additional clinical benefit by activation of BKCa in the lung, yet may lead to adverse events through BKCa activation, elsewhere.

Activation of the epithelial sodium channel (ENaC) by the alkaline protease from Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that significantly contributes to the mortality of patients with cystic fibrosis. Chronic infection by Pseudomonas induces sustained immune and inflammatory responses and damage to the airway. The ability of Pseudomonas to resist host defenses is aided, in part, by secreted proteases, which act as virulence factors in multiple modes of infection. Recent studies suggest that misregulation of protease activity in the cystic fibrosis lung may alter fluid secretion and pathogen clearance by proteolytic activation of the epithelial sodium channel (ENaC). To evaluate the possibility that proteolytic activation of ENaC may contribute to the virulence of Pseudomonas, primary human bronchial epithelial cells were exposed to P. aeruginosa and ENaC function was assessed by short circuit current measurements. Apical treatment with a strain known to express high levels of alkaline protease (AP) resulted in an increase in basal ENaC current and a loss of trypsin-inducible ENaC current, consistent with sustained activation of ENaC. To further characterize this AP-induced ENaC activation, AP was purified, and its folding, activity, and ability to activate ENaC were assessed. AP folding was efficient under pH and calcium conditions thought to exist in the airway surface liquid of normal and cystic fibrosis (CF) lungs. Short circuit measurements of ENaC in polarized monolayers indicated that AP activated ENaC in immortalized cell lines as well as post-transplant, primary human bronchial epithelial cells from both CF and non-CF patients. This activation was mapped to the γ-subunit of ENaC. Based on these data, patho-mechanisms associated with AP in the CF lung are proposed wherein secretion of AP leads to decreased airway surface liquid volume and a corresponding decrease in mucocilliary clearance of pulmonary pathogens.

Rab11b regulates the trafficking and recycling of the epithelial sodium channel (ENaC).

Expression of the epithelial sodium channel (ENaC) at the apical membrane of cortical collecting duct (CCD) principal cells is modulated by regulated trafficking mediated by vesicle insertion and retrieval. Small GTPases are known to facilitate vesicle trafficking, recycling, and membrane fusion events; however, little is known about the specific Rab family members that modify ENaC surface density. Using a mouse CCD cell line that endogenously expresses ENaC (mpkCCD), the channel was localized to both Rab11a- and Rab11b-positive endosomes by immunoisolation and confocal fluorescent microscopy. Expression of a dominant negative (DN) form of Rab11a or Rab11b significantly reduced the basal and cAMP-stimulated ENaC-dependent sodium (Na(+)) transport. The greatest reduction in Na(+) transport was observed with the expression of DN-Rab11b. Furthermore, small interfering RNA-mediated knockdown of each Rab11 isoform demonstrated the requirement for Rab11b in ENaC surface expression. These data indicate that Rab11b, and to a lesser extent Rab11a, is involved in establishing the constitutive and cAMP-stimulated Na(+) transport in mpkCCD cells.

Regulation of the epithelial sodium channel (ENaC) by membrane trafficking.

The epithelial Na(+) channel (ENaC) is a major regulator of salt and water reabsorption in a number of epithelial tissues. Abnormalities in ENaC function have been directly linked to several human disease states including Liddle syndrome, psuedohypoaldosteronism, and cystic fibrosis and may be implicated in salt-sensitive hypertension. ENaC activity in epithelial cells is regulated both by open probability and channel number. This review focuses on the regulation of ENaC in the cells of the kidney cortical collecting duct by trafficking and recycling. The trafficking of ENaC is discussed in the broader context of epithelial cell vesicle trafficking. Well-characterized pathways and protein interactions elucidated using epithelial model cells are discussed, and the known overlap with ENaC regulation is highlighted. In following the life of ENaC in CCD epithelial cells the apical delivery, internalization, recycling, and destruction of the channel will be discussed. While a number of pathways presented still need to be linked to ENaC regulation and many details of the regulation of ENaC trafficking remain to be elucidated, knowledge of these mechanisms may provide further insights into ENaC activity in normal and disease states.

Non-coding RNAs and the mineralocorticoid receptor in the kidney.

The final steps in the Renin-Angiotensin-Aldosterone signaling System (RAAS) involve binding of the corticosteroid hormone, aldosterone to its mineralocorticoid receptor (MR). The bound MR interacts with response elements to induce or repress the transcription of aldosterone-regulated genes. Along with the classic genomic targets of aldosterone that alter mRNA and protein expression, aldosterone also regulates the expression of non-coding RNAs (ncRNAs). Short ncRNAs termed microRNAs (miRs) have been shown to play a role in transducing aldosterone's actions via MR signaling. The role of miRs in homeostatic regulation of aldosterone signaling, and the potential for aldosterone-regulated miRs to act as feedback regulators of MR have been recently reported. In this review, the role of miRs in RAAS signaling and feedback regulation of MR in kidney epithelial cells will be discussed.

Acute regulation of the epithelial sodium channel in airway epithelia by proteases and trafficking.

Effective clearance of inhaled pathogens is the primary innate defense mechanism in the lung, and requires the maintenance of a proper airway surface liquid (ASL) volume to facilitate ciliary beat and optimize mucociliary clearance. Na(+) absorption via the epithelial sodium channel (ENaC) is tightly regulated and, together with chloride movement, provides the optimal osmotic gradients to absorb excessive fluid in the airway lumen while preventing excessive ASL dehydration, which would compromise mucus clearance from the lung. To absorb excessive fluid from the luminal surface, a local mechanism of ENaC activation allows for an increase in Na(+) absorption at times when the ASL volume is expanded. To help define these regulatory mechanisms, we examined the effects of ASL volume expansion on ENaC activity in primary human bronchial epithelial (HBE) cell cultures. We found that ENaC activity increases dramatically after rapid dilution of endogenous ASL. Approximately 35% of the increase in Na(+) absorption was attributable to activation of ENaC by proteases. The remainder of the increase in Na(+) current was prevented when membrane trafficking was disrupted with brefeldin A, nocodazole, or myosin light chain kinase inhibitors, demonstrating that trafficking is involved with ENaC regulation in the airway. These findings demonstrate that Na(+) absorption in the airway is acutely modulated by the coordinated trafficking of channels to the luminal surface and by the proteolytic activation of ENaC in response to ASL volume expansion.

Regulation of Aldosterone Signaling by MicroRNAs.

The mineralocorticoid hormone aldosterone is released by the adrenal glands in a homeostatic mechanism to regulate blood volume. Several cues elicit aldosterone release, and the long-term action of the hormone is to restore blood pressure and/or increase the retrieval of sodium from filtered plasma in the kidney. While the signaling cascade that results in aldosterone release is well studied, the impact of this hormone on tissues and cells in various organ systems is pleotropic. Emerging evidence indicates aldosterone may alter non-coding RNAs (ncRNAs) to integrate the hormonal response, and these ncRNAs may contribute to the heterogeneity of signaling outcomes in aldosterone target tissues. The best studied of the ncRNAs in aldosterone action are the small ncRNAs, microRNAs. MicroRNA expression is regulated by aldosterone stimulation, and microRNAs are able to modulate protein expression at all steps in the renin-angiotensin-aldosterone-signaling system. The discovery and synthesis of microRNAs will be briefly covered followed by a discussion of the reciprocal role of aldosterone/microRNA regulation, including misregulation of microRNA signaling in aldosterone-linked disease states.

The Lhx1-Ldb1 complex interacts with Furry to regulate microRNA expression during pronephric kidney development.

The molecular events driving specification of the kidney have been well characterized. However, how the initial kidney field size is established, patterned, and proportioned is not well characterized. Lhx1 is a transcription factor expressed in pronephric progenitors and is required for specification of the kidney, but few Lhx1 interacting proteins or downstream targets have been identified. By tandem-affinity purification, we isolated FRY like transcriptional coactivator (Fryl), one of two paralogous genes, fryl and furry (fry), have been described in vertebrates. Both proteins were found to interact with the Ldb1-Lhx1 complex, but our studies focused on Lhx1/Fry functional roles, as they are expressed in overlapping domains. We found that Xenopus embryos depleted of fry exhibit loss of pronephric mesoderm, phenocopying the Lhx1-depleted animals. In addition, we demonstrated a synergism between Fry and Lhx1, identified candidate microRNAs regulated by the pair, and confirmed these microRNA clusters influence specification of the kidney. Therefore, our data shows that a constitutively-active Ldb1-Lhx1 complex interacts with a broadly expressed microRNA repressor, Fry, to establish the kidney field.

Acute ENaC stimulation by cAMP in a kidney cell line is mediated by exocytic insertion from a recycling channel pool.

Acute hormonal regulation of the epithelial sodium channel (ENaC) in tight epithelia increases transcellular Na(+) transport via trafficking of intracellular channels to the apical surface. The fate of the channels removed from the apical surface following agonist washout is less clear. By repetitively stimulating polarized mouse cortical collecting duct (mCCD, (MPK)CCD(14)) epithelia, we evaluated the hypothesis that ENaC recycles through an intracellular pool to be available for reinsertion into the apical membrane. Short circuit current (I(SC)), membrane capacitance (C(T)), and conductance (G(T)) were recorded from mCCD epithelia mounted in modified Ussing chambers. Surface biotinylation of ENaC demonstrated an increase in channel number in the apical membrane following cAMP stimulation. This increase was accompanied by a 83 +/- 6% (n = 31) increase in I(SC) and a 15.3 +/- 1.5% (n = 15) increase in C(T). Selective membrane permeabilization demonstrated that the C(T) increase was due to an increase in apical membrane capacitance. I(SC) and C(T) declined to basal levels on stimulus washout. Repetitive cAMP stimulation and washout (approximately 1 h each cycle) resulted in response fatigue; DeltaI(SC) decreased approximately 10% per stimulation-recovery cycle. When channel production was blocked by cycloheximide, DeltaI(SC) decreased approximately 15% per stimulation cycle, indicating that newly synthesized ENaC contributed a relatively small fraction of the channels mobilized to the apical membrane. Selective block of surface ENaC by benzamil demonstrated that channels inserted from a subapical pool made up >90% of the stimulated I(SC), and that on restimulation a large proportion of channels retrieved from the apical surface were reinserted into the apical membrane. Channel recycling was disrupted by brefeldin A, which inhibited ENaC exocytosis, by chloroquine, which inhibited ENaC endocytosis and recycling, and by latrunculin A, which blocked ENaC exocytosis. A compartment model featuring channel populations in the apical membrane and intracellular recycling pool provided an adequate kinetic description of the I(SC) responses to repetitive stimulation. The model supports the concept of ENaC recycling in response to repetitive cAMP stimulation.

Expression of a Diverse Array of Ca2+-Activated K+ Channels (SK1/3, IK1, BK) that Functionally Couple to the Mechanosensitive TRPV4 Channel in the Collecting Duct System of Kidney.

The voltage- and Ca2+-activated, large conductance K+ channel (BK, maxi-K) is expressed in the collecting duct system of kidney where it underlies flow- and Ca2+-dependent K+ excretion. To determine if other Ca2+-activated K+ channels (KCa) may participate in this process, mouse kidney and the K+-secreting mouse cortical collecting duct (CCD) cell line, mCCDcl1, were assessed for TRPV4 and KCa channel expression and cross-talk. qPCR mRNA analysis and immunocytochemical staining demonstrated TRPV4 and KCa expression in mCCDcl1 cells and kidney connecting tubule (CNT) and CCD. Three subfamilies of KCa channels were revealed: the high Ca2+-binding affinity small-conductance SK channels, SK1and SK3, the intermediate conductance channel, IK1, and the low Ca2+-binding affinity, BK channel (BKα subunit). Apparent expression levels varied in CNT/CCD where analysis of CCD principal cells (PC) and intercalated cells (IC) demonstrated differential staining: SK1:PCIC, IK1:PC>IC, BKα:PC = IC, and TRPV4:PC>IC. Patch clamp analysis and fluorescence Ca2+ imaging of mCCDcl1 cells demonstrated potent TRPV4-mediated Ca2+ entry and strong functional cross-talk between TRPV4 and KCa channels. TRPV4-mediated Ca2+ influx activated each KCa channel, as evidenced by selective inhibition of KCa channels, with each active KCa channel enhancing Ca2+ entry (due to membrane hyperpolarization). Transepithelial electrical resistance (TEER) analysis of confluent mCCDcl1 cells grown on permeable supports further demonstrated this cross-talk where TRPV4 activation induce a decrease in TEER which was partially restored upon selective inhibition of each KCa channel. It is concluded that SK1/SK3 and IK1 are highly expressed along with BKα in CNT and CCD and are closely coupled to TRPV4 activation as observed in mCCDcl1 cells. The data support a model in CNT/CCD segments where strong cross talk between TRPV4-mediated Ca2+ influx and each KCa channel leads to enhance Ca2+ entry which will support activation of the low Ca2+-binding affinity BK channel to promote BK-mediated K+ secretion.