Role of calcineurin in regulating renal potassium (K+) excretion: Mechanisms of calcineurin inhibitor-induced hyperkalemia.

Calcineurin, protein phosphatase 2B (PP2B) or protein phosphatase 3 (PP3), is a calcium-dependent serine/threonine protein phosphatase. Calcineurin is widely expressed in the kidney and regulates renal Na+ and K+ transport. In the thick ascending limb, calcineurin plays a role in inhibiting NKCC2 function by promoting the dephosphorylation of the cotransporter and an intracellular sorting receptor, called sorting-related-receptor-with-A-type repeats (SORLA), is involved in modulating the effect of calcineurin on NKCC2. Calcineurin also participates in regulating thiazide-sensitive NaCl-cotransporter (NCC) in the distal convoluted tubule. The mechanisms by which calcineurin regulates NCC include directly dephosphorylation of NCC, regulating Kelch-like-3/CUL3 E3 ubiquitin-ligase complex, which is responsible for WNK (with-no-lysin-kinases) ubiquitination, and inhibiting Kir4.1/Kir5.1, which determines NCC expression/activity. Finally, calcineurin is also involved in regulating ROMK (Kir1.1) channels in the cortical collecting duct and Cyp11 2 expression in adrenal zona glomerulosa. In summary, calcineurin is involved in the regulation of NKCC2, NCC, and inwardly rectifying K+ channels in the kidney, and it also plays a role in modulating aldosterone synthesis in adrenal gland, which regulates epithelial-Na+-channel expression/activity. Thus, application of calcineurin inhibitors (CNIs) is expected to abrupt calcineurin-mediated regulation of transepithelial Na+ and K+ transport in the kidney. Consequently, CNIs cause hypertension, compromise renal K+ excretion, and induce hyperkalemia.

[Recording and identification of depolarization-activated current in intercalated cells].

The depolarization-activated current of intercalated cells in the distal nephron was detected for the first time, and the type of ion channel mediating the current was identified based on electrophysiological and pharmacological properties. The whole-cell current of distal nephron in kidney of C57BL/6J mice was recorded by Axon MultiClamp 700B patch-clamp system, and the effects of several K+ channel inhibitors on the depolarization-activated current in intercalated cells were observed. In addition, the immunofluorescence technique was used to investigate the localization of the channel in intercalated cells. The results showed that when K+ concentration of the bath solution was equal to intracellular fluid (140 mmol/L K+), the depolarization-activated current could be recorded in intercalated cells, but this current was not observed in the principal cells. The depolarization-activated current detected in the intercalated cells could be blocked by Kv4.1 inhibitors. The immunofluorescence experiment showed that the fluorescence of Kv4.1 protein was only present in intercalated cells and not observed in principal cells. Kv4.1 protein immunofluorescence was observed in the luminal and basolateral membrane of intercalated cells, but the fluorescence intensity of luminal membrane was higher than that of basolateral membrane. We conclude that the depolarization-activated current detected in intercalated cells is mediated by Kv4.1 and this channel is mainly expressed in the luminal membrane of intercalated cells.

Adenosine stimulates the basolateral 50 pS K+ channel in renal proximal tubule via adenosine-A1 receptor.

Background: The basolateral potassium channels play an important role in maintaining the membrane transport in the renal proximal tubules (PT) and adenosine receptors have been shown to regulate the trans-epithelial Na+ absorption in the PT. The aim of the present study is to explore whether adenosine also regulates the basolateral K+ channel of the PT and to determine the adenosine receptor type and the signaling pathway which mediates the effect of adenosine on the K+ channel. Methods: We have used the single channel recording to examine the basolateral K+ channel activity in the proximal tubules of the mouse kidney. All experiments were performed in cell-attached patches. Results: Single channel recording has detected a 50 pS inwardly-rectifying K+ channel with high channel open probability and this 50 pS K+ channel is a predominant type K+ channel in the basolateral membrane of the mouse PT. Adding adenosine increased 50 pS K+ channel activity in cell-attached patches, defined by NPo (a product of channel Numbers and Open Probability). The adenosine-induced stimulation of the 50 pS K+ channel was absent in the PT pretreated with DPCPX, a selective inhibitor of adenosine A1 receptor. In contrast, adenosine was still able to stimulate the 50 pS K+ channel in the PT pretreated with CP-66713, a selective adenosine A2 receptor antagonist. This suggests that the stimulatory effect of adenosine on the 50 pS K+ channel of the PT was mediated by adenosine-A1 receptor. Moreover, the effect of adenosine on the 50 pS K+ channel was blocked in the PT pretreated with U-73122 or Calphostin C, suggesting that adenosine-induced stimulation of the 50 pS K+ channels of the PT was due to the activation of phospholipase C (PLC) and protein kinase C (PKC) pathway. In contrast, the inhibition of phospholipase A2 (PLA2) with AACOCF3 or inhibition of protein kinase A (PKA) with H8 failed to block the adenosine-induced stimulation of the 50 pS K+ channel of the PT. Conclusion: We conclude that adenosine activates the 50 pS K+ channels in the basolateral membrane of PT via adenosine-A1 receptor. Furthermore, the effect of adenosine on the 50 pS K+ channel is mediated by PLC-PKC signaling pathway.

Alkali and Heavy Metal Copoisoning Resistant Catalytic Reduction of NOx via Liberating Lewis Acid Sites.

The catalyst deactivation caused by the coexistence of alkali and heavy metals remains an obstacle for selective catalytic reduction of NOx with NH3. Moreover, the copoisoning mechanism of alkali and heavy metals is still unclear. Herein, the copoisoning mechanism of K and Cd was revealed from the adsorption and variation of reaction intermediates at a molecular level through time-resolved in situ spectroscopy combined with theoretical calculations. The alkali metal K mainly decreased the adsorption of NH3 on Lewis acid sites and altered the reaction more depending on the formation of the NH4NO3 intermediate, which is highly related to NOx adsorption and activation. However, Cd further inhibited the generation of active nitrate intermediates and thus decreased the NOx abatement about 60% on potassium-poisoned CeTiOx catalysts. Physically mixing with acid additives for CeTiOx catalysts could significantly liberate the active Lewis acid sites from the occupation of alkali metals and relieve the high dependence on NOx adsorption and activation, thus recovering the NOx removal rate to the initial state. This work revealed the copoisoning mechanism of K and Cd on Ce-based de-NOx catalysts and developed a facile anti-poisoning strategy, which paves a way for the development of durable catalysts among alkali and heavy metal copoisoning resistant catalytic reduction of NOx.

Macrolides selectively inhibit mutant KCNJ5 potassium channels that cause aldosterone-producing adenoma.

Aldosterone-producing adenomas (APAs) are benign tumors of the adrenal gland that constitutively produce the salt-retaining steroid hormone aldosterone and cause millions of cases of severe hypertension worldwide. Either of 2 somatic mutations in the potassium channel KCNJ5 (G151R and L168R, hereafter referred to as KCNJ5MUT) in adrenocortical cells account for half of APAs worldwide. These mutations alter channel selectivity to allow abnormal Na+ conductance, resulting in membrane depolarization, calcium influx, aldosterone production, and cell proliferation. Because APA diagnosis requires a difficult invasive procedure, patients often remain undiagnosed and inadequately treated. Inhibitors of KCNJ5MUT could allow noninvasive diagnosis and therapy of APAs carrying KCNJ5 mutations. Here, we developed a high-throughput screen for rescue of KCNJ5MUT-induced lethality and identified a series of macrolide antibiotics, including roxithromycin, that potently inhibit KCNJ5MUT, but not KCNJ5WT. Electrophysiology demonstrated direct KCNJ5MUT inhibition. In human aldosterone-producing adrenocortical cancer cell lines, roxithromycin inhibited KCNJ5MUT-induced induction of CYP11B2 (encoding aldosterone synthase) expression and aldosterone production. Further exploration of macrolides showed that KCNJ5MUT was similarly selectively inhibited by idremcinal, a macrolide motilin receptor agonist, and by synthesized macrolide derivatives lacking antibiotic or motilide activity. Macrolide-derived selective KCNJ5MUT inhibitors thus have the potential to advance the diagnosis and treatment of APAs harboring KCNJ5MUT.

ENaC and ROMK activity are inhibited in the DCT2/CNT of TgWnk4PHAII mice.

Mice transgenic for genomic segments harboring PHAII (pseudohypoaldosteronism type II) mutant Wnk4 (with-No-Lysine kinase 4) (TgWnk4PHAII) have hyperkalemia which is currently believed to be the result of high activity of Na-Cl cotransporter (NCC). This leads to decreasing Na+ delivery to the distal nephron segment including late distal convoluted tubule (DCT) and connecting tubule (CNT). Since epithelial Na+ channel (ENaC) and renal outer medullary K+ channel (ROMK or Kir4.1) are expressed in the late DCT and play an important role in mediating K+ secretion, the aim of the present study is to test whether ROMK and ENaC activity in the DCT/CNT are also compromised in the mice expressing PHAII mutant Wnk4. Western blot analysis shows that the expression of ßENaC and γENaC subunits but not αENaC subunit was lower in TgWnk4PHAII mice than that in wild-type (WT) and TgWnk4WT mice. Patch-clamp experiments detected amiloride-sensitive Na+ currents and TPNQ-sensitive K+ currents in DCT2/CNT, suggesting the activity of ENaC and ROMK. However, both Na+ and ROMK currents in DCT2/CNT of TgWnk4PHAII mice were significantly smaller than those in WT and TgWnk4WT mice. In contrast, the basolateral K+ currents in the DCT were similar among three groups, despite higher NCC expression in TgWnk4PHAII mice than those of WT and TgWnk4WTmice. An increase in dietary K+ intake significantly increased both ENaC and ROMK currents in the DCT2/CNT of all three groups. However, high-K+ (HK) intake-induced stimulation of Na+ and K+ currents was smaller in TgWnk4PHAII mice than those in WT and TgWnk4WT mice. We conclude that ENaC and ROMK channel activity in DCT2/CNT are inhibited in TgWnk4PHAII mice and that Wnk4PHAII-induced inhibition of ENaC and ROMK may contribute to the suppression of K+ secretion in the DCT2/CNT in addition to increased NCC activity.

PGF2α regulates the basolateral K channels in the distal convoluted tubule.

Our aim is to examine the role of PGF2α receptor (FP), a highly expressed prostaglandin receptor in the distal convoluted tubule (DCT) in regulating the basolateral 40-pS K channel. The single-channel studies demonstrated that PGF2α had a biphasic effect on the 40-pS K channel in the DCT-PGF2α stimulated at low concentrations (less than 500 nM), while at high concentrations (above 1 µM), it inhibited the 40-pS K channels. Moreover, neither 13,14-dihydro-15-keto-PGF2α (a metabolite of PGF2α) nor PGE2 was able to mimic the effect of PGF2α on the 40-pS K channel in the DCT. The inhibition of PKC had no significant effect on the 40-pS K channel; however, it abrogated the inhibitory effect of 5 µM PGF2α on the K channel. Moreover, stimulation of PKC inhibited the 40-pS K channel in the DCT, suggesting that PKC mediates the inhibitory effect of PGF2α on the 40-pS K channel. Conversely, the stimulatory effect of PGF2α on the 40-pS K channel was absent in the DCT treated with DPI, a NADPH oxidase (NOX) inhibitor. Also, adding 100 µM H2O2 mimicked the stimulatory effect of PGF2α and increased the 40-pS K channel activity in DCT. Moreover, the stimulatory effect of 500 nM PGF2α and H2O2 was not additive, suggesting the role of superoxide-related species in mediating the stimulatory effect of PGF2α on the 40-pS K channel. The inhibition of Src family tyrosine protein kinase (SFK) not only inhibited the 40-pS K channel in the DCT but also completely abolished the stimulatory effects of PGF2α and H2O2 on the 40-pS K channel. We conclude that PGF2α at low doses stimulates the basolateral 40-pS K channel by a NOX- and SFK-dependent mechanism, while at high concentrations, it inhibits the K channel by a PKC-dependent pathway.

Caveolin-1 regulates corneal wound healing by modulating Kir4.1 activity.

The expression of caveolin-1 (Cav1) in corneal epithelium is associated with regeneration potency. We used Cav1(-/-) mice to study the role of Cav1 in modulating corneal wound healing. Western blot and whole cell patch clamp were employed to study the effect of Cav1 deletion on Kir4.1 current density in corneas. We found that Ba(2+)-sensitive K(+) currents in primary cultured murine corneal epithelial cells (pMCE) from Cav1(-/-) were dramatically reduced (602 pA) compared with those from wild type (WT; 1,300 pA). As a consequence, membrane potential was elevated in pMCE from Cav1(-/-) compared with that from WT (-43 ± 7.5 vs. -58 ± 4.0 mV, respectively). Western blot showed that either inhibition of Cav1 expression or Ba(2+) incubation stimulated phosphorylation of the EGFR. The transwell migration assay showed that Cav1 genetic inactivation accelerated cell migration. The regrowth efficiency of human corneal epithelial cells (HCE) transfected with siRNA-Cav1 or negative control was evaluated by scrape injury assay. With the presence of mitomycin C (10 µg/ml) to avoid the influence of cell proliferation, Cav1 inhibition with siRNA significantly increased migration compared with control siRNA in HCE. This promoting effect by siRNA-Cav1 could not be further enhanced by cotransfection with siRNA-Kcnj10. By using corneal debridement, we found that wound healing was significantly accelerated in Cav1(-/-) compared with WT mice (70 ± 10 vs. 36 ± 3%, P < 0.01). Our findings imply that the mechanism by which Cav-1 knockout promotes corneal regrowth is, at least partially, due to the inhibition of Kir4.1 which stimulates EGFR signaling.

Disruption of KCNJ10 (Kir4.1) stimulates the expression of ENaC in the collecting duct.

Kcnj10 encodes the inwardly rectifying K(+) channel 4.1 (Kir4.1) and is expressed in the basolateral membrane of late thick ascending limb, distal convoluted tubule (DCT), connecting tubule (CNT), and cortical collecting duct (CCD). In the present study, we perform experiments in postneonatal day 9 Kcnj10(-/-) or wild-type mice to examine the role of Kir.4.1 in contributing to the basolateral K(+) conductance in the CNT and CCD, and to investigate whether the disruption of Kir4.1 upregulates the expression of the epithelial Na(+) channel (ENaC). Immunostaining shows that Kir4.1 is expressed in the basolateral membrane of CNT and CCD. Patch-clamp studies detect three types of K(+) channels (23, 40, and 60 pS) in the basolateral membrane of late CNT and initial CCD in wild-type (WT) mice. However, only 23- and 60-pS K(+) channels but not the 40-pS K(+) channel were detected in Kcnj10(-/-) mice, suggesting that Kir.4.1 is a key component of the 40-pS K(+) channel in the CNT/CCD. Moreover, the depletion of Kir.4.1 did not increase the probability of finding the 23- and 60-pS K(+) channel in the CNT/CCD. We next used the perforated whole cell recording to measure the K(+) reversal voltage in the CNT/CCD as an index of cell membrane potential. Under control conditions, the K(+) reversal potential was -69 mV in WT mice and -61 mV in Kcnj10(-/-) mice, suggesting that Kir4.1 partially participates in generating membrane potential in the CNT/CCD. Western blotting and immunostaining showed that the expression of ENaCß and ENaCγ subunits from a renal medulla section of Kcnj10(-/-) mice was significantly increased compared with that of WT mice. Also, the disruption of Kir4.1 increased aquaporin 2 expression. We conclude that Kir4.1 is expressed in the CNT and CCD and partially participates in generating the cell membrane potential. Also, increased ENaC expression in medullary CD of Kcnj10(-/-) mice is a compensatory action in response to the impaired Na(+) transport in the DCT.

Vasopressin-induced stimulation of the Na(+)-activated K(+) channels is responsible for maintaining the basolateral K(+) conductance of the thick ascending limb (TAL) in EAST/SeSAME syndrome.

The renal phenotype of EAST syndrome, a disease caused by the loss-of-function-mutations of Kcnj10 (Kir4.1), is a reminiscence of Gitelman's syndrome characterized by the defective function in the distal convoluted tubule (DCT). The aim of the present study is to test whether antidiuretic hormone (vasopressin)-induced stimulation of the Na(+)-activated 80-150pS K(+) channel is responsible for compensating the lost function of Kcnj10 in the thick ascending limb (TAL) of subjects with EAST syndrome. Immunostaining and western blot showed that the expression of aquaporin 2 (AQP2) was significantly higher in Kcnj10(-/-) mice than those of WT littermates, suggesting that the disruption of Kcnj10 stimulates vasopressin response in the kidney. The role of vasopressin in stimulating the basolateral K(+) conductance of the TAL was strongly indicated by the finding that the application of arginine-vasopressin (AVP) hyperpolarized the membrane in the TAL of Kcnj10(-/-) mice. Application of AVP significantly stimulated the 80-150pS K(+) channel in the TAL and this effect was blocked by tolvaptan (V2 receptor antagonist) or by inhibiting PKA. Moreover, the water restriction for 24h significantly increased the probability of finding the 80-150pS K(+) channel and the K(+) channel open probability in the TAL. The application of a membrane permeable cAMP analog also mimicked the effect of AVP and activated this K(+) channel, suggesting that cAMP-PKA pathway stimulates the 80-150pS K(+) channels. The role of the basolateral K(+) conductance in maintaining transcellular Cl(-) transport is further suggested by the finding that the inhibition of basolateral K(+) channels significantly diminished the AVP-induced stimulation of the basolateral 10pS Cl(-) channels. We conclude that vasopressin stimulates the 80-150pS K(+) channel in the TAL via a cAMP-dependent mechanism. The vasopressin-induced stimulation of K(+) channels is responsible for compensating lost function of Kcnj10 thereby rescuing the basolateral K(+) conductance which is essential for the transport function in the TAL.

Caveolin-1 Deficiency Inhibits the Basolateral K+ Channels in the Distal Convoluted Tubule and Impairs Renal K+ and Mg2+ Transport.

Kcnj10 encodes the inwardly rectifying K(+) channel Kir4.1 in the basolateral membrane of the distal convoluted tubule (DCT) and is activated by c-Src. However, the regulation and function of this K(+) channel are incompletely characterized. Here, patch-clamp experiments in Kcnj10-transfected HEK293 cells demonstrated that c-Src-induced stimulation of Kcnj10 requires coexpression of caveolin-1 (cav-1), and immunostaining showed expression of cav-1 in the basolateral membrane of parvalbumin-positive DCT. Patch-clamp experiments detected a 40-pS inwardly rectifying K(+) channel, a heterotetramer of Kir4.1/Kir5.1, in the basolateral membrane of the early DCT (DCT1) in both wild-type (WT) and cav-1-knockout (KO) mice. However, the activity of this basolateral 40-pS K(+) channel was lower in KO mice than in WT mice. Moreover, the K(+) reversal potential (an indication of membrane potential) was less negative in the DCT1 of KO mice than in the DCT1 of WT mice. Western blot analysis demonstrated that cav-1 deficiency decreased the expression of the Na(+)/Cl(-) cotransporter and Ste20-proline-alanine-rich kinase (SPAK) but increased the expression of epithelial Na(+) channel-α. Furthermore, the urinary excretion of Mg(2+) and K(+) was significantly higher in KO mice than in WT mice, and KO mice developed hypomagnesemia, hypocalcemia, and hypokalemia. We conclude that disruption of cav-1 decreases basolateral K(+) channel activity and depolarizes the cell membrane potential in the DCT1 at least in part by suppressing the stimulatory effect of c-Src on Kcnj10. Furthermore, the decrease in Kcnj10 and Na(+)/Cl(-) cotransporter expression induced by cav-1 deficiency may underlie the compromised renal transport of Mg(2+), Ca(2+), and K(+).

KCNJ10 (Kir4.1) is expressed in the basolateral membrane of the cortical thick ascending limb.

The aim of the present study is to examine the role of Kcnj10 (Kir.4.1) in contributing to the basolateral K conductance in the cortical thick ascending limb (cTAL) using Kcnj10(+/+) wild-type (WT) and Kcnj10(-/-) knockout (KO) mice. The patch-clamp experiments detected a 40- and an 80-pS K channel in the basolateral membrane of the cTAL. Moreover, the probability of finding the 40-pS K was significantly higher in the late part of the cTAL close to the distal convoluted tubule than those in the initial part. Immunostaining showed that Kcnj10 staining was detected in the basolateral membrane of the cTAL but the expression was not uniformly distributed. The disruption of Kcnj10 completely eliminated the 40-pS K channel but not the 80-pS K channel, suggesting the role of Kcnj10 in forming the 40-pS K channel of the cTAL. Also, the disruption of Kcnj10 increased the probability of finding the 80-pS K channel in the cTAL, especially in the late part of the cTAL. Because the channel open probability of the 80-pS K channel in KO was similar to those of WT mice, the increase in the 80-pS K channel may be achieved by increasing K channel number. The whole cell recording further showed that K reversal potential measured with 5 mM K in the bath and 140 mM K in the pipette was the same in the WT and KO mice. Moreover, Western blot and immunostaining showed that the disruption of Kcnj10 did not affect the expression of Na-K-Cl cotransporter 2 (NKCC2). We conclude that Kir.4.1 is expressed in the basolateral membrane of cTAL and that the disruption of Kir.4.1 has no significant effect on the membrane potential of the cTAL and NKCC2 expression.

Potassium modulates electrolyte balance and blood pressure through effects on distal cell voltage and chloride.

Dietary potassium deficiency, common in modern diets, raises blood pressure and enhances salt sensitivity. Potassium homeostasis requires a molecular switch in the distal convoluted tubule (DCT), which fails in familial hyperkalemic hypertension (pseudohypoaldosteronism type 2), activating the thiazide-sensitive NaCl cotransporter, NCC. Here, we show that dietary potassium deficiency activates NCC, even in the setting of high salt intake, thereby causing sodium retention and a rise in blood pressure. The effect is dependent on plasma potassium, which modulates DCT cell membrane voltage and, in turn, intracellular chloride. Low intracellular chloride stimulates WNK kinases to activate NCC, limiting potassium losses, even at the expense of increased blood pressure. These data show that DCT cells, like adrenal cells, sense potassium via membrane voltage. In the DCT, hyperpolarization activates NCC via WNK kinases, whereas in the adrenal gland, it inhibits aldosterone secretion. These effects work in concert to maintain potassium homeostasis.

Kcnj10 is a major type of K+ channel in mouse corneal epithelial cells and plays a role in initiating EGFR signaling.

We used primary mouse corneal epithelial cells (pMCE) to examine the role of Kcnj10 in determining membrane K(+) conductance and cell membrane potential and in regulating EGF/TGFA release. Western blot, immunostaining, and RT-PCR detected the expression of Kcnj10 in mouse cornea. The single channel recording identified the 20-pS inwardly rectifying K(+) channels in pMCE of WT mice, but these channels were absent in Kcnj10(-/-). Moreover, the whole cell recording demonstrates that deletion of Kcnj10 largely abolished the inward K(+) currents and depolarized the cell membrane K(+) reversal potential (an index of the cell membrane potential). This suggests that Kcnj10 is a main contributor to the cell K(+) conductance and it is pivotal in generating membrane potential in cornea. Furthermore, to test the hypothesis that Kcnj10 expression plays a key role in the stimulation of growth factors release, we employed an immortalized human corneal epithelial cell line (HCE) transfected with siRNA-Kcnj10 or siRNA-control. Levels of TGFA and EGF secreted in the medium were measured by ELISA. Coimmunoprecipitation, biotinylation, and pull-down assay were used to examine the expression of EGFR and the GTP bound form of Rac1 (active Rac1). Downregulation of Kcnj10 activated Rac1 and enhanced EGF/TGFA release, which further contributed to the upregulation of EGFR phosphorylation and surface expression. We conclude that Kcnj10 is a main K(+) channel expressed in corneal epithelial cells and the inhibition of Kcnj10 resulted in depolarization, which in turn induced an EGF-like effect.

KCNJ10 determines the expression of the apical Na-Cl cotransporter (NCC) in the early distal convoluted tubule (DCT1).

The renal phenotype induced by loss-of-function mutations of inwardly rectifying potassium channel (Kir), Kcnj10 (Kir4.1), includes salt wasting, hypomagnesemia, metabolic alkalosis and hypokalemia. However, the mechanism by which Kir.4.1 mutations cause the tubulopathy is not completely understood. Here we demonstrate that Kcnj10 is a main contributor to the basolateral K conductance in the early distal convoluted tubule (DCT1) and determines the expression of the apical Na-Cl cotransporter (NCC) in the DCT. Immunostaining demonstrated Kcnj10 and Kcnj16 were expressed in the basolateral membrane of DCT, and patch-clamp studies detected a 40-pS K channel in the basolateral membrane of the DCT1 of p8/p10 wild-type Kcnj10(+/+) mice (WT). This 40-pS K channel is absent in homozygous Kcnj10(-/-) (knockout) mice. The disruption of Kcnj10 almost completely eliminated the basolateral K conductance and decreased the negativity of the cell membrane potential in DCT1. Moreover, the lack of Kcnj10 decreased the basolateral Cl conductance, inhibited the expression of Ste20-related proline-alanine-rich kinase and diminished the apical NCC expression in DCT. We conclude that Kcnj10 plays a dominant role in determining the basolateral K conductance and membrane potential of DCT1 and that the basolateral K channel activity in the DCT determines the apical NCC expression possibly through a Ste20-related proline-alanine-rich kinase-dependent mechanism.

Cyp2c44 epoxygenase in the collecting duct is essential for the high K+ intake-induced antihypertensive effect.

Cytochrome P-450, family 2, subfamily c, polypeptide 44 (Cyp2c44) epoxygenase metabolizes arachidonic acid (AA) to epoxyeicosatrienoic acids (EETs) in kidney and vascular tissues. In the present study, we used real-time quantitative PCR techniques to examine the effect of high salt or high K(+) (HK) intake on the expression of Cyp2c44, a major Cyp2c epoxygenase in the mouse kidney. We detected Cyp2c44 in the proximal convoluted tubule, thick ascending limb, distal convoluted tubule (DCT)/connecting tubule (CNT), and collecting duct (CD). A high-salt diet increased the expression of Cyp2c44 in the thick ascending limb and DCT/CNT but not in the proximal convoluted tubule and CD. In contrast, an increase in dietary K(+) intake augmented Cyp2c44 expression only in the DCT/CNT and CD. Neither high salt nor HK intake had a significant effect on the blood pressure (BP) of wild-type mice. However, HK but not high salt intake increased BP in CD-specific, Cyp2c44 conditional knockout (KO) mice. Amiloride, an epithelial Na(+) channel (ENaC) inhibitor, normalized the BP of KO mice fed HK diets, suggesting that lack of Cyp2c44 in the CD enhances ENaC activity and increases Na(+) absorption in KO mice fed HK diets. This notion was supported by metabolic cage experiments demonstrating that renal Na(+) excretion was compromised in KO mice fed HK diets. Also, patch-clamp experiments demonstrated that 11,12-EET, a major Cyp2c44 product, but not AA inhibited ENaC activity in the cortical CD of KO mice. We conclude that Cyp2c44 in the CD is required for preventing the excessive Na(+) absorption induced by HK intake by inhibition of ENaC and facilitating renal Na(+) excretion.

MicroRNA-194 (miR-194) regulates ROMK channel activity by targeting intersectin 1.

The aim of the study is to explore the role of miR-194 in mediating the effect of high-K (HK) intake on ROMK channel. Northern blot analysis showed that miR-194 was expressed in kidney and that HK intake increased while low-K intake decreased the expression of miR-194. Real-time PCR analysis further demonstrated that HK intake increased the miR-194 expression in the cortical collecting duct. HK intake decreased the expression of intersectin 1 (ITSN1) which enhanced With-No-Lysine Kinase (WNK)-induced endocytosis of ROMK. Expression of miR-194 mimic decreased luciferase reporter gene activity in HEK293 T cells transfected with ITSN-1-3'UTR containing the complementary seed sequence for miR-194. In contrast, transfection of miR-194 inhibitor increased the luciferase activity. This effect was absent in the cells transfected with mutated 3'UTR of ITSN1 in which the complimentary seed sequence was deleted. Moreover, the inhibition of miR-194 expression increased the protein level of endogenous ITSN1 in HEK293T cells. Expression of miR-194 mimic also decreased the translation of exogenous ITSN1 in the cells transfected with the ITSN1 containing 3'UTR but not with 3'UTR-free ITSN1. Expression of pre-miR-194 increased K currents and ROMK expression in the plasma membrane in ROMK-transfected cells. Coexpression of ITSN1 reversed the stimulatory effect of miR-194 on ROMK channels. This effect was reversed by coexpression of ITSN1. We conclude that miR-194 regulates ROMK channel activity by modulating ITSN1 expression thereby enhancing ITSN1/WNK-dependent endocytosis. It is possible that miR-194 is involved in mediating the effect of a HK intake on ROMK channel activity.

Inhibition of miR-205 impairs the wound-healing process in human corneal epithelial cells by targeting KIR4.1 (KCNJ10).

PURPOSE: The aim of the study was to test the hypotheses that injury stimulates the expression of miR-205, which in turn inhibits KCNJ10 channels by targeting its 3' UTR, thereby facilitating the wound-healing process in human corneal epithelial cells (HCECs). METHODS: A stem-loop qRT-PCR was used to examine the miR-205 expression. BrdU cell proliferation assay and wound scratch assay were applied to measure the effect of miR-205 mimic or antagomer in HCECs. The patch-clamp technique, dual luciferase reporter assay, and Western blot analysis were employed to test whether miR-205 regulates KCNJ10, one of the target genes of miR-205. Both of the primary human and mouse corneal epithelial cells (pH/MCECs) were employed to further confirm the observations obtained in HCECs. RESULTS: The scratch injury in pH/MCECs increased the expression of miR-205 and decreased the expression of KCNJ10 within 24 hours. The notion that miR-205 may target KCNJ10 was supported by dual luciferase reporter assay showing an inhibition effect of miR-205 on 3' UTR of KCNJ10. Application of miR-205 antagomer significantly delayed the regrowth in wounded HCECs. However, inhibition of KCNJ10 partially abolished the effect from miR-205 antagomer and restored the healing process. Moreover, overexpression miR-205 antagomer enhanced the protein expression of KCNJ10 but not KCNJ16. In addition, patch-clamp demonstrated that inhibition of endogenous miR-205 expression increased Ba²âº-sensitive inwardly rectifying K⁺ channels. In addition, an electrophysiological study of pHCECs showed the presence of KCNJ10-like 20 pS K⁺ channels and scratch injury significantly decreased the Ba²âº-sensitive inwardly rectifying K⁺ currents. CONCLUSIONS: miR-205 stimulates wound healing by inhibiting its target gene KCNJ10.