Angiotensin-II-Type-1a Receptor (AT1aR) and Renal K+ Wasting during Overnight Low-Na+ Intake.

BACKGROUND: Chronic Angiotensin-II (Ang-II) perfusion stimulates Kir4.1/Kir5.1 of the DCT via angiotensin-II-type-1a-receptor (AT1aR) and low-sodium-intake also stimulates Kir4.1/Kir5.1. However, it is not explored the role of AT1aR in mediating the effect of LS on Kir4.1/Kir5.1. METHODS: We used patch-clamp-technique to examine Kir4.1/Kir5.1 activity of the DCT, employed immunoblotting to examine NCC expression/activity, and used in vivo perfusion-technique to measure renal-Na+ and renal-K+-excretion in control, kidney-tubule-specific-AT1aR-knockout (Ks-AT1aR-KO) and DCT-specific-AT1aR-knockout mice (DCT-AT1aR- KO). RESULTS: Ang-II acutely stimulated 40-pS-K+ channel (Kir4.1/Kir5.1-heterotetramer), increased whole-cell Kir4.1/Kir5.1-mediated K+-currents and the negativity of DCT-membrane-potential only in late-DCT2 but not in early-DCT. Acute Ang-II increased thiazide-induced renal Na+-excretion (ENa). The effect of Ang-II on Kir4.1/Kir5.1 and HCTZ-induced-ENa was absent in Ks-AT1aR-KO mice. Overnight-low-salt stimulated the expression of Agtr1a mRNA in DCT, increased whole-cell Kir4.1/Kir5.1-mediated K+-currents in late-DCT, hyperpolarized late-DCT membrane, augmented the expression of phosphor-Na-Cl-cotransporter (pNCC) and enhanced thiazide-induced renal-ENa in the control mice. However, the effect of overnight-low-salt on Kir4.1/Kir5.1-activity, DCT membrane potential and NCC activity/expression was abolished in DCT-AT1aR-KO or Ks-AT1aR-KO mice. Overnight-low-salt had no effect on baseline renal K+-excretion (EK) and plasma K+-concentrations in the control mice but it increased baseline renal-EK and decreased plasma K+-concentrations in DCT-AT1aR-KO or in Ks-AT1aR-KO mice. CONCLUSIONS: Acute Ang-II or overnight-LS stimulated Kir4.1/Kir5.1 in late-DCT and that AT1aR was responsible for acute Ang-II or overnight-low-salt-induced stimulation of Kir4.1/Kir5.1 and NCC. AT1aR of the DCT plays a role in maintaining adequate baseline renal-EK and plasma K+ concentrations during overnight-LS.

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.

Role of Angiotensin II Type 1a Receptor (AT1aR) of Renal Tubules in Regulating Inwardly Rectifying Potassium Channels 4.2 (Kir4.2), Kir4.1, and Epithelial Na+ Channel (ENaC).

BACKGROUND: Kir4.2 and Kir4.1 play a role in regulating membrane transport in the proximal tubule (PT) and in the distal-convoluted-tubule (DCT), respectively. METHODS: We generated kidney-tubule-specific-AT1aR-knockout (Ks-AT1aR-KO) mice to examine whether renal AT1aR regulates Kir4.2 and Kir4.1. RESULTS: Ks-AT1aR-KO mice had a lower systolic blood pressure than Agtr1aflox/flox (control) mice. Ks-AT1aR-KO mice had a lower expression of NHE3 (Na+/H+-exchanger 3) and Kir4.2, a major Kir-channel in PT, than Agtr1aflox/flox mice. Whole-cell recording also demonstrated that the membrane potential in PT of Ks-AT1aR-KO mice was lesser negative than Agtr1aflox/flox mice. The expression of Kir4.1 and Kir5.1, Kir4.1/Kir5.1-mediated K+ currents of DCT and DCT membrane potential in Ks-AT1aR-KO mice, were similar to Agtr1aflox/flox mice. However, angiotensin II perfusion for 7 days hyperpolarized the membrane potential in PT and DCT of the control mice but not in Ks-AT1aR-KO mice, while angiotensin II perfusion did not change the expression of Kir4.1, Kir4.2, and Kir5.1. Deletion of AT1aR did not significantly affect the expression of αENaC (epithelial Na+ channel) and ßENaC but increased cleaved γENaC expression. Patch-clamp experiments demonstrated that deletion of AT1aR increased amiloride-sensitive Na+-currents in the cortical-collecting duct but not in late-DCT. However, tertiapin-Q sensitive renal outer medullary potassium channel currents were similar in both genotypes. CONCLUSIONS: AT1aR determines the baseline membrane potential of PT by controlling Kir4.2 expression/activity but AT1aR is not required for determining the baseline membrane potential of the DCT and Kir4.1/Kir5.1 activity/expression. However, AT1aR is required for angiotensin II-induced hyperpolarization of basolateral membrane of PT and DCT. Deletion of AT1aR had no effect on baseline renal outer medullary potassium channel activity but increased ENaC activity in the CCD.

Calcineurin inhibitors stimulate Kir4.1/Kir5.1 of the distal convoluted tubule to increase NaCl cotransporter.

We examine whether calcineurin or protein phosphatase 2B (PP2B) regulates the basolateral inwardly rectifying potassium channel Kir4.1/Kir5.1 in the distal convoluted tubule (DCT). Application of tacrolimus (FK506) or cyclosporine A (CsA) increased whole-cell Kir4.1/Kir5.1-mediated K+ currents and hyperpolarized the DCT membrane. Moreover, FK506-induced stimulation of Kir4.1/Kir5.1 was absent in kidney tubule-specific 12 kDa FK506-binding protein-knockout mice (Ks-FKBP-12-KO). In contrast, CsA stimulated Kir4.1/Kir5.1 of the DCT in Ks-FKBP-12-KO mice, suggesting that FK506-induced stimulation of Kir4.1/Kir5.1 was due to inhibiting PP2B. Single-channel patch-clamp experiments demonstrated that FK506 or CsA stimulated the basolateral Kir4.1/Kir5.1 activity of the DCT, defined by NPo (a product of channel number and open probability). However, this effect was absent in the DCT treated with Src family protein tyrosine kinase (SFK) inhibitor or hydroxyl peroxide. Fluorescence imaging demonstrated that CsA treatment increased membrane staining intensity of Kir4.1 in the DCT of Kcnj10fl/fl mice. Moreover, CsA treatment had no obvious effect on phosphorylated NaCl cotransporter (pNCC) expression in Ks-Kir4.1-KO mice. Immunoblotting showed acute FK506 treatment increased pNCC expression in Kcnj10fl/fl mice, but this effect was attenuated in Ks-Kir4.1-KO mice. In vivo measurement of thiazide-induced renal Na+ excretion demonstrated that FK506 enhanced thiazide-induced natriuresis. This effect was absent in Ks-FKBP-12-KO mice and blunted in Ks-Kir4.1-KO mice. We conclude that inhibition of PP2B stimulates Kir4.1/Kir5.1 of the DCT and NCC and that PP2B inhibition-induced stimulation of NCC is partially achieved by stimulation of the basolateral Kir4.1/Kir5.1.

Role of inwardly rectifying K+ channel 5.1 (Kir5.1) in the regulation of renal membrane transport.

PURPOSE OF REVIEW: Kir5.1 interacts with Kir4.2 in proximal tubule and with Kir4.1 in distal convoluted tubule (DCT), connecting tubule (CNT) and cortical collecting duct (CCD) to form basolateral-K+-channels. Kir4.2/Kir5.1 and Kir4.1/Kir5.1 play an important role in regulating Na+/HCO3--transport of the proximal tubule and Na+/K+ -transport in the DCT/CNT/CCD. The main focus of this review is to provide an overview of the recent development in the field regarding the role of Kir5.1 regulating renal electrolyte transport in the proximal tubule and DCT. RECENT FINDINGS: Loss-of-function-mutations of KCNJ16 cause a new form of tubulopathy, characterized by hypokalaemia, Na+-wasting, acid-base-imbalance and metabolic-acidosis. Abnormal bicarbonate transport induced by loss-of-function of KCNJ16-mutants is recapitulated in Kir4.2-knockout-(Kir4.2 KO) mice. Deletion of Kir5.1 also abolishes the effect of dietary Na+ and K+-intakes on the basolateral membrane voltage and NCC expression/activity. Long-term high-salt intake or high-K+-intake causes hyperkalaemic in Kir5.1-deficient mice. SUMMARY: Kir4.2/Kir5.1 activity in the proximal tubule plays a key role in regulating Na+, K+ and bicarbonate-transport through regulating electrogenic-Na+-bicarbonate-cotransporter-(NBCe1) and type 3-Na+/H+-exchanger-(NHE3). Kir4.1/Kir5.1 activity of the DCT plays a critical role in mediating the effect of dietary-K+ and Na+-intakes on NCC activity/expression. As NCC determines the Na+ delivery rate to the aldosterone-sensitive distal nephron (ASDN), defective regulation of NCC during high-salt and high-K+ compromises renal K+ excretion and K+ homeostasis.

Multiple molecular mechanisms are involved in the activation of the kidney sodium-chloride cotransporter by hypokalemia.

Low potassium intake activates the kidney sodium-chloride cotransporter (NCC) whose phosphorylation and activity depend on the With-No-Lysine kinase 4 (WNK4) that is inhibited by chloride binding to its kinase domain. Low extracellular potassium activates NCC by decreasing intracellular chloride thereby promoting chloride dissociation from WNK4 where residue L319 of WNK4 participates in chloride coordination. Since the WNK4-L319F mutant is constitutively active and chloride-insensitive in vitro, we generated mice harboring this mutation that displayed slightly increased phosphorylated NCC and mild hyperkalemia when on a 129/sv genetic background. On a low potassium diet, upregulation of phosphorylated NCC was observed, suggesting that in addition to chloride sensing by WNK4, other mechanisms participate which may include modulation of WNK4 activity and degradation by phosphorylation of the RRxS motif in regulatory domains present in WNK4 and KLHL3, respectively. Increased levels of WNK4 and kidney-specific WNK1 and phospho-WNK4-RRxS were observed in wild-type and WNK4L319F/L319F mice on a low potassium diet. Decreased extracellular potassium promoted WNK4-RRxS phosphorylation in vitro and ex vivo as well. These effects might be secondary to intracellular chloride depletion, as reduction of intracellular chloride in HEK293 cells increased phospho-WNK4-RRxS. Phospho-WNK4-RRxS levels were increased in mice lacking the Kir5.1 potassium channel, which presumably have decreased distal convoluted tubule intracellular chloride. Similarly, phospho-KLHL3 was modulated by changes in intracellular chloride in HEK293 cells. Thus, our data suggest that multiple chloride-regulated mechanisms are responsible for NCC upregulation by low extracellular potassium.

Inwardly rectifying K+ channels 4.1 and 5.1 (Kir4.1/Kir5.1) in the renal distal nephron.

The inwardly rectifying potassium channel (Kir) 4.1 (encoded by KCNJ10) interacts with Kir5.1 (encoded by KCNJ16) to form a major basolateral K+ channel in the renal distal convoluted tubule (DCT), connecting tubule (CNT), and the cortical collecting duct (CCD). Kir4.1/Kir5.1 heterotetramer plays an important role in regulating Na+ and K+ transport in the DCT, CNT, and CCD. A recent development in the field has firmly established the role of Kir4.1/Kir5.1 heterotetramer of the DCT in the regulation of thiazide-sensitive Na-Cl cotransporter (NCC). Changes in Kir4.1/Kir5.1 activity of the DCT are an essential step for the regulation of NCC expression/activity induced by dietary K+ and Na+ intakes and play a role in modulating NCC by type 2 angiotensin II receptor (AT2R), bradykinin type II receptor (BK2R), and ß-adrenergic receptor. Since NCC activity determines the Na+ delivery rate to the aldosterone-sensitive distal nephron (ASDN), a distal nephron segment from late DCT to CCD, Kir4.1/Kir5.1 activity plays a critical role not only in the regulation of renal Na+ absorption but also in modulating renal K+ excretion and maintaining K+ homeostasis. Thus, Kir4.1/Kir5.1 activity serves as an important component of renal K+ sensing mechanism. The main focus of this review is to provide an overview regarding the role of Kir4.1 and Kir5.1 of the DCT and CCD in the regulation of renal K+ excretion and Na+ absorption.

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.

Deletion of renal Nedd4-2 abolishes the effect of high K+ intake on Kir4.1/Kir5.1 and NCC activity in the distal convoluted tubule.

High-dietary K+ (HK) intake inhibits basolateral Kir4.1/Kir5.1 activity in the distal convoluted tubule (DCT), and HK-induced inhibition of Kir4.1/Kir5.1 is essential for HK-induced inhibition of NaCl cotransporter (NCC). Here, we examined whether neural precursor cell expressed developmentally downregulated 4-2 (Nedd4-2) deletion compromises the effect of HK on basolateral Kir4.1/Kir5.1 and NCC in the DCT. Single-channel recording and whole cell recording showed that neither HK decreased nor low-dietary K+ (LK) increased basolateral Kir4.1/Kir5.1 activity of the DCT in kidney tubule-specific Nedd4-2 knockout (Ks-Nedd4-2 KO) mice. In contrast, HK inhibited and LK increased Kir4.1/Kir5.1 activity in control mice [neural precursor cell expressed developmentally downregulated 4-like (Nedd4l)flox/flox]. Also, HK intake decreased the negativity of K+ current reversal potential in the DCT (depolarization) only in control mice but not in Ks-Nedd4-2 KO mice. Renal clearance experiments showed that HK intake decreased, whereas LK intake increased, hydrochlorothiazide-induced renal Na+ excretion only in control mice, but this effect was absent in Ks-Nedd4-2 KO mice. Western blot analysis also demonstrated that HK-induced inhibition of phosphorylated NCC (Thr53) and total NCC was observed only in control mice but not in Ks-Nedd4-2 KO mice. Furthermore, expression of all three subunits of the epithelial Na+ channel in Ks-Nedd4-2 KO mice on HK was higher than in control mice. Thus, plasma K+ concentrations were similar between Nedd4lflox/flox and Ks-Nedd4-2 KO mice on HK for 7 days despite high NCC expression. We conclude that Nedd4-2 plays a role in regulating HK-induced inhibition of Kir4.1/Kir5.1 and NCC in the DCT.NEW & NOTEWORTHY Basolateral Kir4.1/Kir5.1 in the distal convoluted tubule plays an important role as a "K+ sensor" in the regulation of renal K+ excretion after high K+ intake. We found that neural precursor cell expressed developmentally downregulated 4-2 (Nedd4-2) a role in mediating the effect of K+ diet on Kir4.1/Kir5.1 and NaCl cotransporter because high K+ intake failed to inhibit basolateral Kir4.1/Kir5.1 and NaCl cotransporter in kidney tubule-specific Nedd4-2 knockout mice.

Deletion of renal Nedd4-2 abolishes the effect of high sodium intake (HS) on Kir4.1, ENaC, and NCC and causes hypokalemia during high HS.

Neural precursor cell expressed developmentally downregulated protein 4-2 (Nedd4-2) regulates the expression of Kir4.1, thiazide-sensitive NaCl cotransporter (NCC), and epithelial Na+ channel (ENaC) in the aldosterone-sensitive distal nephron (ASDN), and Nedd4-2 deletion causes salt-sensitive hypertension. We now examined whether Nedd4-2 deletion compromises the effect of high-salt (HS) diet on Kir4.1, NCC, ENaC, and renal K+ excretion. Immunoblot analysis showed that HS diet decreased the expression of Kir4.1, Ca2+-activated large-conductance K+ channel subunit-α (BKα), ENaCß, ENaCγ, total NCC, and phospho-NCC (at Thr53) in floxed neural precursor cell expressed developmentally downregulated gene 4-like (Nedd4lfl/fl) mice, whereas these effects were absent in kidney-specific Nedd4-2 knockout (Ks-Nedd4-2 KO) mice. Renal clearance experiments also demonstrated that Nedd4-2 deletion abolished the inhibitory effect of HS diet on hydrochlorothiazide-induced natriuresis. Patch-clamp experiments showed that neither HS diet nor low-salt diet had an effect on Kir4.1/Kir5.1 currents of the distal convoluted tubule in Nedd4-2-deficient mice, whereas we confirmed that HS diet inhibited and low-salt diet increased Kir4.1/Kir5.1 activity in Nedd4lflox/flox mice. Nedd4-2 deletion increased ENaC currents in the ASDN, and this increase was more robust in the cortical collecting duct than in the distal convoluted tubule. Also, HS-induced inhibition of ENaC currents in the ASDN was absent in Nedd4-2-deficient mice. Renal clearance experiments showed that HS intake for 2 wk increased the basal level of renal K+ excretion and caused hypokalemia in Ks-Nedd4-2-KO mice but not in Nedd4lflox/flox mice. In contrast, plasma Na+ concentrations were similar in Nedd4lflox/flox and Ks-Nedd4-2 KO mice on HS diet. We conclude that Nedd4-2 plays an important role in mediating the inhibitory effect of HS diet on Kir4.1, ENaC, and NCC and is essential for maintaining normal renal K+ excretion and plasma K+ ranges during long-term HS diet.NEW & NOTEWORTHY The present study suggests that Nedd4-2 is involved in mediating the inhibitory effect of high salt (HS) diet on Kir4.1/kir5.1 in the distal convoluted tubule, NaCl cotransporter function, and epithelial Na+ channel activity and that Nedd4-2 plays an essential role in maintaining K+ homeostasis in response to a long-term HS diet. This suggests the possibility that HS intake could lead to hypokalemia in subjects lacking proper Nedd4-2 E3 ubiquitin ligase activity in aldosterone-sensitive distal nephron.

Deletion of Kir5.1 abolishes the effect of high Na+ intake on Kir4.1 and Na+-Cl- cotransporter.

High sodium (HS) intake inhibited epithelial Na+ channel (ENaC) in the aldosterone-sensitive distal nephron and Na+-Cl- cotransporter (NCC) by suppressing basolateral Kir4.1/Kir5.1 in the distal convoluted tubule (DCT), thereby increasing renal Na+ excretion but not affecting K+ excretion. The aim of the present study was to explore whether deletion of Kir5.1 compromises the inhibitory effect of HS on NCC expression/activity and renal K+ excretion. Patch-clamp experiments demonstrated that HS failed to inhibit DCT basolateral K+ channels and did not depolarize K+ current reversal potential of the DCT in Kir5.1 knockout (KO) mice. Moreover, deletion of Kir5.1 not only increased the expression of Kir4.1, phospho-NCC, and total NCC but also abolished the inhibitory effect of HS on the expression of Kir4.1, phospho-NCC, and total NCC and thiazide-induced natriuresis. Also, low sodium-induced stimulation of NCC expression/activity and basolateral K+ channels in the DCT were absent in Kir5.1 KO mice. Deletion of Kir5.1 decreased ENaC currents in the late DCT, and HS further inhibited ENaC activity in Kir5.1 KO mice. Finally, measurement of the basal renal K+ excretion rate with the modified renal clearance method demonstrated that long-term HS inhibited the renal K+ excretion rate and steadily increased plasma K+ levels in Kir5.1 KO mice but not in wild-type mice. We conclude that Kir5.1 plays an important role in mediating the effect of HS intake on basolateral K+ channels in the DCT and NCC activity/expression. Kir5.1 is involved in maintaining renal ability of K+ excretion during HS intake. NEW & NOTEWORTHY Kir5.1 plays an important role in mediating the effect of high sodium intake on basolateral K+ channels in the distal convoluted tubule and Na+-Cl- cotransporter activity/expression.

Renal Tubule Nedd4-2 Deficiency Stimulates Kir4.1/Kir5.1 and Thiazide-Sensitive NaCl Cotransporter in Distal Convoluted Tubule.

BACKGROUND: The potassium channel Kir4.1 forms the Kir4.1/Kir5.1 heterotetramer in the basolateral membrane of the distal convoluted tubule (DCT) and plays an important role in the regulation of the thiazide-sensitive NaCl cotransporter (NCC). Kidney-specific deletion of the ubiquitin ligase Nedd4-2 increases expression of NCC, and coexpression of Nedd4-2 inhibits Kir4.1/Kir5.1 in vitro. Whether Nedd4-2 regulates NCC expression in part by regulating Kir4.1/Kir5.1 channel activity in the DCT is unknown. METHODS: We used electrophysiology studies, immunoblotting, immunostaining, and renal clearance to examine Kir4.1/Kir5.1 activity in the DCT and NCC expression/activity in wild-type mice and mice with kidney-specific knockout of Nedd4-2, Kir4.1, or both. RESULTS: Deletion of Nedd4-2 increased the activity/expression of Kir4.1 in the DCT and also, hyperpolarized the DCT membrane. Expression of phosphorylated NCC/total NCC and thiazide-induced natriuresis were significantly increased in the Nedd4-2 knockout mice, but these mice were normokalemic. Double-knockout mice lacking both Kir4.1/Kir5.1 and Nedd4-2 in the kidney exhibited increased expression of the epithelial sodium channel α-subunit, largely abolished basolateral potassium ion conductance (to a degree similar to that of kidney-specific Kir4.1 knockout mice), and depolarization of the DCT membrane. Compared with wild-type mice, the double-knockout mice displayed inhibited expression of phosphorylated NCC and total NCC and had significantly blunted thiazide-induced natriuresis as well as renal potassium wasting and hypokalemia. However, NCC expression/activity was higher in the double-knockout mice than in Kir4.1 knockout mice. CONCLUSIONS: Nedd4-2 regulates Kir4.1/Kir5.1 expression/activity in the DCT and modulates NCC expression by Kir4.1-dependent and Kir4.1-independent mechanisms. Basolateral Kir4.1/Kir5.1 activity in the DCT partially accounts for the stimulation of NCC activity/expression induced by deletion of Nedd4-2.

Effect of Angiotensin II on ENaC in the Distal Convoluted Tubule and in the Cortical Collecting Duct of Mineralocorticoid Receptor Deficient Mice.

Background Angiotensin II stimulates epithelial Na+ channel (ENaC) by aldosterone-independent mechanism. We now test the effect of angiotensin II on ENaC in the distal convoluted tubule (DCT) and cortical collecting duct (CCD) of wild-type (WT) and kidney-specific mineralocorticoid receptor knockout mice (KS-MR-KO). Methods and Results We used electrophysiological, immunoblotting and renal-clearance methods to examine the effect of angiotensin II on ENaC in KS-MR-KO and wild-type mice. High K+ intake stimulated ENaC in the late DCT/early connecting tubule (DCT2/CNT) and in the CCD whereas low sodium intake stimulated ENaC in the CCD but not in the DCT2/CNT. The deletion of MR abolished the stimulatory effect of high K+ and low sodium intake on ENaC, partially inhibited ENaC in DCT2/CNT but almost abolished ENaC activity in the CCD. Application of losartan inhibited ENaC only in DCT2/CNT of both wild-type and KS-MR-KO mice but not in the CCD. Angiotensin II infusion for 3 days has a larger stimulatory effect on ENaC in the DCT2/CNT than in the CCD. Three lines of evidence indicate that angiotensin II can stimulate ENaC by MR-independent mechanism: (1) angiotensin II perfusion augmented ENaC expression in KS-MR-KO mice; (2) angiotensin II stimulated ENaC in the DCT2/CNT but to a lesser degree in the CCD in KS-MR-KO mice; (3) angiotensin II infusion augmented benzamil-induced natriuresis, increased the renal K+ excretion and corrected hyperkalemia of KS-MR-KO mice. Conclusions Angiotensin II-induced stimulation of ENaC occurs mainly in the DCT2/CNT and to a lesser degree in the CCD and MR plays a dominant role in determining ENaC activity in the CCD but to a lesser degree in the DCT2/CNT.

Inhibition of AT2R and Bradykinin Type II Receptor (BK2R) Compromises High K+ Intake-Induced Renal K+ Excretion.

The inhibition of Type II angiotensin II receptor (AT2R) or BK2R (bradykinin type II receptor) stimulates basolateral Kir4.1/Kir5.1 in the distal convoluted tubule (DCT) and activates thiazide-sensitive NCC (Na-Cl cotransporter). The aim of the present study is to examine the role of AT2R and BK2R in mediating the effect of HK (high dietary K+) intake on the basolateral K+ channels, NCC, and renal K+ excretion. Feeding mice (male and female) with HK diet for overnight significantly decreased the basolateral K+ conductance, depolarized the DCT membrane, diminished the expression of pNCC (phosphorylated NCC) and tNCC (total NCC), and decreased thiazide-sensitive natriuresis. Overnight HK intake also increased the expression of cleaved ENaC-α and -γ subunits but had no effect on NKCC2 expression. Pretreatment of the mice (male and female) with PD123319 and HOE140 stimulated the expression of tNCC and pNCC, augmented hydrochlorothiazide-induced natriuresis, and increased the negativity of the DCT membrane. The deletion of Kir4.1 not only decreased the NCC activity but also abolished the stimulatory effect of PD123319 and HOE140 perfusion on NCC activity. Moreover, the effect of overnight HK loading on Kir4.1/Kir5.1 in the DCT and NCC expression/activity was compromised in the mice treated with AT2R/BK2R antagonists. Renal clearance study showed that inhibition of AT2R and BK2R impairs renal K+ excretion in response to overnight HK loading, and the mice pretreated with PD123319 and HOE140 were hyperkalemic during HK intake. We conclude that synergistic activation of AT2R and BK2R is required for the effect of overnight HK diet on Kir4.1/Kir5.1 in the DCT and NCC activity.

Mg2+ restriction downregulates NCC through NEDD4-2 and prevents its activation by hypokalemia.

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

Deletion of Kir5.1 Impairs Renal Ability to Excrete Potassium during Increased Dietary Potassium Intake.

BACKGROUND: The basolateral potassium channel in the distal convoluted tubule (DCT), comprising the inwardly rectifying potassium channel Kir4.1/Kir5.1 heterotetramer, plays a key role in mediating the effect of dietary potassium intake on the thiazide-sensitive NaCl cotransporter (NCC). The role of Kir5.1 (encoded by Kcnj16) in mediating effects of dietary potassium intake on the NCC and renal potassium excretion is unknown. METHODS: We used electrophysiology, renal clearance, and immunoblotting to study Kir4.1 in the DCT and NCC in Kir5.1 knockout (Kcnj16-/- ) and wild-type (Kcnj16+/+ ) mice fed with normal, high, or low potassium diets. RESULTS: We detected a 40-pS and 20-pS potassium channel in the basolateral membrane of the DCT in wild-type and knockout mice, respectively. Compared with wild-type, Kcnj16-/- mice fed a normal potassium diet had higher basolateral potassium conductance, a more negative DCT membrane potential, higher expression of phosphorylated NCC (pNCC) and total NCC (tNCC), and augmented thiazide-induced natriuresis. Neither high- nor low-potassium diets affected the basolateral DCT's potassium conductance and membrane potential in Kcnj16-/- mice. Although high potassium reduced and low potassium increased the expression of pNCC and tNCC in wild-type mice, these effects were absent in Kcnj16-/- mice. High potassium intake inhibited and low intake augmented thiazide-induced natriuresis in wild-type but not in Kcnj16-/- mice. Compared with wild-type, Kcnj16-/- mice with normal potassium intake had slightly lower plasma potassium but were more hyperkalemic with prolonged high potassium intake and more hypokalemic during potassium restriction. CONCLUSIONS: Kir5.1 is essential for dietary potassium's effect on NCC and for maintaining potassium homeostasis.

Kir4.1/Kir5.1 Activity Is Essential for Dietary Sodium Intake-Induced Modulation of Na-Cl Cotransporter.

BACKGROUND: Dietary sodium intake regulates the thiazide-sensitive Na-Cl cotransporter (NCC) in the distal convoluted tubule (DCT). Whether the basolateral, inwardly rectifying potassium channel Kir4.1/Kir5.1 (a heterotetramer of Kir4.1/Kir5.1) in the DCT is essential for mediating the effect of dietary sodium intake on NCC activity is unknown. METHODS: We used electrophysiology, renal clearance techniques, and immunoblotting to examine effects of Kir4.1/Kir5.1 in the DCT and NCC in wild-type and kidney-specific Kir4.1 knockout mice. RESULTS: Low sodium intake stimulated basolateral Kir4.1/Kir5.1 activity, increased basolateral K+ conductance, and hyperpolarized the membrane. Conversely, high sodium intake inhibited the potassium channel, decreased basolateral K+ currents, and depolarized the membrane. Low sodium intake increased total and phosphorylated NCC expression and augmented hydrochlorothiazide-induced natriuresis; high sodium intake had opposite effects. Thus, elevated NCC activity induced by low sodium intake was associated with upregulation of Kir4.1/Kir5.1 activity in the DCT, whereas inhibition of NCC activity by high sodium intake was associated with diminished Kir4.1/Kir5.1 activity. In contrast, dietary sodium intake did not affect NCC activity in knockout mice. Further, Kir4.1 deletion not only abolished basolateral K+ conductance and depolarized the DCT membrane, but also abrogated the stimulating effects induced by low sodium intake on basolateral K+ conductance and hyperpolarization. Finally, dietary sodium intake did not alter urinary potassium excretion rate in hypokalemic knockout and wild-type mice. CONCLUSIONS: Stimulation of Kir4.1/Kir5.1 by low intake of dietary sodium is essential for NCC upregulation, and inhibition of Kir4.1/Kir5.1 induced by high sodium intake is a key step for downregulation of NCC.

Norepinephrine-Induced Stimulation of Kir4.1/Kir5.1 Is Required for the Activation of NaCl Transporter in Distal Convoluted Tubule.

The stimulation of ß-adrenergic receptor increases thiazide-sensitive NaCl cotransporter (NCC), an effect contributing to salt-sensitive hypertension by sympathetic stimulation. We now test whether the stimulation of ß-adrenergic receptor-induced activation of NCC is achieved through activating basolateral Kir4.1 in the distal convoluted tubule (DCT). Application of norepinephrine increased the basolateral 40 pS K+ channel (Kir4.1/Kir5.1 heterotetramer) in the DCT. The stimulatory effect of norepinephrine on the K+ channel was mimicked by cAMP analogue but abolished by inhibiting PKA (protein kinase A). Also, the effect of norepinephrine on the K+ channel in the DCT was recapitulated by isoproterenol but not by α-adrenergic agonist and blocked by propranolol, suggesting that norepinephrine effect on the K+ channel was mediated by ß-adrenergic receptor. The whole-cell recording shows that norepinephrine and isoproterenol increased DCT K+ currents and shifted the K+ current ( IK) reversal potential to negative range (hyperpolarization). Continuous norepinephrine perfusion (7 days) increased DCT K+ currents, hyperpolarized IK reversal potential, and increased the expression of total NCC/phosphorylated NCC, but it had no significant effect on the expression of NKCC2 (type 2 Na-Cl-K cotransporter) and ENaC-α (epithelial Na channel-α subunit). Renal clearance study demonstrated that norepinephrine perfusion augmented thiazide-induced urinary Na+ excretion only in wild-type but not in kidney-specific Kir4.1 knockout mice, suggesting that Kir4.1 is required for mediating the effect of norepinephrine on NCC. However, norepinephrine perfusion did not affect urinary K+ excretion. We conclude that the stimulation of ß-adrenergic receptor activates the basolateral Kir4.1 in the DCT and that the activation of Kir4.1 is required for norepinephrine-induced stimulation of NCC.

Bradykinin Stimulates Renal Na+ and K+ Excretion by Inhibiting the K+ Channel (Kir4.1) in the Distal Convoluted Tubule.

Stimulation of BK2R (bradykinin [BK] B2 receptor) has been shown to increase renal Na+ excretion. The aim of the present study is to explore the role of BK2R in regulating Kir4.1 and NCC (NaCl cotransporter) in the distal convoluted tubule (DCT). Immunohistochemical studies demonstrated that BK2R was highly expressed in both apical and lateral membrane of Kir4.1-positive tubules, such as DCT. Patch-clamp experiments demonstrated that BK inhibited the basolateral 40-pS K+ channel (a Kir4.1/5.1 heterotetramer) in the DCT, and this effect was blocked by BK2R antagonist but not by BK1R (BK B1 receptor) antagonist. Whole-cell recordings also demonstrated that BK decreased the basolateral K+ conductance of the DCT and depolarized the membrane. Renal clearance experiments showed that BK increased urinary Na+ and K+ excretion. However, the BK-induced natriuretic effect was completely abolished in KS-Kir4.1 KO (kidney-specific conditional Kir4.1 knockout) mice, suggesting that Kir4.1 activity is required for BK-induced natriuresis. The continuous infusion of BK with osmotic pump for 3 days decreased the basolateral K+ conductance and the negativity of the DCT membrane. Western blot showed that infusion of BK decreased the expression of total NCC and phosphorylated NCC. Renal clearance experiments demonstrated that thiazide-induced natriuresis was blunted in the mice receiving BK infusion, suggesting that BK inhibited NCC function. Consequently, mice receiving BK infusion for 3 days were hypokalemic. We conclude that stimulation of BK2R inhibits NCC activity, increases urinary K+ excretion, and causes mice hypokalemia and that Kir4.1 is required for BK2R-mediated stimulation of urinary Na+ and K+ excretion.