scholarly journals With-No-Lysine Kinase 1 (WNK1) Augments TRPV4 Function in the Aldosterone-Sensitive Distal Nephron

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1482
Author(s):  
Viktor N. Tomilin ◽  
Kyrylo Pyrshev ◽  
Naghmeh Hassanzadeh Khayyat ◽  
Oleg Zaika ◽  
Oleh Pochynyuk
Keyword(s):  
Collecting Duct ◽  
Chinese Hamster ◽  
Dominant Negative ◽  
K Channel ◽  
Apical Plasma Membrane ◽  
Dependent Manner ◽  
Distal Nephron ◽  
Potassium Homeostasis ◽  
Wnk Kinases ◽  
K Secretion

Kidneys play a central role in regulation of potassium homeostasis and maintenance of plasma K+ levels within a narrow physiological range. With-no-lysine (WNK) kinases, specifically WNK1 and WNK4, have been recognized to regulate K+ balance, in part, by orchestrating maxi K+ channel (BK)-dependent K+ secretion in the aldosterone-sensitive distal nephron (ASDN), which includes the connecting tubule and collecting duct. We recently demonstrated that the Ca2+-permeable TRPV4 channel is essential for BK activation in the ASDN. Furthermore, high K+ diet increases TRPV4 activity and expression largely in an aldosterone-dependent manner. In the current study, we aimed to test whether WNK kinases contribute to regulation of TRPV4 activity and its stimulation by aldosterone. Systemic inhibition of WNK with WNK463 (1 mg/kgBW for 3 days) markedly decreased TRPV4-dependent Ca2+ influx in freshly isolated split-opened collecting ducts. Aldosterone greatly increased TRPV4 activity and expression in cultured mpkCCDc14 cells and this effect was abolished in the presence of WNK463. Selective inhibition of WNK1 with WNK-in-11 (400 nM, 24 h) recapitulated the effects of WNK463 on TRPV4-dependent Ca2+ influx. Interestingly, WNK-in-11 did not interfere with up-regulation of TRPV4 expression by aldosterone, but prevented translocation of the channel to the apical plasma membrane. Furthermore, co-expression of TRPV4 and WNK1 into Chinese hamster ovary (CHO) cells increased the macroscopic TRPV4-dependent cation currents. In contrast, over-expression of TRPV4 with a dominant negative WNK1 variant (K233M) decreased the whole-cell currents, suggesting both stimulatory and permissive roles of WNK1 in regulation of TRPV4 activity. Overall, we show that WNK1 is essential for setting functional TRPV4 expression in the ASDN at the baseline and in response to aldosterone. We propose that this new mechanism contributes to regulation of K+ secretion and, by extension, urinary K+ levels to maintain systemic potassium homeostasis.

Effect of insulin on potassium secretion in rabbit cortical collecting ducts

1992 ◽  
Vol 262 (1) ◽  
pp. F30-F35 ◽  
Author(s):  
H. Furuya ◽  
K. Tabei ◽  
S. Muto ◽  
Y. Asano
Keyword(s):  
Collecting Duct ◽  
Dependent Manner ◽  
Cortical Collecting Duct ◽  
Plasma Insulin Concentration ◽  
Concentration Dependent Manner ◽  
K Secretion ◽  
Potassium Secretion ◽  
Transepithelial Voltage ◽  
K Transport

Insulin is known to play an important role in the regulation of extrarenal K homeostasis. Previous clearance studies have shown that insulin decreases urinary K excretion, but the responsible nephron segments have not been identified. In this microperfusion study, in vitro, the effect of insulin on K transport in the cortical collecting duct (CCD), which is thought to be an important segment for regulation of the final urinary K excretion, was investigated. Basolateral insulin (10(-6) M) significantly inhibited net K secretion by 20% (mean JK = -26.2 +/- 4.2 peq.mm-1.min-1 for controls compared with -21.1 +/- 3.4 with insulin, P less than 0.001) and depolarized the transepithelial voltage (VT, from -14.6 +/- 3.5 to -10.8 +/- 3.5 mV, P less than 0.005), recovery did not occur over 60 min. Insulin (10(-11)-10(-5) M) depressed K secretion and depolarized the VT in a concentration-dependent manner. The half-maximal concentration was 5 x 10(-10) M, which is within the physiological range of plasma insulin concentration. In tubules of deoxycorticosterone acetate-treated rabbits, insulin also produced a significant fall in K secretion (from -43.4 +/- 7.5 to -36.1 +/- 5.7 peq.mm-1.min-1, P less than 0.05). Although luminal Ba (2 mM) decreased K secretion (from -14.4 +/- 2.9 to -7.0 +/- 1.7 peq.mm-1.min-1), basolateral insulin (10(-6) M) inhibited K secretion further (to -4.7 +/- 1.3 peq.mm-1.min-1, P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Arachidonic acid inhibits basolateral K channels in the cortical collecting duct via cytochrome P-450 epoxygenase-dependent metabolic pathways

2008 ◽  
Vol 294 (6) ◽  
pp. F1441-F1447 ◽  
Author(s):  
ZhiJian Wang ◽  
Yuan Wei ◽  
John R. Falck ◽  
Krishnam Raju Atcha ◽  
Wen-Hui Wang
Keyword(s):  
Arachidonic Acid ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Inhibitory Effect ◽  
K Channel ◽  
Dependent Manner ◽  
Cortical Collecting Duct ◽  
Patch Clamp Technique ◽  
K Channels ◽  
Cytochrome P 450

We used the patch-clamp technique to study the effect of arachidonic acid (AA) on basolateral 18-pS K channels in the principal cell of the cortical collecting duct (CCD) of the rat kidney. Application of AA inhibited the 18-pS K channels in a dose-dependent manner and 10 μM AA caused a maximal inhibition. The effect of AA on the 18-pS K channel was specific because application of 11,14,17-eicosatrienoic acid had no effect on channel activity. Also, the inhibitory effect of AA on the 18-pS K channels was abolished by blocking cytochrome P-450 (CYP) epoxygenase with N-methylsulfonyl-6-(propargyloxyphenyl)hexanamide (MS-PPOH) but was not affected by inhibiting CYP ω-hydroxylase or cyclooxygenase. The notion that the inhibitory effect of AA was mediated by CYP epoxygenase-dependent metabolites was further supported by the observation that application of 100 nM 11,12-epoxyeicosatrienoic acid (EET) mimicked the effect of AA and inhibited the basolateral 18-pS K channels. In contrast, addition of either 5,6-, 8,9-, or 14,15-EET failed to inhibit the 18-pS K channels. Moreover, application of 11,12-EET was still able to inhibit the 18-pS K channels in the presence of MS-PPOH. This suggests that 11,12-EET is a mediator for the AA-induced inhibition of the 18-pS K channels. We conclude that AA inhibits basolateral 18-pS K channels by a CYP epoxygenase-dependent pathway and that 11,12-EET is a mediator for the effect of AA on basolateral K channels in the CCD.

scholarly journals WNK4 kinase inhibits Maxi K channel activity by a kinase-dependent mechanism

2011 ◽  
Vol 301 (2) ◽  
pp. F410-F419 ◽  
Author(s):  
Jieqiu Zhuang ◽  
Xuemei Zhang ◽  
Dexuan Wang ◽  
Juan Li ◽  
Bo Zhou ◽  
...  
Keyword(s):  
Protein Expression ◽  
Surface Protein ◽  
Surface Expression ◽  
Inhibitory Effect ◽  
Dominant Negative ◽  
K Channel ◽  
Control Group ◽  
Channel Activity ◽  
Wnk Kinases ◽  
K Activity

WNK [with no lysine (k)] kinase is a serine/threonine kinase subfamily. Mutations in two of the WNK kinases result in pseudohypoaldosteronism type II (PHA II) characterized by hypertension, hyperkalemia, and metabolic acidosis. Recent studies showed that both WNK1 and WNK4 inhibit ROMK activity. However, little is known about the effect of WNK kinases on Maxi K, a large-conductance Ca2+ and voltage-activated potassium (K) channel. Here, we report that WNK4 wild-type (WT) significantly inhibits Maxi K channel activity in HEK αBK stable cell lines compared with the control group. However, a WNK4 dead-kinase mutant, D321A, has no inhibitory effect on Maxi K activity. We further found that WNK4 inhibits total and cell surface protein expression of Maxi K equally compared with control groups. A dominant-negative dynamin mutant, K44A, did not alter the WNK4-mediated inhibitory effect on Maxi K surface expression. Treatment with bafilomycin A1 (a proton pump inhibitor) and leupeptin (a lysosomal inhibitor) reversed WNK4 WT-mediated inhibition of Maxi K total protein expression. These findings suggest that WNK4 WT inhibits Maxi K activity by reducing Maxi K protein at the membrane, but that the inhibition is not due to an increase in clathrin-mediated endocytosis of Maxi K, but likely due to enhancing its lysosomal degradation. Also, WNK4's inhibitory effect on Maxi K activity is dependent on its kinase activity.

scholarly journals Differential regulation of ROMK (Kir1.1) in distal nephron segments by dietary potassium

2011 ◽  
Vol 300 (6) ◽  
pp. F1385-F1393 ◽  
Author(s):  
James B. Wade ◽  
Liang Fang ◽  
Richard A. Coleman ◽  
Jie Liu ◽  
P. Richard Grimm ◽  
...  
Keyword(s):  
Apical Membrane ◽  
Collecting Duct ◽  
Differential Regulation ◽  
Regulatory Mechanisms ◽  
Cortical Collecting Duct ◽  
Distal Nephron ◽  
High K ◽  
Dietary Potassium ◽  
Intracellular Compartments ◽  
K Secretion

ROMK channels are well-known to play a central role in renal K secretion, but the absence of highly specific and avid-ROMK antibodies has presented significant roadblocks toward mapping the extent of expression along the entire distal nephron and determining whether surface density of these channels is regulated in response to physiological stimuli. Here, we prepared new ROMK antibodies verified to be highly specific, using ROMK knockout mice as a control. Characterization with segmental markers revealed a more extensive pattern of ROMK expression along the entire distal nephron than previously thought, localizing to distal convoluted tubule regions, DCT1 and DCT2; the connecting tubule (CNT); and cortical collecting duct (CD). ROMK was diffusely distributed in intracellular compartments and at the apical membrane of each tubular region. Apical labeling was significantly increased by high-K diet in DCT2, CNT1, CNT2, and CD ( P < 0.05) but not in DCT1. Consistent with the large increase in apical ROMK, dramatically increased mature glycosylation was observed following dietary potassium augmentation. We conclude 1) our new antibody provides a unique tool to characterize ROMK channel localization and expression and 2) high-K diet causes a large increase in apical expression of ROMK in DCT2, CNT, and CD but not in DCT1, indicating that different regulatory mechanisms are involved in K diet-regulated ROMK channel functions in the distal nephron.

The Protein Tyrosine Kinase-Dependent Pathway Mediates the Effect of K Intake on Renal K Secretion

Physiology ◽  
2005 ◽  
Vol 20 (2) ◽  
pp. 140-146 ◽  
Author(s):  
Dao-Hong Lin ◽  
Hyacinth Sterling ◽  
Wen-Hui Wang
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Kinase ◽  
Collecting Duct ◽  
K Channel ◽  
Thick Ascending Limb ◽  
Cortical Collecting Duct ◽  
Protein Tyrosine ◽  
K Secretion ◽  
Dependent Pathway

Dietary K intake plays an important role in the regulation of K secretion: a decrease stimulates and an increase suppresses kidney expression of protein tyrosine kinase (PTK), which plays a role in regulating Kir1.1 (ROMK), which is responsible for K secretion in the cortical collecting duct (CCD) and K recycling in the thick ascending limb. Tyrosine phosphorylation of ROMK channels increases with low dietary K and decreases with high dietary K. Moreover, stimulation of tyrosine phosphorylation of ROMK1 enhances ROMK1 internalization and reduces the K channel number in the cell surface in the CCD.

scholarly journals Potassium conservation is impaired in mice with reduced renal expression of Kir4.1

2018 ◽  
Vol 315 (5) ◽  
pp. F1271-F1282 ◽  
Author(s):  
Sundeep Malik ◽  
Emily Lambert ◽  
Junhui Zhang ◽  
Tong Wang ◽  
Heather L. Clark ◽  
...  
Keyword(s):  
Collecting Duct ◽  
K Channel ◽  
Cortical Collecting Duct ◽  
Wild Type ◽  
Distal Nephron ◽  
East Syndrome ◽  
Renal Expression ◽  
Modest Reduction ◽  
Concentrating Defect

To better understand the role of the inward-rectifying K channel Kir4.1 (KCNJ10) in the distal nephron, we initially studied a global Kir4.1 knockout mouse (gKO), which demonstrated the hypokalemia and hypomagnesemia seen in SeSAME/EAST syndrome and was associated with reduced Na/Cl cotransporter (NCC) expression. Lethality by ~3 wk, however, limits the usefulness of this model, so we developed a kidney-specific Kir4.1 “knockdown” mouse (ksKD) using a cadherin 16 promoter and Cre-loxP methodology. These mice appeared normal and survived to adulthood. Kir4.1 protein expression was decreased ~50% vs. wild-type (WT) mice by immunoblotting, and immunofluorescence showed moderately reduced Kir4.1 staining in distal convoluted tubule that was minimal or absent in connecting tubule and cortical collecting duct. Under control conditions, the ksKD mice showed metabolic alkalosis and relative hypercalcemia but were normokalemic and mildly hypermagnesemic despite decreased NCC expression. In addition, the mice had a severe urinary concentrating defect associated with hypernatremia, enlarged kidneys with tubulocystic dilations, and reduced aquaporin-3 expression. On a K/Mg-free diet for 1 wk, however, ksKD mice showed marked hypokalemia (serum K: 1.5 ± 0.1 vs. 3.0 ± 0.1 mEq/l for WT), which was associated with renal K wasting (transtubular K gradient: 11.4 ± 0.8 vs. 1.6 ± 0.4 in WT). Phosphorylated-NCC expression increased in WT but not ksKD mice on the K/Mg-free diet, suggesting that loss of NCC adaptation underlies the hypokalemia. In conclusion, even modest reduction in Kir4.1 expression results in impaired K conservation, which appears to be mediated by reduced expression of activated NCC.

scholarly journals Flow-dependent K+ secretion in the cortical collecting duct is mediated by a maxi-K channel

2001 ◽  
Vol 280 (5) ◽  
pp. F786-F793 ◽  
Author(s):  
Craig B. Woda ◽  
Alvina Bragin ◽  
Thomas R. Kleyman ◽  
Lisa M. Satlin
Keyword(s):  
Collecting Duct ◽  
K Channel ◽  
Sk Channels ◽  
Cortical Collecting Duct ◽  
Flow Rates ◽  
K Secretion ◽  
Tubular Flow ◽  
Flow Stimulation ◽  
Small Conductance

K+ secretion by the cortical collecting duct (CCD) is stimulated at high flow rates. Patch-clamp analysis has identified a small-conductance secretory K+ (SK) and a high-conductance Ca2+-activated K+ (maxi-K) channel in the apical membrane of the CCD. The SK channel, encoded by ROMK, is believed to mediate baseline K+ secretion. The role of the stretch- and Ca2+-activated maxi-K channel is still uncertain. The purpose of this study was to identify the K+ channel mediating flow-dependent K+ secretion in the CCD. Segments isolated from New Zealand White rabbits were microperfused in the absence and presence of luminal tetraethylammonium (TEA) or charybdotoxin, both inhibitors of maxi-K but not SK channels, or apamin, an inhibitor of small-conductance maxi-K+ channels. Net K+ secretion and Na+ absorption were measured at varying flow rates. In the absence of TEA, net K+ secretion increased from 8.3 ± 1.0 to 23.4 ± 4.7 pmol · min−1 · mm−1( P < 0.03) as the tubular flow rate was increased from 0.5 to 6 nl · min−1 · mm−1. Flow stimulation of net K+ secretion was blocked by luminal TEA (8.2 ± 1.2 vs. 9.9 ± 2.7 pmol · min−1 · mm−1 at 0.6 and 6 nl · min−1 · mm−1 flow rates, respectively) or charybdotoxin (6.8 ± 1.6 vs. 8.3 ± 1.6 pmol · min−1 · mm−1 at 1 and 4 nl · min−1 · mm−1 flow rates, respectively) but not by apamin. These results suggest that flow-dependent K+ secretion is mediated by a maxi-K channel, whereas baseline K+ secretion occurs through a TEA- and charybdotoxin-insensitive SK (ROMK) channel.

Ca-activated K channels in apical membrane of mammalian CCT, and their role in K secretion

1987 ◽  
Vol 252 (3) ◽  
pp. F458-F467 ◽  
Author(s):  
G. Frindt ◽  
L. G. Palmer
Keyword(s):  
Apical Membrane ◽  
K Channel ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
K Channels ◽  
Voltage Dependent ◽  
K Secretion ◽  
Excised Patches ◽  
Cytoplasmic Side ◽  
High Conductance

High conductance, Ca-activated K channels were studied in the apical membrane of the rat cortical collecting tubule (CCT) using the patch-clamp technique. In cell-attached patches the channels were found mainly in the closed state at the spontaneous apical membrane potential. They spent progressively more time in the open state as the pipette potential was made negative relative to the bath. In excised patches these channels had a high selectivity for K over Na and were activated by micromolar concentrations of Ca2+ on the cytoplasmic side of the membrane in a voltage-dependent manner. They had a low conductance to Rb and were blocked by Ba (1-100 microM) from the cytoplasmic side and tetraethylammonium (TEA) (0.2-1 mM) from the luminal side. Block by external TEA and small conductance to Rb were used to investigate the role of these channels in K transport by the isolated perfused rabbit CCT. Ba (2.5 mM), a well-studied blocker of apical K conductance in this segment, hyperpolarized the transepithelial voltage (VT) by 3.7 +/- 0.9 mV when added to the luminal solution of the perfused tubule. Addition of TEA (5 mM) to the luminal solution has no effect on VT. When Na transport was abolished by luminal amiloride, perfusion with 30 mM K (replacing Na) resulted in a lumen-negative VT (18-34 mV). Under these conditions, VT was reduced by 6.0 +/- 1.5 mV by 2.5 mM Ba, whereas TEA had no effect. Perfusion with 30 mM Rb (replacing Na) also caused a lumen-negative VT that was approximately 50% of that observed with 30 mM K. The apical K conductance of the perfused CCT appears to be insensitive to luminal TEA and only modestly selective for K over Rb. This conductance, at least under the conditions of our studies, is probably not mediated by the high conductance Ca-activated K channel.

Electrophysiological characterization of RACTK1 K+ channel in stable cell line

1996 ◽  
Vol 270 (3) ◽  
pp. C964-C968 ◽  
Author(s):  
M. Suzuki ◽  
M. Murata ◽  
M. Ikeda ◽  
T. Miyoshi ◽  
M. Imai
Keyword(s):  
Protein Kinase ◽  
Cell Line ◽  
Collecting Duct ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Stable Cell Line ◽  
K Channel ◽  
Established Cell Line ◽  
Open Probability ◽  
Ovary Cells

RACTK1 is a pH-sensitive K+ channel cloned from rabbit renal collecting tubule cells. To characterize the function of this K+ channel in more detail, RACTK1 was transfected to an established cell line and the patch-clamp study was performed. cDNA of RACTK1 was inserted in the pMAM vector and transfected to Chinese hamster ovary cells. In one of 36 cell lines, the channel protein as expressed by the dexamethasone-induced mRNA and was detected by the specific antibody. The RACTK1 K+ channel with 80 pS was consistently observed. In inside-out patch, Ca2+ at concentrations higher than 500 nM activated the channel. Open probability was decreased by protein kinase A (from 45 to 4.2%, n+6) but not by protein kinase C. Whole cell currents of the transformed cells represented K+ conductance that was not blocked by an addition of charybdotoxin but by apamin. RACTK1 K+ channel has similar, though not identical, characteristics to the Ca2+ -activated K+ channel. RACTK1 might therefore encode a subunit of the intermediate conductance Ca2+ -activated K+ channel observed in the apical membrane of the rabbit renal collecting duct.

Ontogeny of flow-stimulated potassium secretion in rabbit cortical collecting duct: functional and molecular aspects

2003 ◽  
Vol 285 (4) ◽  
pp. F629-F639 ◽  
Author(s):  
Craig B. Woda ◽  
Nobuyuki Miyawaki ◽  
Santhanam Ramalakshmi ◽  
Mohan Ramkumar ◽  
Raul Rojas ◽  
...  
Keyword(s):  
Collecting Duct ◽  
Cell Labeling ◽  
K Channel ◽  
Urinary Flow ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Α Subunit ◽  
K Channels ◽  
K Secretion ◽  
High Conductance

High urinary flow rates stimulate K secretion in the fully differentiated but not neonatal or weanling rabbit cortical collecting duct (CCD). Both small-conductance secretory K and high-conductance Ca2+/stretch-activated maxi-K channels have been identified in the apical membrane of the mature CCD by patch-clamp analysis. We reported that flow-stimulated net K secretion in the adult rabbit CCD is 1) blocked by TEA and charybdotoxin, inhibitors of intermediate- and high-conductance (maxi-K) Ca2+-activated K channels, and 2) associated with increases in net Na absorption and intracellular Ca2+ concentration ([Ca2+]i). The present study examined whether the absence of flow-stimulated K secretion early in life is due to a 1) limited flow-induced rise in net Na absorption and/or [Ca2+]i and/or 2) paucity of apical maxi-K channels. An approximately sixfold increase in tubular fluid flow rate in CCDs isolated from 4-wk-old rabbits and microperfused in vitro led to an increase in net Na absorption and [Ca2+]i, similar in magnitude to the response observed in 6-wk-old tubules, but it failed to generate an increase in net K secretion. By 5 wk of age, there was a small, but significant, flow-stimulated rise in net K secretion that increased further by 6 wk of life. Luminal perfusion with iberiotoxin blocked the flow stimulation of net K secretion in the adult CCD, confirming the identity of the maxi-K channel in this response. Maxi-K channel α-subunit message was consistently detected in single CCDs from animals ≥4 wk of age by RT-PCR. Indirect immunofluorescence microscopy using antibodies directed against the α-subunit revealed apical labeling of intercalated cells in cryosections from animals ≥5 wk of age; principal cell labeling was generally intracellular and punctate. We speculate that the postnatal appearance of flow-dependent K secretion is determined by the transcriptional/translational regulation of expression of maxi-K channels. Furthermore, our studies suggest a novel function for intercalated cells in mediating flow-stimulated K secretion.

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