[Activation of renal outer medullary potassium channel in the renal distal convoluted tubule by high potassium diet].

Renal outer medullary potassium (ROMK) channel is an important K+ excretion channel in the body, and K+ secreted by the ROMK channels is most or all source of urinary potassium. Previous studies focused on the ROMK channels of thick ascending limb (TAL) and collecting duct (CD), while there were few studies on the involvement of ROMK channels of the late distal convoluted tubule (DCT2) in K+ excretion. The purpose of the present study was mainly to record the ROMK channels current in renal DCT2 and observe the effect of high potassium diet on the ROMK channels by using single channel and whole-cell patch-clamp techniques. The results showed that a small conductance channel current with a conductance of 39 pS could be recorded in the apical membrane of renal DCT2, and it could be blocked by Tertiapin-Q (TPNQ), a ROMK channel inhibitor. The high potassium diet significantly increased the probability of ROMK channel current occurrence in the apical membrane of renal DCT2, and enhanced the activity of ROMK channel, compared to normal potassium diet (P < 0.01). Western blot results also demonstrated that the high potassium diet significantly up-regulated the protein expression levels of ROMK channels and epithelial sodium channel (ENaC), and down-regulated the protein expression level of Na+-Cl- cotransporter (NCC). Moreover, the high potassium diet significantly increased urinary potassium excretion. These results suggest that the high potassium diet may activate the ROMK channels in the apical membrane of renal DCT2 and increase the urinary potassium excretion by up-regulating the expression of renal ROMK channels.

[The mechanism of blood pressure regulation by high potassium diet in the kidney].

Hypertension is one of the strongest risk factors for cardiovascular diseases, cerebral stroke, and kidney failure. Lifestyle and nutrition are important factors that modulate blood pressure. Hypertension can be controlled by increasing physical activity, decreasing alcohol and sodium intake, and stopping tobacco smoking. Chronic kidney disease patients often have increased blood pressure, which indicates that kidney is one of the major organs responsible for blood pressure homeostasis. The decrease of renal sodium reabsorption and increase of diuresis induced by high potassium intake is critical for the blood pressure reduction. The beneficial effect of a high potassium diet on hypertension could be explained by decreased salt reabsorption by sodium-chloride cotransporter (NCC) in the distal convoluted tubule (DCT). In DCT cells, NCC activity is controlled by with-no-lysine kinases (WNKs) and its down-stream target kinases, Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress-responsive 1 (OSR1). The kinase activity of WNKs is inhibited by intracellular chloride ([Cl-]i) and WNK4 is known to be the major WNK positively regulating NCC. Based on our previous studies, high potassium intake reduces the basolateral potassium conductance, decreases the negativity of DCT basolateral membrane (depolarization), and increases [Cl-]i. High [Cl-]i inhibits WNK4-SPAK/OSR1 pathway, and thereby decreases NCC phosphorylation. In this review, we discuss the role of DCT in the blood pressure regulation by dietary potassium intake, which is the mechanism that has been best dissected so far.

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.

[The function and regulation of basolateral Kir4.1 and Kir4.1/Kir5.1 in renal tubules].

Basolateral inwardly-rectifying K+ channels (Kir) play an important role in the control of resting membrane potential and transepithelial voltage, thereby modulating water and electrolyte transport in the distal part of nephron. Kir4.1 and Kir4.1/Kir5.1 heterotetramer are abundantly expressed in the basolateral membrane of late thick ascending limb (TAL), distal convoluted tubule (DCT), connecting tubule (CNT) and cortical collecting duct (CCD). Loss-of-function mutations in KCNJ10 cause EAST/SeSAME syndrome in humans associated with epilepsy, ataxia, sensorineural deafness and water-electrolyte metabolism imbalance, which is characterized by salt wasting, hypomagnesaemia, hypokalaemia and metabolic alkalosis. In contrast, mice lacking Kir5.1 have severe renal phenotype apart from hypokalaemia such as high chlorine metabolic acidosis and hypercalcinuria. The genetic knockout or functional inhibition of Kir4.1 suppresses Na-Cl cotransporter (NCC) expression and activity in the DCT. However, the downregulation of Kir4.1 increases epithelial Na+ channel (ENaC) expression in the collecting duct. Recently, factors regulating expression and activity of Kir4.1 and Kir4.1/Kir5.1 were identified, such as cell acidification, dopamine, insulin and insulin-like growth factor-1. The involved mechanisms include PKC, PI3K, Src family protein tyrosine kinases and WNK-SPAK signal transduction pathways. Here we review the progress of renal tubule basolateral Kir, and mainly discuss the function and regulation of Kir4.1 and Kir4.1/Kir5.1.

CYP-omega-hydroxylation-dependent metabolites of arachidonic acid inhibit the basolateral 10 pS chloride channel in the rat thick ascending limb.

Metabolites of arachidonic acid influence sodium chloride (NaCl) transport in the thick ascending limb. Because a 10 pS Cl channel is the major type of chloride channel in the basolateral membrane of this nephron segment, we explored the effect of arachidonic acid on this channel in cell-attached patches. Addition of 5 micromol arachidonic acid significantly decreased channel activity (a product of channel number and open probability) while linoleic acid had no effect. To determine if this was mediated by acachidonic acid per se or by its metabolites, we measured channel activity in the presence of the cyclooxygenase inhibitor indomethacin, the selective lipoxygenase inhibitor nordihydroguaiaretic acid, and the cytochrome P-450 (CYP)-omega-hydroxylation inhibitor 17-octadecynoic acid. Neither cyclooxygenase nor lipoxygenase inhibition had an effect on basal chloride channel activity; further they failed to abolish the inhibitory effect of arachidonate on the 10 pS channel. However, inhibition of CYP-omega-hydroxylation completely abolished the effect of arachidonic acid. The similarity of the effects of 20-hydroxyeicosatetraenoic acid (20-HETE) and arachidonic acid suggests that the effect of arachidonic acid was mediated by CYP-omega-hydroxylation-dependent metabolites. We conclude that arachidonic acid inhibits the 10 pS chloride channel in the basolateral membrane of the medullary thick ascending limb, an effect mediated by the CYP-omega-hydroxylation-dependent metabolite 20-HETE.

Mechanisms underlying low [Ca(2+)](o)-induced increased excitability of hippocampal neurons.

OBJECTIVE: Concentration of extracellular calcium ([Ca(2+)](o)) in the central nervous system decreases substantially in different conditions. It results in facilitating neuronal excitability. The goal of this study is to examine the mechanisms of enhanced neuronal excitation in low [Ca(2+)](o) in order to provide new clues to treat the hyperexcitability diseases in clinic. METHODS: Whole-cell patch-clamp technique and neuron culture were used in the study. RESULTS: The firing threshold of cultured hippocampal neurons decreased markedly in low [Ca(2+)](o) saline. Unexpectedly, apamine and isoprenaline, antagonists of medium afterhyperpolarization (mAHP) and slow AHP (sAHP) respectively, had no statistic significant effect on excitability of neurons. TTX at a low concentration was sufficient to inhibit I(NaP), which blocked the increase of firing frequency in low [Ca(2+)](o). It also reduced the number of spikes in normal [Ca(2+)](o). CONCLUSION: These results suggest that in cultured hippocampal neurons, modulation of spiking threshold but not AHP may cause the increased excitability in low [Ca(2+)](o).

Chlorzoxazone inhibits contraction of rat thoracic aorta.

Chlorzoxazone has been reported to activate the intermediate-conductance, Ca(2+)-activated K(+) channels in aortic endothelial cells and to relax the artery. The aim of the present study was to characterize the chlorzoxazone-induced relaxation of rat thoracic artery. Chlorzoxazone did not affect the tension of the thoracic artery rings at rest, but relaxed the precontraction induced by 1 muM noradrenaline in an endothelium independent manner. Preincubation with chlorzoxazone also antagonized the contraction induced by 1 microM noradrenaline or 25 mM KCl. The chlorzoxazone-induced relaxation of the thoracic artery pre-contracted by noradrenaline was suppressed by 5 mM tetraethylammonium, 75 mM ethanol and 2 microM paxilline, but not by 2 microM clotrimazole. Chlorzoxazone relaxed the 4-aminopyridine-induced contraction. The pattern of chlorzoxazone-induced relaxation was different from that of verapamil, the L-type Ca(2+) channel blocker. The inhibition of the noradrenaline-induced contraction by chlorzoxazone was attenuated when chlorzoxazone treatment was prolonged to 4 h. No difference in the contraction-relaxation was found between the artery rings from normal rats and those from rats that received 100 mg/kg chlorzoxazone for 7 days. We conclude that chlorzoxazone abolishes the contraction of rat thoracic artery induced by noradrenaline and that the effect of chlorzoxazone is endothelium independent and also not mediated by intermediate-conductance, Ca(2+)-activated K(+) channels.

Indapamide induces apoptosis of GH3 pituitary cells independently of its inhibition of voltage-dependent K+ currents.

Indapamide blocks multiple voltage-dependent K+ currents (Kv) in the heart and Kv have an important role in cell proliferation and apoptosis, so the aim of this work was to study the effects of indapamide on Kv and the viability of GH3 cells. Indapamide inhibited Kv of GH3 cells and the inhibition was irreversible after a 10-min washout when more than 250 microM indapamide was used. Indapamide reduced the viability of GH3 cells in a concentration-dependent manner. The decreased cell viability was because indapamide induced cell apoptosis, or even necrosis at higher concentrations. HepG2 cells, which express no apparent Kv, were used to determine the association between inhibition of Kv and the apoptotic action of indapamide. Indapamide had a similar action on cell viability and apoptosis of HepG2 cells. 4-Aminopyridine, the voltage-dependent K+ channel blocker, inhibited Kv of GH3 cells but did not induce the cell apoptosis. We concluded that while indapamide inhibited Kv and induced apoptosis of GH3 cells, the apoptotic action of indapamide was not associated with its inhibition of Kv.

Arachidonic acid inhibits K channels in basolateral membrane of the thick ascending limb.

We have used the patch-clamp technique to study the effect of arachidonic acid (AA) on the basolateral K channels in the medullary thick ascending limb (mTAL) of rat kidney. An inwardly rectifying 50-pS K channel was identified in cell-attached and inside-out patches in the basolateral membrane of the mTAL. The channel open probability (P(o)) was 0.51 at the spontaneous cell membrane potential and decreased to 0.25 by 30 mV hyperpolarization. The addition of 5 microM AA decreased channel activity, identified as NP(o), from 0.58 to 0.08 in cell-attached patches. The effect of AA on the 50-pS K channel was specific because 10 microM cis-11,14,17-eicosatrienoic acid had no significant effect on channel activity. To determine whether the effect of AA was mediated by AA per se or by its metabolites, we examined the effect of AA on channel activity in the presence of indomethacin, an inhibitor of cyclooxygenase, or N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), an inhibitor of cytochrome P-450 monooxygenase. Inhibition of cyclooxygenase increased channel activity from 0.54 to 0.9. However, indomethacin did not abolish the inhibitory effect of AA on the 50-pS K channel. In contrast, inhibition of cytochrome P-450 metabolism not only increased channel activity from 0.49 to 0.83 but also completely abolished the effect of AA. Moreover, addition of DDMS can reverse the inhibitory effect of AA on channel activity. The notion that the effect of AA was mediated by cytochrome P-450-dependent metabolites of AA is also supported by the observation that addition of 100 nM of 20-hydroxyeicosatetraenoic acid, a main metabolite of AA in the mTAL, can mimic the effect of AA. We conclude that AA inhibits the 50-pS K channel in the basolateral membrane of the mTAL and that the effect of AA is mainly mediated by cytochrome P-450-dependent metabolites of AA.

K depletion enhances the extracellular Ca2+-induced inhibition of the apical K channels in the mTAL of rat kidney.

We have shown previously that raising extracellular Ca(2)+ inhibited the apical 70-pS K channel in the thick ascending limb (TAL; Wang, W.H., M. Lu, and S.C. Hebert. 1996. Am. J. Physiol. 270:C103-C111). We now used the patch-clamp technique to study the effect of increasing the extracellular Ca(2)+ on the 70-pS K channel in the mTAL from rats on a different K diet. Increasing the extracellular Ca(2)+ from 10 microM to 0.5, 1, and to 1.5 mM in the mTAL from rats on a K-deficient (KD) diet inhibited the channel activity by 30, 65, and 90%, respectively. In contrast, raising the extracellular Ca(2)+ to 1.5 mM had no significant effect on channel activity in the mTAL from animals on a high K (HK) diet and further increasing the extracellular Ca(2)+ to 2.5, 3.5, and 5.5 mM decreased the channel activity by 29, 55, and 90%, respectively. Inhibition of the cytochrome P450 monooxygenase completely abolished the effect of the extracellular Ca(2)+ on channel activity in the mTAL from rats on a different K diet. In contrast, blocking cyclooxygenase did not significantly alter the responsiveness of the 70-pS K channel to the extracellular Ca(2)+. Moreover, addition of sodium nitropruside, a nitric oxide (NO) donor, not only increased the channel activity, but also blunted the inhibitory effect of the extracellular Ca(2)+ on the 70-pS K channel and decreased 20-hydroxyeicosatetraenoic acid (20-HETE) concentration in the mTAL from rats on a KD diet. In contrast, inhibiting NOS with L-NAME enhanced the inhibitory effect of the extracellular Ca(2)+ on the channel activity and increased 20-HETE concentration in the mTAL from rats on a high K diet. Western blot has further shown that the expression of inducible NO synthase (iNOS) is significantly higher in the renal medulla from rats on an HK diet than that on a KD diet. Also, addition of S-nitroso-N-acetylpenicillamine abolished the inhibitory effect of arachidonic acid on channel activity in the mTAL, whereas it did not block the inhibitory effect of 20-HETE. We conclude that a low dietary K intake increases the sensitivity of the 70-pS K channel to the extracellular Ca(2)+, and that a decrease in NOS activity is involved in enhancing the inhibitory effect of the extracellular Ca(2)+ on channel activity in the mTAL during K depletion.

Inhibition of protein-tyrosine phosphatase stimulates the dynamin-dependent endocytosis of ROMK1.

We have previously shown that inhibiting protein-tyrosine kinase increased whereas inhibiting protein-tyrosine phosphatase (PTP) decreased renal outer medullary potassium channel 1 (ROMK1) channel activity (1). We have now used confocal microscopy, the patch clamp technique, and biotin labeling to further examine the role of tyrosine phosphorylation in regulating ROMK1 trafficking. Human embryonic kidney 293 cells were cotransfected with c-Src and green fluorescent protein-ROMK1, which has the same biophysical properties as those of ROMK1. Patch clamp studies have shown that phenylarsine oxide (PAO), an inhibitor of PTP, decreased the activity of ROMK1. Moreover, addition of PAO reduced the cell surface localization of green fluorescent protein-ROMK1 detected by confocal microscopy and diminished the surface ROMK1 density by 65% measured by biotin labeling. Also, PAO treatment significantly increased the phosphorylation of ROMK1. The notion that the effect of PAO is mediated by stimulating tyrosine phosphorylation-induced endocytosis of ROMK1 has also been supported by findings that mutating the tyrosine residue 337 of ROMK1 to alanine abolished the effect of PAO. Finally, the inhibitory effect of PAO on ROMK1 was completely blocked in the cells co-transfected with dominant negative dynamin (dynaminK44A). This indicates that the tyrosine phosphorylation-induced endocytosis of ROMK1 is dynamin-dependent. We conclude that inhibiting PTP increases ROMK1 phosphorylation and results in a dynamin-dependent internalization of the channel.