Role of gp91phox-Containing NADPH Oxidase in Mediating the Effect of K Restriction on ROMK Channels and Renal K Excretion
Elisa Babilonia,
Daohong Lin,
Yan Zhang,
Yuan Wei,
Peng Yue and
Wen-Hui Wang
Department of Pharmacology, New York Medical College, Valhalla, New York
Correspondence: Dr. Wen-Hui Wang, Department of Pharmacology, New York Medical College, Valhalla, NY 10595. Phone: 914-594-4139; Fax: 914-347-4956; E-mail: wenhui_wang{at}nymc.edu
Received for publication December 8, 2006.
Accepted for publication April 1, 2007.
Previous study has demonstrated that superoxide and the relatedproducts are involved in mediating the effect of low K intakeon renal K secretion and ROMK channel activity in the corticalcollecting duct (CCD). This study investigated the role of gp91phox-containingNADPH oxidase (NOXII) in mediating the effect of low K intakeon renal K excretion and ROMK channel activity in gp91(–/–)mice. K depletion increased superoxide levels, phosphorylationof c-Jun, expression of c-Src, and tyrosine phosphorylationof ROMK in renal cortex and outer medulla in wild-type (WT)mice. In contrast, tempol treatment in WT mice abolished whereasdeletion of gp91 significantly attenuated the effect of lowK intake on superoxide production, c-Jun phosphorylation, c-Srcexpression, and tyrosine phosphorylation of ROMK. Patch-clampexperiments demonstrated that low K intake decreased mean productof channel number (N) and open probability (P) (NPo) of ROMKchannels from 1.1 to 0.4 in the CCD. However, the effect oflow K intake on ROMK channel activity was significantly attenuatedin the CCD from gp91(–/–) mice and completely abolishedby tempol treatment. Immunocytochemical staining also was usedto examine the ROMK distribution in WT, gp91(–/–),and WT mice with tempol treatment in response to K restriction.K restriction decreased apical staining of ROMK in WT mice.In contrast, a sharp apical ROMK staining was observed in thetempol-treated WT or gp91(–/–) mice. Metabolic cagestudy further showed that urinary K loss is significantly higherin gp91(–/–) mice than in WT mice. It is concludedthat superoxide anions play a key role in suppressing K secretionduring K restriction and that NOXII is involved in mediatingthe effect of low K intake on renal K secretion and ROMK channelactivity.
Maintaining plasma K in a normal range is essential for thefunctions of a variety of cells, including neuron, cardiac myocytes,and skeletal muscle.1 The kidney plays a key role in maintainingK homeostasis2,3: A high K enhances whereas a low K intake suppressesrenal K secretion. We previously reported that low K intakeincreased the superoxide levels in the renal cortex and outermedulla.4 Superoxide or related products have been shown toactivate mitogen-activated protein kinase (MAPK) such as P38and extracellular signal–regulated kinase (ERK),5 stimulateprotein tyrosine kinase (PTK) activity,6 and augment the expressionof Src family PTK in the kidney.7 We have also demonstratedthat P38 and ERK MAPK and PTK inhibited ROMK channels in thecortical collecting duct (CCD).7,8 The role of superoxide inmediating the effect of K-depletion on ROMK channels and renalK secretion is best indicated by experiments in which decreasingsuperoxide levels with tempol treatment increased ROMK channelactivity and renal K loss.4
The superoxide anions are generated by the univalent reductionof triple-state molecular oxygen mediated by NADPH and xanthineoxidase.5 However, it is generally believed that superoxideanions that are generated by NADPH oxidase are a major sourceof reactive oxygen species in different types of cells.9,10A large body of evidence indicates that gp91phox-containingNADPH oxidase (NOXII) and NOXIV are expressed in the kidneyand that NOXII is expressed in the connecting tubule (CT) andCCD.11,12 It is possible that NOXII could be activated by lowK intake and partially responsible for increased productionof superoxide anions that is induced by K restriction. Thus,inactivation of NOXII should decrease the superoxide productionand impair the renal ability to preserve K during K restriction.In this study, we used gp91phox null mice to test the hypothesisthat superoxide anions that are generated by NOXII are involvedin mediating the inhibitory effect of low K intake on renalK secretion.
We previously demonstrated that K restriction increased superoxideproduction and that the low K intake–induced increasesin superoxide were abolished by tempol treatment in the ratkidney.4 In this study, we first examined the effect of K restrictionon superoxide production in wild-type (WT) mice to determinewhether the response of mouse kidney to low K intake was thesame as that of rat kidney. Data summarized in Figure 1 demonstratethat low K intake significantly increased superoxide productionin renal cortex and outer medulla by 100 ± 10% (n = 5;P < 0.01) in comparison with those on a normal-K diet. Also,tempol treatment significantly reduced production of superoxidemediated by low K intake by 90 ± 7% (n = 5) but had nosignificant effect on superoxide production in mice that werefed a normal-K diet. Thus, the response of mouse kidney to lowK intake was the same as that observed in rats. In contrast,the effect of low K intake on superoxide production was significantlyattenuated in kidneys from gp91phox (–/–) mice becausea decrease in dietary K content increased superoxide formationonly by 46 ± 5% (n = 5; P < 0.05). We could not measurethe superoxide production in tempol-treated gp91phox (–/–)mice that were on a K-deficient (KD) diet because they weredead within 5 d after tempol injection plus low K diet (n =8 mice). This indicates that NOXII is an important source forgenerating superoxide induced by K restriction.
Figure 1. Superoxide levels in the renal cortex and outer medulla from wild-type (WT) mice on a normal-K diet (1.1%), normal K + tempol treatment, K-deficient (KD) diet, and KD plus tempol treatment (left set of bars) and in gp91phox (–/–) mice on a control and/or KD diet (right set of bars). #The superoxide level in WT mice that were on a KD diet is significantly higher than that in every other group (P < 0.05). *Significant difference between control [gp91phox (–/–) mice on 1.1% K] and experimental group [gp91phox (–/–) mice on KD diet; P < 0.05].
Superoxide anions and related products have been demonstratedto mediate the effect of low K intake on Src family PTK viastimulation of transcription and translation.4 Thus, diminishedsuperoxide levels are expected to decrease the expression ofSrc family PTK. We examined the effect of low K intake on c-Junphosphorylation, an index for activation of the transcriptionfactor. As observed in rats, low K intake significantly increasedphosphorylation of c-Jun on serine 73 by 110 ± 5% inWT mice (n = 3; P < 0.05; Figure 2). Tempol treatment abolishedthe effect of low K intake on c-Jun phosphorylation and hadno significant effect on c-Jun phosphorylation in mice thatwere on a normal-K diet. However, low K intake did not significantlyincrease the phosphorylation of c-Jun in gp91phox (–/–)mice (15 ± 4%; n = 4). We next examined the effect oflow K intake on expression of c-Src, which serves as a representativemember of Src family PTK in WT and gp91phox (–/–)mice. Figure 3 is a Western blot showing that low K intake increasedthe expression of c-Src in WT mice by 115 ± 17% (n =5; P < 0.01) in comparison with those that were on controlK diet. Similar to that in rats, tempol treatment abolishedthe stimulatory effect of low K intake on c-Src expression (n= 5) and had no significant effect on c-Src expression in micethat were on a normal-K diet. Also, the effect of low K intakeon c-Src expression was significantly smaller in gp91phox (–/–)mice than in WT mice because K restriction increased the c-Srcexpression only by 55 ± 6% (n = 5; P < 0.05).
Figure 2. (A) A Western blot demonstrating the effect of low K intake on cJun phosphorylation (top) and total c-Jun (bottom) in the renal cortex and outer medulla of WT mice on a normal-K diet, KD diet, and KD diet plus tempol treatment and gp91phox (–/–) mice that were fed a normal-K and/or a KD diet. (B) A Western blot showing the cJun phosphorylation in renal cortex and outer medulla in tempol-treated and non–tempol-treated WT mice that were fed a normal-K diet. (C) Bar graphs summarize the effect of K intake on cJun phosphorylation under different experimental conditions. #Phosphorylation of cJun in wt mice on KD diet is significantly higher than that in every other group (P < 0.05).
Figure 3. (A) A Western blot demonstrating the effect of low K intake on c-Src expression in the renal cortex and outer medulla of WT mice on a normal-K diet, KD diet, and KD diet plus tempol treatment and gp91phox (–/–) mice that were fed a normal-K and/or KD diet. (B) A Western blot showing the c-Src expression in renal cortex and outer medulla in WT mice on normal-K diet with or without tempol treatment. (C) Bar graph demonstrating the effect of dietary K intake on c-Src expression under different experimental conditions. #cSrc level in WT mice on KD diet is significantly higher than that in every other group (P < 0.05). *Significant difference between control [gp91phox (–/–] mice on 1.1% K) and experimental group [gp91phox (–/–) mice on KD diet; P < 0.05].
After demonstrating that deletion of NOXII diminished the effectof low K intake on c-Src expression, we examined the effectof low K intake on tyrosine phosphorylation of ROMK. Similarto rat kidney,8,13 low K intake stimulates tyrosine phosphorylationof ROMK in mouse kidney. Figure 4 is a Western blot showingthat K depletion increased the tyrosine phosphorylation of ROMKby 180 ± 10% (n = 6; P < 0.01) in WT mice. The lowK intake–induced increase in tyrosine phosphorylationwas almost completely abolished by tempol. Also, tempol treatmentdid not significantly affect the phosphorylation of ROMK inmice that were on a normal-K diet. Thus, superoxide anions playa key role in mediating the effect of K restriction on tyrosinephosphorylation of ROMK in mouse kidney. This view is also supportedby the observation that the K restriction–induced stimulationof tyrosine phosphorylation was significantly attenuated ingp91phox (–/–) mice. Low K intake increased thetyrosine phosphorylation of ROMK only by 75 ± 6% (n =6; P < 0.05) in comparison with gp91phox (–/–)mice that were fed a normal-K diet.
Figure 4. (A) Western blot showing the effect of low K intake on tyrosine phosphorylation of ROMK (top) and total ROMK expression (bottom) in the renal cortex and outer medulla in WT mice on a normal-K diet, KD diet, and KD diet plus tempol treatment and gp91phox (–/–) mice that were fed a normal-K and/or KD diet. (B) Western blot showing the tyrosine phosphorylation of ROMK in renal cortex and outer medulla in WT mice on normal-K diet with or without tempol treatment. (C) Bar graph showing the effect of K restriction on the tyrosine phosphorylation of ROMK. #Tyrosine phosphorylation of ROMK in WT mice on KD diet is significantly higher than that in every other group (P < 0.05). *Significant difference between control [gp91phox (–/–) mice on 1.1% K] and experimental group [gp91phox (–/–) mice on KD diet; P < 0.05].
Because increased tyrosine phosphorylation of ROMK has beenshown to enhance the internalization of the K channels,8 inhibitionof tyrosine phosphorylation of ROMK was expected to diminishthe low K intake–induced internalization of ROMK in tempol-treatedWT or in gp91phox (–/–) mice. We examined the ROMKstaining in the kidney of mice that were kept on a normal-Kor KD diet (Figure 5). A sharp apical/subapical staining ofROMK was observed in the kidney of WT mice (Figure 5, A throughC) and gp91phox (–/–) mice (Figure 5D) that werefed a normal-K diet. The intensity of apical/subapical stainingof ROMK was apparently diminished in both outer medulla (Figure 5A)and cortex (Figure 5B) of WT mice that were kept on a KD dietfor 7 d. In contrast, a relative sharp apical/subapical stainingof ROMK was still observed in tempol-treated mice that wereon a KD diet for 7 d and also in gp91phox (–/–)mice that were fed a KD diet (Figure 5D). For this set of experiments,we used three mice for each group and repeated the stainingfor each mouse at least twice. We observed a similar stainingpattern in every experiment (at least three sections per mouse,and three mice were used). Also, we conducted experiments inrats and found that tempol treatment increased the apical/subapicalROMK staining in rats that were fed a KD diet in comparisonwith those without tempol treatment (W.-H.W., unpublished observations).
Figure 5. Confocal images demonstrate the effect of dietary K intake on ROMK staining in wt mice (A through C) and gp91phox (–/–) mice (D). Double staining of aquaporin 2 (red) and ROMK (green) in outer medulla (A) or cortex (B) of tempol (T)-treated or non–tempol-treated mice on normal-K (NK, 1.1%) and KD diets, respectively. (C) ROMK staining in outer medulla of WT mice on NK or KD diet. (D) ROMK staining in outer medulla of gp91phox (–/–) mice on NK or KD diet.
We used the patch-clamp technique to examine the ROMK-like SKchannels in the CCD of WT mice on a control K diet, a KD diet,and a KD diet plus tempol. Data summarized in Figure 6 showthat K restriction decreased product of channel number (N) andopen probability (P) (NPo) by 64% from 1.1 ± 0.3 to 0.4± 0.1 (n = 10; P < 0.05) in the CCD of WT mice. Tempoltreatment significantly increased channel activity (NPo = 0.9± 0.2; n = 10) in the CCD of mice that were on a KD diet.The channel activity in the CCD of gp91phox (–/–)mice that were on normal-K diet was not significantly differentfrom that of WT mice (NPo = 1.1 ± 0.3; n = 10). However,K depletion modestly decreased NPo by 37% to 0.7 ± 0.2(n = 6; P < 0.05). Therefore, low K intake–inducedsuppression of SK channels in the CCD was compromised in gp91phox(–/–) mice.
Figure 6. The SK channel activity in the cortical collecting duct of WT mice on a normal-K diet, normal-K diet with tempol treatment, KD diet, and KD diet plus tempol treatment (left set of bars) and gp91phox (–/–) mice that were fed a normal-K and/or KD diet (right set of bars). The experiments were performed in cell-attached patches. Significance between different groups is indicated.
Because ROMK-like SK channels are responsible for K secretion,changes in the channel activity should influence renal K excretion.Therefore, we used the metabolic cage to study renal K excretionin mice that were on control K diet, on a KD diet, or on a KDdiet plus tempol for 7 d. The mean food intake (per mouse perday) was 3.2 g for gp91phox (–/–) mice that wereon a normal-K diet, 3.04 g for gp91phox (–/–) micethat were on a KD diet, 2.6 g for WT mice that were on a normal-Kdiet, 3.5 g for WT mice that were on a KD diet, and 3.4 g fortempol-treated mice that were on a KD diet. Results summarizedin Figure 7, A and B, show renal K excretion of mice that wereon normal-K and KD diets, respectively. Low K intake decreased24-h renal excretion from 0.48 ± 0.08 to 0.02 ±0.01 mEq in WT mice (n = 4; P < 0.001). However, renal Kexcretion in tempol-treated mice that were on a KD diet wassignificantly higher (0.10 ± 0.02 mEq; n = 4; P <0.01) in comparison with that without tempol treatment. Althoughrenal K excretion in gp91phox (–/–) mice that wereon a normal-K diet (0.51 ± 0.04 mEq) was not significantlydifferent from that of WT mice, K depletion caused less inhibitionof renal K excretion (0.05 ± 0.01 mEq) in gp91phox (–/–)mice (Figure 7B). We also measured plasma K concentration inWT and gp91phox (–/–) mice (Figure 7C). Low K intakedecreased plasma K concentration by 30% from 3.9 ± 0.4(control K diet) to 2.7 ± 0.1 mM and had further fallento 2.3 ± 0.1 mM in tempol-treated mice that were on aKD diet (Figure 7C). In gp91phox (–/–) mice, K restrictiondecreased plasma K concentration by approximately 40% from 4.2± 0.3 (control K diet) to 2.6 ± 0.01 mM (Figure 7D).Falls in plasma K concentrations were slightly higher in gp91phox(–/–) mice than in WT mice.
Figure 7. (A) Twenty-four-hour urinary K excretion in WT mice on normal-K diet (1.1%), WT mice on KD diet, tempol-treated WT mice on KD diet, gp91phox (–/–) mice on KD diet, and gp91phox (–/–) mice on normal-K diet. (B) Plasma K concentrations in WT mice on a normal-K diet, KD diet, or KD diet plus tempol for 7 d (left set of bars) and in gp91phox (–/–) mice on a normal-K or KD diet (right set of bars). *Significant difference from the corresponding control (normal K) and experimental groups.
A large body of evidence has indicated that superoxide anionsand the related products not only are involved in the regulationof immunoresponse but also play an important role in mediatinga variety of cell functions.5 Superoxide anions have been demonstratedto modulate the activity of various protein kinases and phosphatases.14–19Hydrogen peroxide has been shown to inhibit protein tyrosinephosphatase17,20,21 and activate several members of Src familyPTK, such as Lck and Fyn.18,19 Stimulation of insulin receptorshas been shown to augment the formation of superoxide,22 andlow concentrations of hydrogen peroxide can potentiate the insulineffect in insulin-responsive tissues.23 Furthermore, high concentrationsof hydrogen peroxide can induce insulin-like effects via stimulationof the tyrosine phosphorylation of the insulin receptor.24 Thisstudy has added another piece of evidence demonstrating therole of superoxide anions in the regulation of renal K excretion.
Renal K excretion is determined by both K secretion in the CTand the CCD and K absorption in the outer medullary collectingduct.3,25 For K secretion, K enters the cell across the basolateralmembrane via Na-K-ATPase and is secreted into the lumen throughapical K channels. It is generally accepted that ROMK channelsare mainly responsible for K secretion under normal K intake.We and others have shown that the K restriction decreased ROMKchannel activity and caused the internalization of ROMK channels.13,26,27Although the molecular mechanism by which low K suppresses ROMKchannel activity and renal K secretion is not completely understood,our previous study suggests that superoxide anions and relatedproducts are signaling molecules that mediate the effect oflow K intake on ROMK channels and renal K secretion4,7,28 Furthermore,we show that P38 and ERK MAPK and PTK are involved in mediatingthe effect of superoxide anions on ROMK channels.4,7,28 Therole of P38 and ERK MAPK in mediating the effect of low K intakeand superoxide anions on ROMK channels is supported by severallines of evidence: (1) Application of hydrogen peroxide stimulatedthe phosphorylation of ERK and P38 MAPK in M-1 cells; (2) inhibitionof superoxide anion production with tempol abolished the stimulatoryeffect of low K intake on the phosphorylation of P38 and ERK;and (3) suppression of P38 and ERK MAPK increased ROMK channelactivity and diminished the inhibition of ROMK channels inducedby hydrogen peroxide in the CCD.28 The evidence to support therole of PTK in mediating the effect of low K intake and superoxideanions on ROMK channel activity26 includes the following: (1)Low K intake significantly increased the expression of Src familyPTK and suppression of superoxide anions abolished the effectof low K intake on PTK expression,4 (2) increased PTK activityenhanced the tyrosine phosphorylation of ROMK channels29 andenhanced the internalization of ROMK channels,8 and (3) inhibitionof PTK attenuated the hydrogen peroxide–induced inhibitionof ROMK channels in the CCD.28 Because low K–induced increasesin PTK expression occurs later than that of the phosphorylationof P38 and ERK MAPK, it is possible that the inhibitory effectof low K intake on ROMK channels and renal K secretion is firstachieved by activation of MAPK.7 However, it is safe to concludethat superoxide anions mediate the effect of low K intake onROMK channels and renal K secretion through MAPK and PTK.
Another important finding of this study is that NOXII participatesin generating superoxide anions that are induced by low K intake.Although NOXII is the most abundant in macrophages among othertypes of cells, several studies demonstrated that NADPH oxidasewas also expressed in the kidney.11,12 It has been reportedthat mRNA for all five components of the phagocyte-type NADPHoxidase in the rat kidney and that immunocytochemical studieshave further confirmed the expression of p22phox, p47phox, andp67phox in the luminal membranes of macula densa, distal convolutedtubule, and the CCD. However, two lines of evidence indicatethat oxidase other than NOXII is also involved in mediatingthe effect of low K intake on renal K secretion and ROMK channelactivity: (1) The tempol treatment–induced K loss wasmore severe in WT mice that were on a KD diet than in gp91 (–/–)mice that were on a KD diet; and (2) low K intake still increasedsuperoxide levels in gp91(–/–) mice, whereas tempoltreatment completely abolished the effect of K restriction onsuperoxide production. It is known that superoxide anions arealso generated through activation of cytochrome P450 oxidase,cyclooxygenase, and xanthine oxidase,30 which are expressedin the renal tissue. Moreover, a renal type of NOXII homolog,NOXIV, has been shown to be expressed in the renal tubules.12We need further experiments to examine the role of other oxidasessuch as NOXIV in mediating the effect of low K intake on renalK secretion and ROMK channel activity.
The mechanism by which low K intake stimulates NADPH oxidaseactivity is not known. Several hormones such as growth factorsand angiotensin II have been shown to stimulate NADPH oxidaseand increase superoxide formation.31,32 It has been shown thatdietary K deficiency in rabbits stimulates mRNA expression andprotein expression of angiotensin II receptors in the renalcortex and in the proximal tubule.33 Stimulation of angiotensinII receptors has been shown to activate NOXII by a protein kinaseC–dependent pathway.34 In this regard, we previously demonstratedthat low K intake stimulates the expression of protein kinaseC.35
Although this study is mainly focused on the role of superoxideanions in the regulation of ROMK channels, the finding thatBK channels are inhibited by P38 and ERK MAPK, which are activatedby superoxide anions, suggests that superoxide anions couldalso inhibit BK channels.36 Several studies have convincinglydemonstrated that BK channels are also involved in K secretionduring high K intake and high tubule flow rate.37–39 Inaddition, BK channels are present also in intercalated cells(IC) and could play a role in K recycling across the apicalmembrane of IC. Because K recycling determines the efficiencyof K absorption via K-H-ATPase,40 superoxide anion may alsobe involved in the regulation of K absorption in the outer medullarycollecting duct.
Superoxide anions and related products play a role in mediatingthe effect of low K intake on renal K secretion through a MAPK-and a PTK-dependent pathway, and NOXII is an important sourceof superoxide generated during K restriction.
Animals
Male gp91phox (–/–) and C57BL/6 (WT) mice (6 wkold) were purchased from Jackson Laboratory (Bar Harbor, ME).C57BL/6 have the same genotype, and they were age-matched withgp91phox (–/–). Three days after they were received,the mice were housed in metabolic cages (two mice per cage)for 3-d adaptation. Mice (WT) were divided into three groups:(1) Control group in which animals were kept on a normal-K (1.1%)diet and had a daily intraperitoneal injection of saline for1 wk, (2) low K intake group in which mice were maintained ona KD diet and received a daily intraperitoneal injection ofsaline for 7 d, and (3) the tempol-treated group in which micewere also fed a KD diet and had a daily intraperitoneal injectionof tempol (15 mg/kg) for 1 wk. The gp91phox (–/–)mice were divided into two groups: Control and low K intake.Data regarding 24-h food intake, body weight, and urine outputwere recorded. After metabolic cage study, the mice were anesthetizedwith pentobarbital (60 mg/kg), blood was drawn from the heart,and the kidneys were removed for tissue preparation. Plasmaand urinary K and Na concentrations were measured using flamephotometry, and daily Na and K excretion was expressed as mEq/24h.
Measurement of Superoxide Anion
We followed the method described previously for measurementof superoxide.41 The tissue from mouse cortex and outer medulla(100 mg) was cut into a small piece with a sharp blade and suspendedin air-equilibrated MOPS-sucrose buffer (pH 7.4) that contained5 µM lucigenin. Although lucigen has been shown to altersuperoxide level in cells, the concentration of lucigen thatwas used in this study (5 µM) has been shown to have nosignificant effect on cell superoxide production.42 The chemiluminescencethat was elicited in the presence of lucigenin was measuredin a liquid scintillation counter with a single active photomultipliertube positioned in out-of-coincidence mode. Blanks were subtractedfrom the average level of the chemiluminescence signal.
Immunoprecipitation and Western Blot
The corresponding antibody was added to the protein samples(500 µg) that were harvested from kidneys at a ratio of5 µl/ml (1:1000) of solution, and IgG antibody was usedas negative control for immunoprecipitation experiment. Themixture was gently rotated at 4°C overnight, followed byincubation with 25 µl of protein A/G agarose (Santa CruzBiotechnology, Santa Cruz, CA) for an additional 2 h at 4°C.The tube that contained the mixture was centrifuged at 3000rpm and washed twice with PBS that contained 10 µl/mlPMSF and 10 µl/ml protease inhibitor cocktail. The agarosepellet was resuspended in 25 µl of 2x SDS sample bufferthat containing 4% SDS, 100 mM Tris-HCl (pH 6.8), 20% glycerol,200 mM dithiothreitol, and 0.2% bromophenol blue. After thesamples were boiled for 5 min, the proteins were separated byelectrophoresis on 10% SDS-polyacrylamide gels and transferredto Immuno-Blot polyvinylidene membrane (Bio-Rad, Hercules, CA).The membrane was blocked with 5% nonfat dry milk in Tris-bufferedsaline and incubated overnight with the primary antibody at4°C, then washed three times for 15 min with Tris-bufferedsaline that contained 0.05% Tween 20, followed by incubationfor 30 min with respective second antibody horseradish peroxidaseconjugate. ECL plus (Amersham Pharmacia Biotech, Piscataway,NJ) was used to detect the protein bands, and the intensityof the bands of interest was determined using Alpha DigiDoc1000 (Alpha Innotech, San Leandro, CA).
Immunostaining for ROMK
Kidneys were perfused in situ with PBS plus heparin (40 U/ml)and fixed with 4% paraformaldehyde. Five-micrometer slices werecut from paraffin-embedded tissue. After performing heat-inducedantigen retrieval, the tissue samples were blocked with 2% goatserum in 1x PBS for 1 h at room temperature. Then, the sliceswere incubated overnight with primary antibody (anti-ROMK antibody;Alamone, Jerusalem, Israel) diluted 1:50 in the blocking solution(4°C). The sections were washed in PBS and incubated withthe second antibody, 488 goat/rabbit (1:200), in PBS for 2 h.The slides were washed with PBS for 30 min. Kidney slices wereplaced in the same slide and treated with identical procedure.Images were acquired at the same gain and contrast.
Preparation of CCD for Patch Clamping
Mouse CCD was isolated and placed in a chamber (1000 µl)mounted on an inverted Nikon microscope. The CCD were superfusedwith HEPES buffered NaCl solution that contained (in mM) 140NaCl, 5 KCl, 1.8 CaCl2, 1.8 MgCl2, and 5 HEPES (pH 7.4). Thepipette solution was composed of (in mM) 140 KCl, 1.8 MgCl2,and 5 HEPES (pH 7.4). The temperature of the chamber was maintainedat 37 ± 1°C by circulating warm water around thechamber. The CCD was cut open with a sharpened micropipetteto expose the apical membrane.
Patch-Clamp Technique
An Axon200A patch-clamp amplifier was used to record channelcurrent. The current was low-pass filtered at 1 KHz by an eight-poleBessel filter (902LPF; Frequency Devices, Haverhill, MA) anddigitized with Axon interface (Digidata 1200, Molecular Devices,Sunnyvale, CA). Data were analyzed using the pClamp softwaresystem 6.04 (Axon Instruments, Burlingame, CA). Channel activitywas defined as NPo, which was calculated from data samples of60-s duration in the steady state as follows:
where ti is the fractional open time spent at eachof the observed current levels.
Experimental Materials
Antibodies to c-Src and -actin were purchased from Santa CruzBiotechnology and anti–tyrosine-phosphorylation antibody(4G10)43 and ROMK antibody were obtained from Upstate USA (Charlottesville,VA) and Alamone, respectively. The specificity of ROMK antibodywas tested previously in ROMK null mouse in which immunostainingwas completely absent in the kidney.44 Antibodies to phospho-c-Jun(serine 73) and c-Jun were obtained from Cell Signaling Technology(Beverly, MA). The 4-hydroxy-tempo was obtained from Sigma (St.Louis, MO).
Statistical Analyses
Data are shown as means ± SEM, and one-way ANOVA statisticalanalysis was used to determine the significance among the threegroups. Statistical significance was taken as P < 0.05.
Babilonia E, Wei Y, Sterling H, Kaminski P, Wolin MS: Superoxide anions are involved in mediating the effect of low K intake on c-Src expression and renal K secretion in the cortical collecting duct.
J Biol Chem 280
: 10790
–10796, 2005[Abstract/Free Full Text]
Droge W: Free radicals in the physiological control of cell function.
Physiol Rev 82
: 47
–95, 2002[Abstract/Free Full Text]
Tang H, Hao Q, Rutherford SA, Low B, Zhao ZJ: Inactivation of Src family tyrosine kinases by reactive oxygen species in vivo.
J Biol Chem 280
: 23918
–23925, 2005[Abstract/Free Full Text]
Babilonia E, Li D, Wang ZJ, Sun P, Lin DH, Wang WH: Mitogen-activated protein kinase (MAPK) inhibits ROMK-like small conductance K channels in the CCD of K-restricted rats.
J Am Soc Nephrol 17
: 2687
–2696, 2006[Abstract/Free Full Text]
Sterling H, Lin DH, Gu RM, Dong K, Hebert SC, Wang WH: Inhibition of protein-tyrosine phosphatase stimulates the dynamin-dependent endocytosis of ROMK1.
J Biol Chem 277
: 4317
–4323, 2002[Abstract/Free Full Text]
Geiszt M, Leto T: The Nox family of NAD(P)H oxidases: Host defense and beyond.
J Biol Chem 279
: 51715
–51718, 2004[Free Full Text]
Haque MZ, Majid DSA: Assessment of renal functional phenotype in mice lacking pp91Phox subunit of NAD(P)H oxidase.
Hypertension 43
: 335
–340, 2004[Abstract/Free Full Text]
Chabrashvili T, Tojo A, Onozato ML, Kitiyakara C, Quinn MT, Fujita A, Welch WJ, Wilcox CS: Expression and cellular location of classic NADPH oxidase subunits in the spontaneously hypertensive rat kidney.
Hypertension 39
: 269
–274, 2002[Abstract/Free Full Text]
Shiose A, Kuroda J, Tsuruya K, Hirai M, Hirakata H, Naito S, Hattori M, Sakaki Y, Sumimoto H: A novel superoxide-producing NAD(P)H oxidase in kidney.
J Biol Chem 276
: 1417
–1423, 2001[Abstract/Free Full Text]
Lin DH, Sterling H, Yang B, Hebert SC, Giebisch G, Wang WH: Protein tyrosine kinase is expressed and regulates ROMK1 location in the cortical collecting duct.
Am J Physiol Renal Physiol 286
: F881
–F892, 2004[Abstract/Free Full Text]
Guyton KZ, Liu Y, Gorospe M, Xu Q, Holbrook NJ: Activation of mitogen-activated protein kinase by H2O2. Role of cell survival following oxidant injury.
J Biol Chem 271
: 4138
–4142, 1996[Abstract/Free Full Text]
Lo YYC, Wong JMS, Cruz TF: Reactive oxygen species mediate cytokine activation of c-jun NH2-terminal kinase.
J Biol Chem 271
: 15703
–15707, 1996[Abstract/Free Full Text]
Baas AS, Berk BC: Differential activation of mitogen-activated protein kinase by H2O2 and O2– in vascular smooth muscle cells.
Circ Res 77
: 29
–36, 1995[Abstract/Free Full Text]
Mahadev K, Zilbering A, Zhu L, Goldstein BJ: Insulin-stimulated hydrogen peroxide reversibly inhibits protein tyrosine phosphatase 1B in vivo and enhances the early insulin action cascade.
J Biol Chem 276
: 21938
–21942, 2001[Abstract/Free Full Text]
Brumell JH, Burkhardt AL, Bolen JB, Grinstein S: Endogenous reactive oxygen intermediates activate tyrosine kinase in human neutrophils.
J Biol Chem 271
: 1455
–1461, 1996[Abstract/Free Full Text]
Nakamura H, Hori T, Sato N, Sugie K, Kawakami T, Yodoi J: Redox regulation of a Src family protein tyrosine kinase p56Lck in T cells.
Oncogene 8
: 3133
–3139, 1993[Medline]
Denu JM, Tanner KG: Specific and reversible inactivation of protein tyrosine phosphatases by hydrogen peroxide: Evidence for a sulfenic acid intermediate and implications for redox regulation.
Biochemistry 37
: 5633
–5642, 1998[CrossRef][Medline]
Barrett DM, Black SM, Todor T, Schmidt-Ullrich RK, Dawson KS, Mikkelsen RB: Inhibition of protein-tyrosine phosphatases by mild oxidative stresses is dependent on s-nitrosylation.
J Biol Chem 280
: 14453
–14461, 2005[Abstract/Free Full Text]
Mahadev K, Wu X, Zilbering A, Zhu L, Lawrence JTR, Goldstein BJ: Hydrogen peroxide generated during cellular insulin stimulation is integral to activation of the distal insulin signaling cascade in 3T3–L1 adipocytes.
J Biol Chem 276
: 48662
–48669, 2001[Abstract/Free Full Text]
Schmid E, Hotz-Wagenblatt A, Hack V, Droege W: Phosphorylation of the insulin receptor kinase by phosphocreatine in combination with hydrogen peroxide. The structure basis of redox priming.
FASEB J 13
: 1491
–1500, 1999[Abstract/Free Full Text]
Hayes GR, Lockwood DH: Role of insulin receptor phosphorylation in the insulinomimetic effects of hydrogen peroxide.
Proc Natl Acad Sci U S A 84
: 8115
–8119, 1987[Abstract/Free Full Text]
Wingo CS, Cain BD: The renal H-K-ATPase: Physiological significance and role in potassium homeostasis.
Annu Rev Physiol 55
: 323
–347, 1993[Medline]
Wei Y, Bloom P, Lin DH, Gu RM, Wang WH: Effect of dietary K intake on the apical small-conductance K channel in the CCD: Role of protein tyrosine kinase.
Am J Physiol Renal Physiol 281
: F206
–F212, 2001[Abstract/Free Full Text]
Chu PY, Quigley R, Babich V, Huang CL: Dietary potassium restriction stimulates endocytosis of ROMK channel in rat cortical collecting duct.
Am J Physiol Renal Physiol 285
: F1179
–F1187, 2003[Abstract/Free Full Text]
Wei Y, Wang ZJ, Babilonia E, Sterling H, Sun P, Wang WH: Effect of hydrogen peroxide on ROMK channels in the cortical collecting duct.
Am J Physiol Renal Physiol 292
: F1151
–F1156, 2007[Abstract/Free Full Text]
Lin DH, Sterling H, Lerea KM, Welling P, Jin L, Giebisch G, Wang WH: K depletion increases the protein tyrosine-mediated phosphorylation of ROMK.
Am J Physiol Renal Physiol 283
: F671
–F677, 2002[Abstract/Free Full Text]
Wolin MS: Reactive oxygen species and vascular signal transduction mechanisms.
Microcirculation 3
: 1
–17, 1996[Medline]
Cai H, Li Z, Dikalov S, Holland SM, Hwang J, Jo H, Dudley SC Jr, Harrison DG: NAD(P)H oxidase-derived hydrogen peroxide mediates endothelial nitric oxide production in response to angiotensin II.
J Biol Chem 277
: 48311
–48317, 2002[Abstract/Free Full Text]
Bae YS, Kang SW, Seo MS, Baines IC, Tekle E, Chock PB, Rhee SG: Epidermal growth factor (EGF)-induced generation of hydrogen peroxide.
J Biol Chem 272
: 217
–221, 1997[Abstract/Free Full Text]
Kevin DB, Smith JB: Potassium depletion stimulates mRNA expression of proximal tubule AT1 angiotensin II receptor.
Nephron 78
: 73
–81, 1998[CrossRef][Medline]
Ohtsu H, Suzuki H, Nakashima H, Dhobale S, Frank GD, Motley ED, Eguchi S: Angiotensin II signal transduction through small GTP-binding proteins: Mechanism and significance in vascular smooth muscle cells.
Hypertension 48
: 534
–540, 2006[Free Full Text]
Sterling H, Lin DH, Chen YJ, Wei Y, Wang ZJ, Lai J, Wang WH: PKC expression is regulated by dietary K intake and mediates internalization of SK channels in the CCD.
Am J Physiol Renal Physiol 286
: F1072
–F1078, 2004[Abstract/Free Full Text]
Li DM, Wang ZJ, Sun P, Jin Y, Lin DH, Hebert SC, Giebisch G, Wang WH: Inhibition of MAPK stimulates the Ca2+-dependent big-conductance K channels in cortical collecting duct.
Proc Natl Acad Sci U S A 103
: 19569
–19574, 2006[Abstract/Free Full Text]
Woda CB, Bragin A, Kleyman T, Satlin LM: Flow-dependent K secretion in the cortical collecting duct (CCD) is mediated by a TEA-sensitive channel.
Am J Physiol Renal Physiol 280
: F786
–F793, 2001[Abstract/Free Full Text]
Satlin LM: Developmental regulation of expression of renal potassium secretory channels.
Curr Opin Nephrol Hypertens 13
: 445
–450, 2004[CrossRef][Medline]
Bailey MA, Cantone A, Yan QS, MacGregor GG, Leng Q, Amorim JB, Wang T, Hebert SC, Giebisch G, Malnic G: Maxi-K channels contribute to urinary potassium excretion in the ROMK-deficient mouse model of type II Bartter's syndrome and in adaptation to a high K diet.
Kidney Int 70
: 51
–59, 2006[CrossRef][Medline]
Zhou X, Lynch IJ, Xia SL, Wingo CS: Activation of H-K-ATPase by CO2 requires a basolateral Ba2+-sensitive pathway during K restriction.
Am J Physiol Renal Physiol 279
: F153
–F160, 2000[Abstract/Free Full Text]
Mohazzab HM, Wolin MS: Sites of superoxide anion production detected by lucigenin in calf pulmonary artery smooth muscle.
Am J Physiol 267
: L815
–L822, 1994[Medline]
Xie YW, Wolin MS: Role of nitric oxide and its interaction with superoxide in the suppression of cardiac muscle mitochondrial respiration.
Circulation 94
: 2580
–2586, 1996[Abstract/Free Full Text]
Butler B, Williams MP, Blystone SD: Ligand-dependent activation of integrin alphav 3.
J Biol Chem 278
: 5264
–5270, 2003[Abstract/Free Full Text]
Lin DH, Sterling H, Yang B, Hebert SC, Giebisch G, Wang WH: Protein tyrosine kinase is expressed and regulates ROMK1 location in the cortical collecting duct.
Am J Physiol Renal Physiol 286
: F881
–F892, 2004[Abstract/Free Full Text]
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Abstract
Introduction
.35 
-actin were purchased from Santa Cruz Biotechnology and anti–tyrosine-phosphorylation antibody (4G10)43 and ROMK antibody were obtained from Upstate USA (Charlottesville, VA) and Alamone, respectively. The specificity of ROMK antibody was tested previously in ROMK null mouse in which immunostaining was completely absent in the kidney.44 Antibodies to phospho-c-Jun (serine 73) and c-Jun were obtained from Cell Signaling Technology (Beverly, MA). The 4-hydroxy-tempo was obtained from Sigma (St. Louis, MO). 
