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(Pro)renin receptor (PRR) is highly expressed in the distal nephron, but it has an unclear functional implication. The present study was conducted to explore a potential role of renal PRR during high K+ (HK) loading. In normal Sprague-Dawley rats, a 1-wk HK intake increased renal expression of full-length PRR and urinary excretion of soluble PRR (sPRR). Administration of PRO20, a decoy peptide antagonist of PRR, in K+-loaded animals elevated plasma K+ level and decreased urinary K+ excretion, accompanied with suppressed urinary aldosterone excretion and intrarenal aldosterone levels. HK downregulated Na+-Cl− cotransporter (NCC) expression but upregulated CYP11B2 (cytochrome P-450, family 11, subfamily B, polypeptide 2), renal outer medullary K+ channel (ROMK), calcium-activated potassium channel subunit α1 (α-BK), α-Na+-K+-ATPase (α-NKA), and epithelial Na+ channel subunit β (β-ENaC), all of which were blunted by PRO20. After HK loading was completed, urinary, but not plasma renin, was upregulated, which was blunted by PRO20. The same experiments that were performed using adrenalectomized (ADX) rats yielded similar results. Interestingly, spironolactone treatment in HK-loaded ADX rats attenuated kaliuresis but promoted natriuresis, which was associated with the suppressed responses of β-ENaC, α-NKA, ROMK, and α-BK protein expression. Taken together, we discovered a novel role of renal PRR in regulation of K+ homeostasis through a local mechanism involving intrarenal renin-angiotensin-aldosterone system and coordinated regulation of membrane Na+- and K+-transporting proteins. [ABSTRACT FROM AUTHOR]

With-no-Lysine kinase 4 (WNK4) inhibited ROMK (Kir1.1) channels and the inhibitory effect of WNK4 was abolished by serumglucocorticoid- induced kinase 1 (SGK1) but restored by c-Src. The aim of the present study is to explore the mechanism by which Src-family tyrosine kinase (SFK) modulates the effect of SGK1 on WNK4 and to test the role of SFK-WNK4-SGK1 interaction in regulating ROMK channels in the kidney. Immunoprecipitation demonstrated that protein phosphatase 1 (PP1) binds to WNK4 at amino acid (aa) residues 695-699 (PP1#1) and at aa 1211-1215 (PP1#2). WNK4-PP1#1 and WNK4-PP1#2, in which the PP1#1 or PP1#2 binding site was deleted or mutated, inhibited ROMK channels as potently as WNK4. However, c-Src restored the inhibitory effect of WNK4 but not WNK4-PP1#1 on ROMK channels in the presence of SGK1. Moreover, expression of c-Src inhibited SGK1-induced phosphorylation of WNK4 but not WNK4-PP1#1 at serine residue 1196 (Ser1196). In contrast, coexpression of c-Src restored the inhibitory effect of WNK4-PP1#2 on ROMK in the presence of SGK1 and diminished SGK1-induced WNK4 phosphorylation at Ser1196 in cells transfected with WNK4-PP1#2. This suggests the possibility that c-Src regulates the interaction between WNK4 and SGK1 through activating PP1 binding to aa 695-9 thereby decreasing WNK4 phosphorylation and restoring the inhibitory effect of WNK4. This mechanism plays a role in suppressing ROMK channel activity during the volume depletion because inhibition of SFK or serine/threonine phosphatases increases ROMK channel activity in the cortical collecting duct of rats on a low-Na diet. We conclude that regulation of phosphatase activity by SFK plays a role in determining the effect of aldosterone on ROMK channels and on renal K secretion. [ABSTRACT FROM AUTHOR]

We examined the effect of angiotensin II (ANG II) on epithelial Na+ channel (ENaC) in the rat cortical collecting duct (CCD) with single-channel and the perforated whole cell patch-clamp recording. Application of 50 nM ANG II increased ENaC activity, defined by NPo (a product of channel numbers and open probability), and the amiloride-sensitive whole cell Na currents by twofold. The stimulatory effect of ANG II on ENaC was absent in the presence of losartan, suggesting that the effect of ANG II on ENaC was mediated by ANG II type 1 receptor. Moreover, depletion of intracellular Ca2+ with 1,2-bis(2-aminophenoxy) ethane-N,N,N=,N=-tetraacetic acid (BAPTA)-AM failed to abolish the stimulatory effect of ANG II on ENaC but inhibiting protein kinase C (PKC) abolished the effect of ANG II, suggesting that the effect of ANG II was the result of stimulating Ca2+-independent PKC. This notion was also suggested by the experiments in which stimulation of PKC with phorbol ester derivative mimicked the effect of ANG II and increased amiloride-sensitive Na currents in the principal cell, an effect that was not abolished by treatment of the CCD with BAPTA-AM. Also, inhibition of NADPH oxidase (NOX) with diphenyleneiodonium chloride abolished the stimulatory effect of ANG II on ENaC and application of superoxide donors, pyrogallol or xanthine and xanthine oxidase, significantly increased ENaC activity. Moreover, addition of ANG II or H2O2 diminished the arachidonic acid (AA)-induced inhibition of ENaC in the CCD. We conclude that ANG II stimulates ENaC in the CCD through a Ca2+- independent PKC pathway that activates NOX thereby increasing superoxide generation. The stimulatory effect of ANG II on ENaC may be partially the result of blocking AA-induced inhibition of ENaC. [ABSTRACT FROM AUTHOR]

We previously demonstrated that K depletion inhibited ROMK-like small-conductance K channels (SK) in the cortical collecting duct (CCD) and that the effect was mediated by superoxide anions that stimulated Src family protein tyrosine kinase (PTK) and mitogen-activated protein kinase (MAPK) (51). However, because animals on a K-deficient diet had a severe hypokalemia, superoxide-dependent signaling may not regulate ROMK channels under physiological conditions with a normal plasma K concentration. In the present study, we used the patch-clamp technique and Western blot to examine the effect of a moderate K restriction on ROMK-like SK channels and the role of PTK and MAPK in regulating apical K channels in the CCD of animals on a low-K diet (LK: 0.1% K), Rats and mice fed a LK diet for 7 days had a normal plasma K concentration. However, a LK intake increased the expression of angiotensin II type 1 receptor in the kidney. Moreover, patch-clamp experiments demonstrated that LK intake decreased the probability finding SK channels and channel activity defined by NPo (a product of channel number and open probability) in the CCD of both rat and mouse kidneys. Also, LK intake significantly stimulated the production of superoxide anions in the renal cortex and outer medulla in both rats and mice and increased superoxide level in the rat CCD. Moreover, LK intake augments the phosphorylation of p38 and ERK MAPK, the expression of c-Src and cytosine phosphorylation of ROMK channels. However, treatment of animals with tempol abolished the effect of LK intake on MAPK and c-Src and increased ROMK channel activity in comparing with those of nontreated rats on a LK diet. Inhibiting p38 and ERK with SB202190 and PD98059 significantly stimulated SK in the CCD in rats on a LK diet. In addition, inhibition of PTK with herbimycin A activated SK channels in the CCD from rats on a LK diet. We conclude that LK intake stimulates the generation of superoxide anion and related products and that MAPK and Src family PTK play a physiological role in inhibiting apical K channels in the principal cells in response to LK intake. [ABSTRACT FROM AUTHOR]

Flow-stimulated net K secretion (Jκ) in the cortical collecting duct (CCD) is mediated by an iberiotoxin (IBX)-sensitive BK channel, and requires an increase in intracellular Ca2+ concentration ([Ca2+],). The a-subunit of the reconstituted BK channel is phosphorylated by PKA and PKC. To test whether the BK channel in the native CCD is regulated by these kinases, Jκ and net Na absorption (JNa) were measured at slow (∼1) and fast (∼5 nl·min-1· mm-1) flow rates in rabbit CCDs microperfused in the presence of mPKI, an inhibitor of PKA; calphostin C, which inhibits diacyiglycerol binding proteins, including PKC; or bisindolylmaleimide (BIM) and Gö6976, inhibitors of classic and novel PKC isoforms, added to luminal (L) and/or basolateral (B) solutions. L but not B mPKI increased Jκ in CCDs perfused at a slow flow rate; a subsequent increase in flow rate augmented Jκ modestly. B mPKI alone or with L inhibitor abolished flow stimulation of Jκ. Similarly, L calphostin C increased Jκ in CCDs perfused at slow flow rates, as did calphostin C in both L and B solutions. The observation that IBX inhibited the L mPKI and calphostin C-mediated increases in Jκ at slow flow rates implicated the BK channel in this K flux, a notion suggested by patch-clamp analysis of principal cells. The kinase inhibited by calphostin C was not PKC as L and/or B BIM and Gö6976 failed to enhance Jκ at the slow flow rate. However, addition of these PKC inhibitors to the B solution alone or with L inhibitor blocked flow stimulation of Jκ. Interpretation of these results in light of the effects of these inhibitors on the flow-induced elevation of [Ca2+]i suggests that the principal cell apical BK channel is tonically inhibited by PKA and that flow stimulation of Jκ in the CCD is PKA and PKC dependent. The specific targets of the kinases remain to be identified. [ABSTRACT FROM AUTHOR]

We used the patch-clamp technique to study the effect of H2O2 on the apical ROMK-like small-conductance K (SK) channel in the cortical collecting duct (CCD). The addition of H2O2 decreased the activity of the SK channels and the inhibitory effect of H2O2 was larger in the CCD from rats on a K-deficient diet than that from rats on a normal-K or a high-K diet. However, application of H2O2 did not inhibit the SK channels in inside-out patches. This suggests that the H2O2-mediated inhibition of SK channels was not due to direct oxidation of the SK channel protein. Because a previous study showed that H2O2 stimulated the expression of Src family protein tyrosine kinase (PTK) which inhibited SK channels (3), we explored the role of PTK in mediating the effect of H2O2 on SK channels. The application of H2O2 stimulated the activity of endogenous PIK in M-1 cells and increased tyrosine phosphorylation of ROMK in HEK293 cells transfected with GFP-ROMK1 and c-Src. However, blockade of PTK only attenuated but did not completely abolish the inhibitory effect of H2O2 on SK channels. Since H2O2 has also been demonstrated to activate mitogen-activated protein kinase, P38, and ERK (3), we examined the role of P38 and ERK in mediating the effect of H2O2 on SK channels. Similar to blockade of PTK, suppression of P38 and ERK did not completely abolish the H2O2-induced inhibition of SK channels. However, combined use of ERK, P38, and P1K inhibitors completely abolished the effect of H2O2 on SK channels. Also, treatment of the CCDs with concanavalin A, an agent which has been shown to inhibit endocytosis (19), abolished the inhibitory effect of H2O2. We conclude that addition of H2O2 inhibited SK channels by stimulating PTK activity, P38, and ERK in the CCD and that H2O2 enhances the internalization of the SK channels. [ABSTRACT FROM AUTHOR]

We previously demonstrated that arachidonic acid (AA) inhibits epithelial Na channels (ENaC) through the cytochrome P-450 (CYP) epoxygenase-dependent pathway (34). In the present study, we tested the hypothesis that low Na intake suppresses the expression of CYP2C23, which is mainly responsible for converting AA to epoxyeicosatrienoic acid (EET) in the kidney (11) and attenuates the AA-induced inhibition of ENaC. Immunostaining showed that CYP2C23 is expressed in the Tamm-Horsfall protein (THP)-positive and aquaporin 2 (AQP2)- positive tubules. This suggests that CYP2C23 is expressed in the thick ascending limb (TAL) and collecting duct (CD). Na restriction significantly suppressed the expression of CYP2C23 in the TAL and CD. Western blot also demonstrated that the expression of CYP2C23 in renal cortex and outer medulla diminished in rats on Na-deficient diet (Na-D) but increased in those on high-Na diet (4%). Moreover, the content of 11, 12-epoxyeicosatrienoic acid (EET) decreased in the isolated cortical CD from rats on Na-D compared with those on a normal-Na diet (0.5%). Patch-clamp study showed that application of 15 µM AA inhibited the activity of ENaC by 77% in the CCD of rats on a Na-D for 3 days. However, the inhibitory effect of AA on ENaC was significantly attenuated in rats on Na-D for 14 days. Furthermore, inhibition of CYP epoxygenase with MS-PPOH increased the ENaC activity in the CCD of rats on a control Na diet. We also used microperfusion technique to examine the effect of MS-PPOH on Na transport in the distal nephron. Application of MS-PPOH significantly increased Na absorption in the distal nephron of control rats but had no significant effect on Na absorption in rats on Na-D for 14 days. We conclude that low Na intake downregulates the activity and expression of CYP2C23 and attenuates the inhibitory effect of AA on Na transport. [ABSTRACT FROM AUTHOR]

We used the patch-clamp technique to examine the effect of adenosine on epithelial sodium channel (ENaC) activity in rat cortical collecting duct (CCD). Application of adenosine inhibits ENaC activity, and the effect of adenosine was mimicked by cyclohexyladenosine (CHA), an A1 adenosine-receptor agonist that reduced channel activity from 1.32 to 0.64. The inhibitory effect of CHA on ENaC was mimicked by cyclopentyladenosine (CPA), which reduced channel activity from 1.1 to 0.55. In contrast, application of CGS-21680, an A2a adenosine-receptor agonist, had no effect on ENaC and increased channel activity from 0.96 to 1.22. This suggests that the inhibitory effect of adenosine analogs resulted from stimulation of the A1 adenosine receptor. Inhibition of PLC with U-73122 failed to abolish the effect of CHA on ENaC. In contrast, the inhibitory effect of CHA on ENaC was absent in the presence of the PLA2 inhibitor arachidonyl trifluoromethyl ketone (AACOCF3). This suggests a role of arachidonic acid (AA) in mediating the effect of adenosine on ENaC. To determine the metabolic pathway of AA responsible for the effect of adenosine, we examined the effect of CHA in the presence of indomethacin or N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH). Inhibition of cytochrome P-450 (CYP) epoxygenase with MS-PPOH blocked the effect of CHA on ENaC. In contrast, CHA reduced ENaC activity in the presence of indomethacin. This suggests that CYP epoxygenase-dependent metabolites of AA mediate the effect of adenosine. Because 11,12-epoxyeicosatrienoic acid (11,12-EET) inhibits ENaC activity in the CCD (Wei Y, Lin DH, Kemp R, Yaddanapudi GSS, Nasjletti A, Falck JR, and Wang WH. J Gen Physiol 124: 719–727, 2004), we examined the role of 11,12-EET in mediating the effect of adenosine on ENaC. Addition of 11,12-EET inhibited ENaC channels in the CCD in which adenosine-induced inhibition was blocked by AACOCF3. We conclude that adenosine inhibits ENaC activity by stimulation of the A1 adenosine receptor in the CCD and that the effect of adenosine is mediated by 11,12-EET. [ABSTRACT FROM AUTHOR]