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ABSTRACT: 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 NP(o) (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 tyrosine 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.
AJP Renal Physiology 03/2010; 298(6):F1515-22. · 4.42 Impact Factor
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ABSTRACT: We have previously demonstrated that ANG II inhibits ROMK-like small-conductance K channels (SK) in the cortical collecting duct from rats on a K-deficient diet (KD) (35). In the present study, we examined the role of angiotensin type 1 receptor (AT(1)R) in mediating the effect of K restriction on K secretion. We confirmed the previous finding that K restriction increased the superoxide anion level, c-Src expression, and the phosphorylation of both p38 and extracellular signal-regulated kinase mitogen-activated protein kinase (MAPK) in renal cortex and outer medulla. However, the effect of K restriction on superoxide anion generation, c-Src expression, and MAPK phosphorylation was significantly attenuated in rats receiving losartan, an inhibitor of AT(1)R. In contrast, losartan treatment had no effect on superoxide anion level, c-Src expression, and MAPK phosphorylation in animals on a normal K diet (NK). K restriction decreased SK channel activity and increased the tyrosine phosphorylation of ROMK. However, inhibiting AT(1)R abolished the effect of K restriction on SK channels and tyrosine phosphorylation of ROMK channels. The notion that AT(1)R is involved in regulating renal K excretion was also supported by the experiments with metabolic cages showing that losartan treatment significantly enhanced urinary K loss in rats on a KD diet while it had no effect in animals on a NK diet. Consequently, losartan-treated animals had severe hypokalemia in response to K restriction compared with rats without losartan intake. We conclude that AT(1)R is involved in mediating the effect of K restriction on superoxide generation, c-Src, and MAPK and that inhibiting AT(1)R impairs renal ability of K conservation in response to K depletion.
American journal of physiology. Renal physiology 03/2009; 296(5):F1179-84. · 3.68 Impact Factor
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ABSTRACT: We used Western blot analysis to examine the effect of dietary K intake on the expression of serine/threonine protein phosphatase in the kidney. K restriction significantly decreased the expression of catalytic subunit of protein phosphatase (PP)2B but increased the expression of PP2B regulatory subunit in both rat and mouse kidney. However, K depletion did not affect the expression of PP1 and PP2A. Treatment of M-1 cells, mouse cortical collecting duct (CCD) cells, or 293T cells with glucose oxidase (GO), which generates superoxide anions through glucose metabolism, mimicked the effect of K restriction on PP2B expression and significantly decreased expression of PP2B catalytic subunits. However, GO treatment increased expression of regulatory subunit of PP2B and had no effect on expression of PP1, PP2A, and protein tyrosine phosphatase 1D. Moreover, deletion of gp91-containing NADPH oxidase abolished the effect of K depletion on PP2B. Thus superoxide anions or related products may mediate the inhibitory effect of K restriction on the expression of PP2B catalytic subunit. We also used patch-clamp technique to study the effect of inhibiting PP2B on renal outer medullary K (ROMK) channels in the CCD. Application of cyclosporin A or FK506, inhibitors of PP2B, significantly decreased ROMK channels, and the effect of PP2B inhibitors was abolished by blocking p38 mitogen-activated protein kinase (MAPK) and ERK. Furthermore, Western blot demonstrated that inhibition of PP2B with cyclosporin A or small interfering RNA increased the phosphorylation of ERK and p38 MAPK. We conclude that K restriction suppresses the expression of PP2B catalytic subunits and that inhibition of PP2B decreases ROMK channel activity through stimulation of MAPK in the CCD.
AJP Cell Physiology 04/2008; 294(3):C765-73. · 3.54 Impact Factor
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ABSTRACT: We have used Western blot analysis and immunocytochemistry to determine the effect of dietary K intake on the expression of protein kinase C (PKC) isoforms in the kidney. Western blot has demonstrated that conventional PKC isoforms (alpha and beta), novel PKC isoforms (delta, epsilon, and eta), and atypical PKC isoforms (zeta) are expressed in the renal cortex and outer medulla. Moreover, a low K intake significantly increases the expression of PKC-epsilon in the renal cortex and outer medulla but does not change the expression of PKC-alpha, PKC-beta, PKC-delta, PKC-eta, and PKC-zeta. Also, immunocytochemistry shows that PKC-epsilon isoform is expressed in the cortical collecting duct (CCD) and outer medullary collecting duct (OMCD) and that the intensity of PKC-epsilon staining is higher in the kidney from rats on a K-deficient diet than those on a control diet. Also, we used the patch-clamp technique to study the role of PKC in mediating internalization of ROMK (Kir 1.1)-like small-conductance K (SK) channels induced by phenylarsine oxide (PAO), an agent that inhibits protein tyrosine phosphatase and has been shown to stimulate the internalization of the SK channel in the CCD (Sterling H, Lin DH, Qu RM, Dong K, Herbert SC, and Wang WH. J Biol Chem 277: 4317-4323, 2002). Inhibition of PKC with calphostin C and GF-109203x had no significant effect on channel activity but abolished the inhibitory effect of PAO on SK channels. In conclusion, a low K intake increases the expression of PKC-epsilon isoform in the renal cortex and outer medulla, and PKC is involved in mediating the internalization of SK channels in the CCD induced by stimulation of protein tyrosine kinase activity.
American journal of physiology. Renal physiology 07/2004; 286(6):F1072-8. · 3.68 Impact Factor
