Decrease in dietary K intake stimulates the generation of superoxide anions
in the kidney and inhibits K secretory channels in the CCD
Zhi-Jian Wang, Peng Sun, WenMing Xing, ChunYang Pan, Dao-Hong Lin, and Wen-Hui Wang
Department of Pharmacology, New York Medical College, Valhalla, New York
Submitted 26 August 2009; accepted in final form 25 March 2010
Wang Z-J, Sun P, Xing WM, Pan CY, Lin D-H, Wang W-H.
Decrease in dietary K intake stimulates the generation of superoxide
anions in the kidney and inhibits K secretory channels in the CCD. Am
J Physiol Renal Physiol 298: F1515–F1522, 2010. First published
March 31, 2010; doi:10.1152/ajprenal.00502.2009.—We previously
demonstrated that K depletion inhibited ROMK-like small-conduc-
tance 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 nor-
mal 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
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.
Ca2?-activated BK channel; ROMK (Kiy1-1); renal K secretion; K
A DECREASE IN THE DIETARY K intake suppresses renal K secre-
tion (41, 43, 53). K restriction-induced decrease in renal K
secretion is achieved by both inhibition of apical ROMK-like
small-conductance K channels (SK) and Ca2?-activated big
conductance K channel (BK) channels in principal cell (PC)
and by stimulating K absorption through K-H-ATPase in
intercalated cell (IC) (57, 58). Several studies demonstrated
that K restriction inhibits ROMK-like SK channels by stimu-
lating endocytosis (12, 55, 56). Although the mechanism by
which K restriction inhibits ROMK channels is not completely
understood, WNK (with-no-lysine kinase) and superoxide an-
ions play an important role in mediating endocytosis (27, 51).
WNK1, 3, and 4 have been shown to be expressed in the
cortical collecting duct (CCD) and inhibit ROMK channels by
stimulating a clathrin-dependent pathway (23, 26–29). More-
over, a kidney-specific WNK1 (KS-WNK1) is also expressed
in the CCD and can antagonize the inhibitory effect of WNK1
on ROMK channels (28). Because K restriction decreases the
KS-WNK1 expression and increases the WNK1 protein level,
increased ratio between the WNK1 and the KS-WNK1 leads to
suppressing ROMK channels in the CCD.
In addition, K restriction increases the superoxide anion
levels in the renal cortex and outer medulla (OM) (3). The role
of superoxide anions in mediating the effect of K depletion on
SK channels and renal K secretion is best suggested by exper-
iments in which decreasing superoxide anion levels with tem-
pol treatment increased SK channel activity in the CCD and
urinary K loss during K restriction (3). Superoxide and its
related products have been shown to activate mitogen-activated
protein kinase (MAPKs) such as p38 and ERK (15) and
stimulate the expression of Src family protein tyrosine kinase
(PTK) in the kidney (2). Increased p38 and ERK activity
inhibit ROMK-like SK channels by a PTK-independent mech-
anism (2). Stimulating Src family PTK phosphorylates ROMK
channels and increases the endocytosis (49). Moreover, c-Src
reversed the effect of SGK1 on WNK4 and restored the
inhibitory effect of WNK4 on ROMK channels. The Src family
PTK not only plays a role in regulating K homeostasis under
extreme K depletion which causes hypokalemia but also under
physiological conditions such as a moderate decrease in dietary
K intake which does not cause hypokalemia. A study per-
formed by Chen et al. (11) showed that decreased dietary K
content from 1 to 0.33% significantly increased the expression
of c-Src in the kidney and enhanced the tyrosine phosphory-
lation of ROMK channels. This indicates that Src family PTK
plays a physiological role in regulating K secretion. However,
it is remained to be determined whether a moderate decrease in
dietary K intake also increases the generation of superoxide
anions and whether superoxide-related products are responsi-
ble for suppressing ROMK channels. Therefore, the aim of the
present study is to examine whether superoxide anion, Src
family PTK, and MAPK are also responsible for maintaining K
homeostasis under physiological conditions.
Animal preparation. Male C57BL/6 mice (6 wk old) and pathogen-
free Sprague-Dawley rats (50–60 g) were used in the experiments and
were purchased from Jackson Laboratory (Bar Habor, ME) and
Taconic Farms (Germantown, NY), respectively. After 1-wk recovery
Address for reprint requests and other correspondence: W.-H. Wang, Dept.
of Pharmacology, New York Medical College, 15 Dana Rd., Valhalla, NY
10595 (e-mail: email@example.com).
Am J Physiol Renal Physiol 298: F1515–F1522, 2010.
First published March 31, 2010; doi:10.1152/ajprenal.00502.2009.
0363-6127/10 Copyright © 2010 the American Physiological Society http://www.ajprenal.orgF1515
21. Haque MZ, Majid DSA. Assessment of renal functional phenotype in
mice lacking pp91Phoxsubunit of NAD(P)H oxidase. Hypertension 43:
22. Hayes GR, Lockwood DH. Role of insulin receptor phosphorylation in
the insulinomimetic effects of hydrogen peroxide. Proc Natl Acad Sci USA
84: 8115–8119, 1987.
23. He G, Wang HR, Huang SK, Huang CL. Intersectin links WNK kinase
to endocytosis of ROMK1. J Clin Invest 117: 1078–1087, 2007.
24. Jin Y, Wang Y, Wang ZJ, Lin DH, Wang WH. Inhibition of angiotensin
type 1 receptor impairs renal ability of K conservation in response to K
restriction. Am J Physiol Renal Physiol 296: F1179–F1184, 2009.
25. Jin Y, Wang Z, Zhang Y, Yang B, Wang WH. PGE2 inhibits apical K
channels in the CCD through activation of the MAPK pathway. Am J
Physiol Renal Physiol 293: F1299–F1307, 2007.
26. Kahle KT, Wilson FH, Leng Q, Lalioti MD, O’Connell AD, Dong K,
Rapson AK, MacGregor GG, Giebisch G, Hebert SC, Lifton RP.
WNK4 regulates the balance between renal NaCl reabsorption and K?
secretion. Nat Genet 35: 372–376, 2003.
27. Kahle KT, Ring AM, Lifton RP. Molecular physiology of the WNK
kinases. Annu Rev Physiol 70: 329–355, 2008.
28. Lazrak A, Liu Z, Huang CL. Antagonistic regulation of ROMK by long
and kidney-specific WNK1 isoforms. Proc Natl Acad Sci USA 103:
29. Leng Q, Kahle KT, Rinehart J, MacGregor GG, Wilson FH, Canessa
CM, Lifton RP, Hebert SC. WNK3, a kinase related to genes mutated in
hereditary hypertension with hyperkaelemia, regulates the K?channel
ROMK1 (Kir1.1). J Physiol 571: 275–286, 2006.
30. Li DM, Wang ZJ, Sun P, Jin Y, Lin DH, Hebert SC, Giebisch G,
Wang WH. Inhibition of mitogen-activated protein kinase stimulates the
Ca2?-dependent big conductance K channels (BK) in cortical collecting
duct. Proc Natl Acad Sci USA 103: 19569–19574, 2006.
31. 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.
32. Linas SL, Peterson LN, Anderson RJ, Aisenbrey GA, Simon FR, Berl
T. Mechanism of renal potassium conservation in the rat. Kidney Int 15:
33. Lo YYC, Wong JMS, Cruz TF. Reactive oxygen species mediate
cytokine activation of c-jun NH2-terminal kinase. J Biol Chem 271:
34. 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.
35. 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:
36. Malnic G, Klose RM, Giebisch G. Micropuncture study of renal potas-
sium excretion in the rat. Am J Physiol 206: 674–686, 1964.
37. Malnic G, Klose RM, Giebisch G. Micropuncture study of distal tubular
potassium and sodium transport in rat nephron. Am J Physiol 211:
38. Mohazzab KM, Wolin MS. Sites of superoxide anion production de-
tected by lucigenin in calf pulmonary artery smooth muscle. Am J Physiol
Lung Cell Mol Physiol 267: L815–L822, 1994.
39. Mohazzab-H KM, Kaminski PM, Fayngersh RP, Wolin MS. Oxygen-
elicited responses in calf coronary arteries: role of H2O2 production via
NADH-derived superoxide. Am J Physiol Heart Circ Physiol 270:
40. 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.
41. Palmer LG. Potassium secretion and the regulation of distal nephron K
channels. Am J Physiol Renal Physiol 277: F821–F825, 1999.
42. Palmer LG, Antonian L, Frindt G. Regulation of apical K and Na
channels and Na/K pumps in rat cortical collecting tubule by dietary K. J
Gen Physiol 104: 693–710, 1994.
43. Palmer LG, Frindt G. Regulation of apical K channels in rat cortical
collecting tubule during changes in dietary K intake. Am J Physiol Renal
Physiol 277: F805–F812, 1999.
44. Ranganathan AC, Nelson KK, Rodriguez AM, Kim KH, Tower GB,
Rutter JL, Brinckerhoff CE, Huang TT, Epstein CJ, Jeffrey JJ,
Melendez JA. Manganese superoxide dismutase signals matrix metallo-
proteinase expression via H2O2-dependent ERK1/2 activation. J Biol
Chem 276: 14264–14270, 2001.
45. Ray PE, Suga S, Liu XH, Huang X, Johnson RJ. Chronic potassium
depletion induces renal injury, salt sensitivity, and hypertension in young
rats. Kidney Int 59: 1850–1858, 2001.
46. Schmid E, Hotz-Wagenblatt A, Hack V, Droege W. Phosphrylation of
the insulin receptor kinase by phosphocreatine in combination with hy-
drogen peroxide. The structure basis of redox priming. FASEB J 13:
47. Schmitz ML, Bacher S, Droge W. Molecular analysis of mitogen-
activated protein kinase signaling pathways induced by reactive oxygen
intermediates. Methods Enzymol 352: 53–61, 2002.
48. Sealey JE, Clark I, Bull MB, Laragh JH. Potassium balance and the
control of renin secretion. J Clin Invest 49: 2119–2127, 1970.
49. Sterling H, Lin DH, Gu RM, Dong K, Hebert SC, Wang WH.
Inhibition of protein tyrosine phosphatase stimulates the dynamin-depen-
dent endocytosis of ROMK1. J Biol Chem 277: 4317–4323, 2002.
50. Suzukawa K, Miura K, Mitsushita J, Resau J, Hirose K, Crystal R,
Kamata T. Nerve growth factor-induced neuronal differentiation requires
generation of Rac1-regulated reactive oxygen species. J Biol Chem 275:
51. Wang W. Regulation of renal K transport by dietary K intake. Annu Rev
Med 66: 547–569, 2004.
52. Wang W, Lerea KM, Chan M, Giebisch G. Protein tyrosine kinase
regulates the number of renal secretory K channels. Am J Physiol Renal
Physiol 278: F165–F171, 2000.
53. Wang WH. Regulation of renal K transport by dietary K intake. Annu Rev
Physiol 66: 547–569, 2004.
54. Wang XT, McCullough KD, Wang XJ, Carpenter G, Holbrook NJ.
Oxidative stress-induced phospholipase C-?1 activation enhances cell
survival. J Biol Chem 276: 28364–28371, 2001.
55. 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.
56. Wei Y, Wang WH. The role of cytoskeleton in mediating the effect of
vasopressin and herbimycin A on the secretory K channels in the CCD.
Am J Physiol Renal Physiol 282: F680–F686, 2001.
57. Wingo CS. Potassium transport by medullary collecting tubule of rabbit:
effects of variation in K intake. Am J Physiol Renal Fluid Electrolyte
Physiol 253: F1136–F1141, 1987.
58. Wingo CS, Armitage FE. Rubidium absorption and proton secretion by
rabbit outer medullary collecting duct via H-K-ATPase. Am J Physiol
Renal Fluid Electrolyte Physiol 263: F849–F857, 1992.
59. 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.
EFFECT OF LOW-K INTAKE ON ROMK
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