Downregulation of the renal outer medullary K+ channel ROMK by the AMP-activated protein kinase

Abstract

The 5'-adenosine monophosphate-activated serine/threonine protein kinase (AMPK) is stimulated by energy depletion, increase in cytosolic Ca(2+) activity, oxidative stress, and nitric oxide. AMPK participates in the regulation of the epithelial Na(+) channel ENaC and the voltage-gated K(+) channel KCNE1/KCNQ1. It is partially effective by decreasing PIP(2) formation through the PI3K pathway. The present study explored whether AMPK regulates the renal outer medullary K(+) channel ROMK. To this end, cRNA encoding ROMK was injected into Xenopus oocytes with and without additional injection of constitutively active AMPK(γR70Q) (AMPK(α1)-HA+AMPK(β1)-Flag+AMPKγ1(R70Q)), or of inactive AMPK(αK45R) (AMPK(α1K45R)+AMPK(β1)-Flag+AMPK(γ1)-HA), and the current determined utilizing two-electrode voltage-clamp and single channel patch clamp. ROMK protein abundance was measured utilizing chemiluminescence in Xenopus oocytes and western blot in whole kidney tissue. Moreover, renal Na(+) and K(+) excretion were determined in AMPK(α1)-deficient mice (ampk ( -/- )) and wild-type mice (ampk ( +/+ )) prior to and following an acute K(+) load (111 mM KCl, 30 mM NaHCO(3), 4.7 mM NaCl, and 2.25 g/dl BSA) at a rate of 500 μl/h. As a result, coexpression of AMPK(γR70Q) but not of AMPK(αK45R) significantly decreased the current in ROMK1-expressing Xenopus oocytes. Injection of phosphatidylinositol PI((4,5))P(2) significantly increased the current in ROMK1-expressing Xenopus oocytes, an effect reversed in the presence of AMPK(γR70Q). Under control conditions, no significant differences between ampk ( -/- ) and ampk ( +/+ ) mice were observed in glomerular filtration rate (GFR), urinary flow rate, serum aldosterone, plasma Na(+), and K(+) concentrations as well as absolute and fractional Na(+) and K(+) excretion. Following an acute K(+) load, GFR, urinary flow rate, serum aldosterone, plasma Na(+), and K(+) concentration were again similar in both genotypes, but renal absolute and fractional Na(+) and K(+) excretion were higher in ampk ( -/- ) than in ampk ( +/+ ) mice. According to micropuncture following a K(+) load, delivery of Na(+) to the early distal tubule but not delivery of K(+) to late proximal and early distal tubules was increased in ampk (-/-) mice. The upregulation of renal ROMK1 protein expression by acute K(+) load was more pronounced in ampk (-/-) than in ampk ( +/+ ) mice. In conclusion, AMPK downregulates ROMK, an effect compromising the ability of the kidney to excrete K(+) following an acute K(+) load.

Kidney-specific genetic deletion of both AMPK α-subunits causes salt and water wasting

“…Only under acute potassium loading a significant difference was found with AMPK␣1 Ϫ/Ϫ showing higher upregulation of ROMK and increased urinary excretion of both K ϩ and Na ϩ . Furthermore, in micropuncture experiments, following a potassium load there was reduced tubular Na ϩ reabsorption between the late proximal and early distal segments of AMPK␣1 Ϫ/Ϫ mice, suggesting that the activity of NKCC2 could be comparatively reduced in these mice after an acute K ϩ load (59). A reduced medullary activity of the NKCC2 cotransporter was also shown in the work of Fraser et al (23) on the AMPK␤1 Ϫ/Ϫ mice.…”

“…A small increase in fractional Na ϩ excretion has been previously described in the AMPK␤1 Ϫ/Ϫ mice, a model in which whole kidney AMPK␣1 activity was found to be reduced by 60% with no appreciable change in AMPK␣2 activity (23). In addition, other studies on the AMPK␣1 Ϫ/Ϫ mice reported increases in fractional urinary excretion of K ϩ , Cl Ϫ (2), and total Na ϩ excretion 30 min after an acute K ϩ load (59). In this study, by using a kidney double knockout mutant, we report for the first time a significant increase in Na ϩ excretion in 24-h samples in both basal condition and under Na ϩ dietary challenges.…”

“…AMPK has been shown to modulate membrane ion transport in epithelial cell models, providing a link between cell energy metabolism and the main energy expenditure process in those tissues (29,33). It is well established that AMPK can inhibit the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel (31,32,34,40,42,43,62), the epithelial sodium channel (ENaC) (7,9,66), and several potassium channels present in epithelia (3,59), among other ion channels. AMPK has also been related to the function of ion pumps (4,30) and transporters (29,52), including the kidney-specific Na-K-Cl cotransporter NKCC2 responsible for sodium reabsorption in the thick ascending limb (24).…”

Renal potassium handling in rats with subtotal nephrectomy: modeling and analysis

“…Conversely, NCC is downregulated (32). We assumed an 80% decrease in NCC expression and a doubling in ROMK expression; these values are comparable, respectively, to the measured 60% NCC decrease (32) and twofold ROMK increase (38). Whereas Palmer and Frindt observed a Ͼ10-fold elevation in the density of ENaC in cell-attached patches (31), no change in ENaC protein expression was seen by Rengarajan et al (32); the present model assumes a 3-fold increase in response to an acute K ϩ load.…”

Lack of Effects of Metformin and AICAR Chronic Infusion on the Development of Hypertension in Dahl Salt-Sensitive Rats

“…Both insulin-dependent and non-insulin-dependent diabetic individuals have been reported to have a significant increase of total exchangeable sodium relative to non-diabetic controls (O'hare et al, 1985; Bickel et al, 2002). In cultured cells, AMPK was shown to regulate renal epithelial currents mediated by ENaC (Bhalla et al, 2006), ROMK (Siraskar et al, 2013), and CFTR (Takiar et al, 2011). Analysis of renal function in mice with genetic deletion of the AMPK α 1 catalytic subunit showed normal renal sodium handling and a moderate antidiuretic state that might indicate a compensatory upregulation in α 2 subunit.…”