High potassium intake enhances the inhibitory effect of 11,12-EET on ENaC

Abstract

High dietary potassium stimulates the renal expression of cytochrome P450 (CYP) epoxygenase 2C23, which metabolizes arachidonic acid (AA). Because the AA metabolite 11,12-epoxyeicosatrienoic acid (11,12-EET) can inhibit the epithelial sodium channel (ENaC) in the cortical collecting duct, we tested whether dietary potassium modulates ENaC function. High dietary potassium increased 11,12-EET in the isolated cortical collecting duct, an effect mimicked by inhibiting the angiotensin II type I receptor with valsartan. In patch-clamp experiments, a high potassium intake or treatment with valsartan enhanced AA-induced inhibition of ENaC, an effect mediated by a CYP-epoxygenase-dependent pathway. Moreover, high dietary potassium and valsartan each augmented the inhibitory effect of 11,12-EET on ENaC. Liquid chromatography/mass spectrometry showed that the rate of EET conversion to dihydroxyeicosatrienoic acids (DHET) was lower in renal tissue obtained from rats on a high-potassium diet than from those on a control diet, but this was not a result of altered expression of soluble epoxide hydrolase (sEH). Instead, suppression of sEH activity seemed to be responsible for the 11,12-EET-mediated enhanced inhibition of ENaC in animals on a high-potassium diet. Patch-clamp experiments demonstrated that 11,12-DHET was a weak inhibitor of ENaC compared with 11,12-EET, whereas 8,9- and 14,15-DHET were not. Furthermore, inhibition of sEH enhanced the 11,12-EET-induced inhibition of ENaC similar to high dietary potassium. In conclusion, high dietary potassium enhances the inhibitory effect of AA and 11,12-EET on ENaC by increasing CYP epoxygenase activity and decreasing sEH activity, respectively.

Figure 1.

HK intake stimulates the expression of rat CYP2C23. (A) Western blot showing the effect of 10% K diet on CYP2C23 in the cortex and OM of rat kidney. (B) 11,12-EET levels in the CCD of rats on a control, 10% HK (7 days), low Na (3 days), and valsartan treatment + low Na (3 days). *Significant difference in comparison with control. (C) Channel recording showing the effect of 100 nM 11S,12R- or 11R,12S-EET on ENaC. The experiments are performed in a cell-attached patch, and the holding potential was 60 mV. Results are summarized in a bar graph shown in the bottom of the figure.

Figure 2.

HK intake enhances the AA-induced inhibition of ENaC. (A and B) A single-channel recording demonstrating the effect of AA on ENaC in rats on a low-Na (A) or on an HK diet (B). The channel closed level is indicated by “C” and a dotted line. The experiments are performed in a cell-attached patch with a holding potential of 60 mV. (C) Dose-response curve of the AA-induced inhibition of ENaC in the CCD of rats on different diets. Each data point represents four to six experiments. *Significant difference in comparison with the corresponding control value (low Na for 3 days). Data are normalized by taking channel activity (NP_o) under control conditions (in the absence of AA) as 100%.

Figure 3.

HK intake enhances the 11,12-EET–induced inhibition of ENaC. (A) A single-channel recording demonstrating the effect of 11,12-EET on ENaC in rats on an HK diet for 7 days (top) or on a low-Na diet for 3 days (bottom). The experiments are performed in a cell-attached patch with a holding potential of 60 mV. (B) Dose-response curve of the 11,12-EET–induced inhibition of ENaC in the CCD of rats on different diets. Each data point represents four to six experiments. *Significant difference in comparison with any other values in the graph. The data are normalized by taking channel activity (NP_o) under control conditions (no 11,12-EET) as 100%.

Figure 4.

HK intake decreases AT1R expression. (A) Western blot demonstrates the expression of AT1R in the mixture of the renal cortex and OM. (B) Western blot shows the effect of valsartan treatment on the expression of CYP2C23 in the renal cortex and OM (mixture) of rats on a low-Na diet for 3 days.

Figure 5.

Inhibiting AT1R enhances the inhibitory effect of AA and 11,12-EET. (A) Dose-response curve of the AA-induced inhibition of ENaC in the CCD of valsartan-treated rats on a low-Na diet for 3 days and on a 10% HK diet for 7 days. (B) Dose-response curve of the 11,12-EET–induced inhibition of ENaC in the CCD of valsartan-treated rats on a low-Na diet for 3 days and on a 10% HK diet for 7 days. The data are normalized by taking channel activity (NP_o) under control conditions (zero AA or 11,12-EET) as 100%.

Figure 6.

HK intake suppresses the activity of sEH. (A) Western blot demonstrating the effect of K intake on the expression of sEH. (B) Rate of EET conversion to DHET in the tissue of the renal cortex and OM from rats on 1 and 10% K diet. *Significant difference.

Figure 7.

11,12-DHET is a weak inhibitor of ENaC. (A) A single-channel recording in a cell-attached patch showing the effect of 100 nM 11,12-DHET on ENaC in the rat CCD. The experiments are performed in a cell-attached patch with a holding potential of 60 mV. (B) Bar graph summarizes the effect of 100 nM 8,9-, 14,15-, and 11,12-DHET on ENaC activity in the rat CCD. Each data point represents four to six measurements. *Significant difference in comparison with the control.

Figure 8.

Inhibition of sEH enhances the inhibitory effect of 11,12-EET. (A) A single-channel recording in a cell-attached patch showing the effect of 50 nM 11,12-EET on ENaC in the rat CCD of animals on an HK diet (top) and a low-Na diet (bottom). The experiments are performed in a cell-attached patch with a holding potential of 60 mV. The channel closed level is indicated by “C” and dotted lines. (B) Dose-response curve of 11,12-EET–induced inhibition of ENaC in AUDA-treated and untreated CCD. Each data point represents four to six measurements. *Value measured in animals on an HK diet is significantly different from the corresponding values measured in animals treated with AUDA. The data are normalized by taking channel activity (NP_o) under control conditions (no 11,12-EET) as 100%.

Figure 9.

A scheme illustrating a possible mechanism by which HK intake enhances the AA- and 11,12-EET–induced inhibition of ENaC.