Depletion of Cholesterol Reduces ENaC Activity by Decreasing Phosphatidylinositol-4,5-Bisphosphate in Microvilli

Abstract

Background/Aims: The epithelial sodium channel (ENaC) in cortical collecting duct (CCD) principal cells plays a critical role in regulating systemic blood pressure. We have previously shown that cholesterol (Cho) in the apical cell membrane regulates ENaC; however, the underlying mechanism remains unclear. Methods: Patch-clamp technique and confocal microscopy were used to evaluate ENaC activity and density. Results: Here we show that extraction of membrane Cho with methyl-β-cyclodextrin (MβCD) significantly reduced amiloride-sensitive current and ENaC single-channel activity. The effects were reproduced by inhibition of Cho synthesis in the cells with lovastatin. We have previously shown that phosphatidylinositol-4,5-bisphosphate (PIP₂), an ENaC activator, is predominantly located in the microvilli, a specialized apical membrane domain. Here, our confocal microscopy data show that α-ENaC was co-localized with PIP₂ in the microvilli and that Cho was also co-localized with PIP₂ in the microvilli. Either extraction of Cho with MβCD or inhibition of Cho synthesis with lovastatin consistently reduced the levels of Cho, PIP₂, and ENaC in the microvilli. Conclusions: Since PIP₂ can directly stimulate ENaC and also affect ENaC trafficking, these data suggest that depletion of Cho reduces ENaC apical density and activity at least in part by decreasing PIP₂ in the microvilli.

Lovastatin attenuates hypertension induced by renal tubule‐specific knockout of ATP‐binding cassette transporter A1, by inhibiting epithelial sodium channels

“…We have shown that pharmacological blockade of ABCA1 with CsA elevates intracellular cholesterol, increases ENaC activity, and induces hypertension (Wang et al, ; Zhai et al, , https://doi.org/10.1016/j.bbadis.2018.08.027). To further determine the role of ABCA1, ABCA1 flox mice were crossed with AQP‐2 Cre mice to selectively delete ABCA1 in CCD principal cells.…”

“…Therefore, loss of the cilium‐controlled ATP release should account for the increased ENaC P O in ABCA1 KO mice. Using a cell culture model, our recent studies have already shown that depletion of cholesterol by lovastatin reduces ENaC activity by decreasing PIP 2 in microvilli in cultured distal nephron cells (Zhai et al, ). These studies suggest that principal cells have a mechanism by which they control sodium reabsorption that depends upon distal nephron flow, the primary cilia, and ATP release from the cells.…”

“…Our studies have shown that statins also inhibit cholesterol synthesis in the cortical collecting ducts (CCD) principal cells (Liu, Song, et al, ). We have also shown that reducing cholesterol levels by http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=2739 decreases ENaC activity whereas exogenous cholesterol increases ENaC activity in excised inside‐out patches (Zhai et al, , https://doi.org/10.1016/j.bbadis.2018.08.027), indicating that intracellular cholesterol is important for ENaC activity. In this study, we show that selective deletion of ABCA1 in CCD principal cells elevates intracellular cholesterol and causes hypertension by stimulating ENaC.…”

“…We have shown that pharmacological blockade of ABCA1 with CsA elevates intracellular cholesterol, increases ENaC activity, and induces hypertension (Wang et al, 2009;Zhai et al, 2018Zhai et al, , 10.1016Zhai et al, /j. bbadis.2018.…”

“…Our studies have shown that statins also inhibit cholesterol synthesis in the cortical collecting ducts (CCD) principal cells (Liu, Song, et al, 2013). We have also shown that reducing cholesterol levels by lovastatin decreases ENaC activity whereas exogenous cholesterol increases ENaC activity in excised inside-out patches (Zhai et al, 2018(Zhai et al, , 10.1016(Zhai et al, /j.bbadis.2018 What is the clinical significance…”