scholarly journals Deletion of the mechanistic‐target‐of‐rapamycin (mTOR) from collecting duct principal associated with increased ubiquitinylation of epithelial sodium channel (ENaC)

2020 ◽  
Vol 34 (S1) ◽  
pp. 1-1
Author(s):  
Bruce Chen ◽  
Maurice B. Fluitt ◽  
Narayan Shivapurkar ◽  
Aaron Brown ◽  
Carolyn Mary Ecelbarger
Keyword(s):  
Sodium Channel ◽  
Collecting Duct ◽  
Epithelial Sodium Channel ◽  
Target Of Rapamycin ◽  
Mechanistic Target Of Rapamycin ◽  
Epithelial Sodium Channel Enac

scholarly journals Selective Deletion of the Mechanistic Target of Rapamycin From the Renal Collecting Duct Principal Cell in Mice Down-Regulates the Epithelial Sodium Channel

2022 ◽  
Vol 12 ◽  
Author(s):  
Bruce Chen ◽  
Maurice B. Fluitt ◽  
Aaron L. Brown ◽  
Samantha Scott ◽  
Anirudh Gadicherla ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Collecting Duct ◽  
Epithelial Sodium Channel ◽  
Principal Cell ◽  
Target Of Rapamycin ◽  
Mtor Inhibition ◽  
Sodium Homeostasis ◽  
Mechanistic Target Of Rapamycin ◽  
Data Support ◽  
Body Sodium

The mechanistic target of rapamycin (mTOR), a serine-threonine-specific kinase, is a cellular energy sensor, integrating growth factor and nutrient signaling. In the collecting duct (CD) of the kidney, the epithelial sodium channel (ENaC) essential in the determination of final urine Na+ losses, has been demonstrated to be upregulated by mTOR, using cell culture and mTOR inhibition in ex vivo preparations. We tested whether CD-principal cell (PC) targeted deletion of mTOR using Cre-lox recombination would affect whole-body sodium homeostasis, blood pressure, and ENaC regulation in mice. Male and female CD-PC mTOR knockout (KO) mice and wild-type (WT) littermates (Cre-negative) were generated using aquaporin-2 (AQP2) promoter to drive Cre-recombinase. Under basal conditions, KO mice showed a reduced (∼30%) natriuretic response to benzamil (ENaC) antagonist, suggesting reduced in vivo ENaC activity. WT and KO mice were fed normal sodium (NS, 0.45% Na+) or a very low Na+ (LS, <0.02%) diet for 7-days. Switching from NS to LS resulted in significantly higher urine sodium losses (relative to WT) in the KO with adaptation occurring by day 2. Blood pressures were modestly (∼5–10 mm Hg) but significantly lower in KO mice under both diets. Western blotting showed KO mice had 20–40% reduced protein levels of all three subunits of ENaC under LS or NS diet. Immunohistochemistry (IHC) of kidney showed enhanced apical-vs.-cellular localization of all three subunits with LS, but a reduction in this ratio for γ-ENaC in the KO. Furthermore, the KO kidneys showed increased ubiquitination of α-ENaC and reduced phosphorylation of the serum and glucocorticoid regulated kinase, type 1 [serum glucocorticoid regulated kinase (SGK1)] on serine 422 (mTOR phosphorylation site). Taken together this suggests enhanced degradation as a consequence of reduced mTOR kinase activity and downstream upregulation of ubiquitination may have accounted for the reduction at least in α-ENaC. Overall, our data support a role for mTOR in ENaC activity likely via regulation of SGK1, ubiquitination, ENaC channel turnover and apical membrane residency. These data support a role for mTOR in the collecting duct in the maintenance of body sodium homeostasis.

scholarly journals The Epithelial Sodium Channel (ENaC) Traffics to Apical Membrane in Lipid Rafts in Mouse Cortical Collecting Duct Cells

2007 ◽  
Vol 282 (52) ◽  
pp. 37402-37411 ◽  
Author(s):  
Warren G. Hill ◽  
Michael B. Butterworth ◽  
Huamin Wang ◽  
Robert S. Edinger ◽  
Jonathan Lebowitz ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Lipid Rafts ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Epithelial Sodium Channel ◽  
Cortical Collecting Duct ◽  
Duct Cells ◽  
Collecting Duct Cells ◽  
Epithelial Sodium Channel Enac

Aldosterone-dependent and -independent regulation of the epithelial sodium channel (ENaC) in mouse distal nephron

2012 ◽  
Vol 303 (9) ◽  
pp. F1289-F1299 ◽  
Author(s):  
Viatcheslav Nesterov ◽  
Anke Dahlmann ◽  
Bettina Krueger ◽  
Marko Bertog ◽  
Johannes Loffing ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Single Channel ◽  
Collecting Duct ◽  
Epithelial Sodium Channel ◽  
Cortical Collecting Duct ◽  
Distal Nephron ◽  
Salt Diet ◽  
Low Salt ◽  
Immunohistochemical Evidence ◽  
Epithelial Sodium Channel Enac

Aldosterone is thought to be the main hormone to stimulate the epithelial sodium channel (ENaC) in the aldosterone-sensitive distal nephron (ASDN) comprising the late distal convoluted tubule (DCT2), the connecting tubule (CNT) and the entire collecting duct (CD). There is immunohistochemical evidence for an axial gradient of ENaC expression along the ASDN with highest expression in the DCT2 and CNT. However, most of our knowledge about renal ENaC function stems from studies in the cortical collecting duct (CCD). Here we investigated ENaC function in the transition zone of DCT2/CNT or CNT/CCD microdissected from mice maintained on different sodium diets to vary plasma aldosterone levels. Single-channel recordings demonstrated amiloride-sensitive Na+ channels in DCT2/CNT with biophysical properties typical for ENaC previously described in CNT/CCD. In animals maintained on a standard salt diet, the average ENaC-mediated whole cell current (Δ Iami) was higher in DCT2/CNT than in CNT/CCD. A low salt diet increased Δ Iami in CNT/CCD but had little effect on Δ Iami in DCT2/CNT. To investigate whether aldosterone is necessary for ENaC activity in the DCT2/CNT, we used aldosterone synthase knockout (AS−/−) mice that lack aldosterone. In CNT/CCD of AS−/− mice, Δ Iami was lower than that in wild-type (WT) animals and was not stimulated by a low salt diet. In contrast, in DCT2/CNT of AS−/− mice, Δ Iami was similar to that in DCT2/CNT of WT animals both on a standard and on a low salt diet. We conclude that ENaC function in the DCT2/CNT is largely independent of aldosterone which is in contrast to its known aldosterone sensitivity in CNT/CCD.

scholarly journals Apelin-13 Decreases Epithelial Sodium Channel (ENaC) Expression and Activity in Kidney Collecting Duct Cells

2022 ◽  
Vol 56 (1) ◽  
pp. 1-12
Keyword(s):  
Sodium Channel ◽  
Collecting Duct ◽  
Water Channel ◽  
Epithelial Sodium Channel ◽  
Sodium Balance ◽  
Principal Cells ◽  
Wide Range ◽  
Expression And Activity ◽  
Epithelial Sodium Channel Enac

BACKGROUND/AIMS: Apelin and its G protein-coupled receptor APLNR (also known as APJ) are widely expressed within the central nervous system and peripheral organs including heart, lung and kidney. Several studies have shown that the apelin/APJ system is involved in various important physiological processes such as energy metabolism, cardiovascular functions and fluid homeostasis. In the kidney, the apelin/APJ system performs a wide range of activities. We recently demonstrated that apelin antagonises the hydro-osmotic effect of vasopressin on aquaporin-2 water channel (AQP-2) expression by reducing its mRNA and protein levels in collecting duct principal cells. The central role of these cells in water and sodium transport is governed by AQP-2 and the epithelial sodium channel (ENaC). The coordination of these channels is essential for the control of extracellular fluid volume, sodium homeostasis and blood pressure. This study aimed at investigating the role of apelin in the regulation of sodium balance in the distal nephron, and more specifically its involvement in modulating the expression and activity of ENaC in collecting duct principal cells. METHODS: mpkCCD cells were incubated in the presence of aldosterone and treated with or without apelin-13. Transepithelial Na+ current was measured and the changes in ENaC expression determined by RT-PCR and immunoblotting. RESULTS: Our data show that apelin-13 reduces the transepithelial sodium amiloride-sensitive current in collecting duct principal cells after 8h and 24h treatment. This effect was associated with a decrease in αENaC subunit expression and mediated through the ERK pathway as well as SGK1 and Nedd4-2. CONCLUSION: Our findings indicate that apelin is involved in the fine regulation of sodium balance in the renal collecting duct by opposing the effects of aldosterone, likely by activation of ENaC ubiquitination.

Regulation of blood pressure, the epithelial sodium channel (ENaC), and other key renal sodium transporters by chronic insulin infusion in rats

2006 ◽  
Vol 290 (5) ◽  
pp. F1055-F1064 ◽  
Author(s):  
Jian Song ◽  
Xinqun Hu ◽  
Shahla Riazi ◽  
Swasti Tiwari ◽  
James B. Wade ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Sodium Channel ◽  
Insulin Infusion ◽  
Collecting Duct ◽  
Epithelial Sodium Channel ◽  
Kidney Cortex ◽  
Sodium Reabsorption ◽  
Cortical Collecting Duct ◽  
Sprague Dawley ◽  
Epithelial Sodium Channel Enac

Hyperinsulinemia is associated with hypertension. Dysregulation of renal distal tubule sodium reabsorption may play a role. We evaluated the regulation of the epithelial sodium channel (ENaC) and the thiazide-sensitive Na-Cl cotransporter (NCC) during chronic hyperinsulinemia in rats and correlated these changes to blood pressure as determined by radiotelemetry. Male Sprague-Dawley rats (∼270 g) underwent one of the following three treatments for 4 wk ( n = 6/group): 1) control; 2) insulin-infused plus 20% dextrose in drinking water; or 3) glucose water-drinking (20% dextrose in water). Mean arterial pressures were increased by insulin and glucose (mmHg at 3 wk): 98 ± 1 (control), 107 ± 2 (insulin), and 109 ± 3 (glucose), P < 0.01. Insulin (but not glucose) increased natriuretic response to benzamil (ENaC inhibitor) and hydrochlorothiazide (NCC inhibitor) on average by 125 and 60%, respectively, relative to control rats, suggesting increased activity of these reabsorptive pathways. Neither insulin nor glucose affected the renal protein abundances of NCC or the ENaC subunits (α, β, and γ) in kidney cortex, outer medulla, or inner medulla in a major way, as determined by immunoblotting. However, insulin and to some extent glucose increased apical localization of these subunits in cortical collecting duct principal cells, as determined by immunoperoxidase labeling. In addition, insulin decreased cortical “with no lysine” kinase (WNK4) abundance (by 16% relative to control), which may have increased NCC activity. Overall, insulin infusion increased blood pressure, and NCC and ENaC activity in rats. Increased apical targeting of ENaC and decreased WNK4 expression may be involved.

scholarly journals Rab11b regulates the trafficking and recycling of the epithelial sodium channel (ENaC)

2012 ◽  
Vol 302 (5) ◽  
pp. F581-F590 ◽  
Author(s):  
Michael B. Butterworth ◽  
Robert S. Edinger ◽  
Mark R. Silvis ◽  
Luciana I. Gallo ◽  
Xiubin Liang ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Collecting Duct ◽  
Fluorescent Microscopy ◽  
Epithelial Sodium Channel ◽  
Surface Expression ◽  
Small Gtpases ◽  
Dominant Negative ◽  
Cortical Collecting Duct ◽  
Interfering Rna ◽  
Epithelial Sodium Channel Enac

Expression of the epithelial sodium channel (ENaC) at the apical membrane of cortical collecting duct (CCD) principal cells is modulated by regulated trafficking mediated by vesicle insertion and retrieval. Small GTPases are known to facilitate vesicle trafficking, recycling, and membrane fusion events; however, little is known about the specific Rab family members that modify ENaC surface density. Using a mouse CCD cell line that endogenously expresses ENaC (mpkCCD), the channel was localized to both Rab11a- and Rab11b-positive endosomes by immunoisolation and confocal fluorescent microscopy. Expression of a dominant negative (DN) form of Rab11a or Rab11b significantly reduced the basal and cAMP-stimulated ENaC-dependent sodium (Na+) transport. The greatest reduction in Na+ transport was observed with the expression of DN-Rab11b. Furthermore, small interfering RNA-mediated knockdown of each Rab11 isoform demonstrated the requirement for Rab11b in ENaC surface expression. These data indicate that Rab11b, and to a lesser extent Rab11a, is involved in establishing the constitutive and cAMP-stimulated Na+ transport in mpkCCD cells.

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