The Role of Epithelial Sodium Channel ENaC and the Apical Cl-/HCO3- Exchanger Pendrin in Compensatory Salt Reabsorption in the Setting of Na-Cl Cotransporter (NCC) Inactivation

Abstract

BackgroundThe absence of NCC does not cause significant salt wasting in NCC deficient mice under basal conditions. We hypothesized that ENaC and pendrin play important roles in compensatory salt absorption in the setting of NCC inactivation, and their inhibition and/or downregulation can cause significant salt wasting in NCC KO mice.MethodsWT and NCC KO mice were treated with a daily injection of either amiloride, an inhibitor of ENaC, or acetazolamide (ACTZ), a blocker of salt and bicarbonate reabsorption in the proximal tubule and an inhibitor of carbonic anhydrases in proximal tubule and intercalated cells, or a combination of acetazolamide plus amiloride for defined durations. Animals were subjected to daily balance studies. At the end of treatment, kidneys were harvested and examined. Blood samples were collected for electrolytes and acid base analysis.ResultsAmiloride injection significantly increased the urine output (UO) in NCC KO mice (from 1.3 ml/day before to 2.5 ml/day after amiloride, p<0.03, n = 4) but caused only a slight change in UO in WT mice (p>0.05). The increase in UO in NCC KO mice was associated with a significant increase in sodium excretion (from 0.25 mmol/24 hrs at baseline to 0.35 mmol/24 hrs after amiloride injection, p<0.05, n = 4). Daily treatment with ACTZ for 6 days resulted in >80% reduction of kidney pendrin expression in both WT and NCC KO mice. However, ACTZ treatment noticeably increased urine output and salt excretion only in NCC KO mice (with urine output increasing from a baseline of 1.1 ml/day to 2.3 ml/day and sodium excretion increasing from 0.22 mmole/day before to 0.31 mmole/day after ACTZ) in NCC KO mice; both parameters were significantly higher than in WT mice. Western blot analysis demonstrated significant enhancement in ENaC expression in medulla and cortex of NCC KO and WT mice in response to ACTZ injection for 6 days, and treatment with amiloride in ACTZ-pretreated mice caused a robust increase in salt excretion in both NCC KO and WT mice. Pendrin KO mice did not display a significant increase in urine output or salt excretion after treatment with amiloride or ACTZ.Conclusion1. ENaC plays an important role in salt reabsorption in NCC KO mice. 2. NCC contributes to compensatory salt reabsorption in the setting of carbonic anhydrase inhibition, which is associated with increased delivery of salt from the proximal tubule and the down regulation of pendrin. 3. ENaC is upregulated by ACTZ treatment and its inhibition by amiloride causes significant diuresis in NCC KO and WT mice. Despite being considered mild agents individually, we propose that the combination of acetazolamide and amiloride in the setting of NCC inhibition (i.e., hydrochlorothiazide) will be a powerful diuretic regimen.

Probenecid Pre-treatment Downregulates the Kidney Cl-/HCO3- Exchanger (Pendrin) and Potentiates Hydrochlorothiazide-Induced Diuresis

“…Our studies demonstrate that treatment with probenecid at either 250 or 100 mg/kg body weight for 10 days causes significant perturbation in the expression of transporters in the collecting duct, including the downregulation of pendrin and, at the higher probenecid dosing (250 mg/kg/day), the AQP2. The downregulation of pendrin enhances salt and water excretion by thiazides, consistent with the indispensable role of pendrin in compensatory salt absorption in response to inactivation/inhibition of NCC ( Soleimani et al, 2012 ; Zahedi et al, 2013 ; Patel-Chamberlin et al, 2016 ; Alshahrani et al, 2017 ). We did not examine the effect of probenecid on the expression of the sodium dependent Cl - /HCO 3 - exchanger, NDCBE (SLC4A8) ( Leviel et al, 2010 ), as a recent report indicates its absence in CCD ( Chen et al, 2017 ).…”

“…Despite being a strong inhibitor of NCC, thiazide derivatives in general are mild diuretics, likely due to compensatory salt absorption by transporters in other nephron segments. Both the sodium channel ENaC and Cl - /HCO 3 - exchanger pendrin are activated in the CNT and CCD; therefore, blunting the diuresis caused by the inhibition or inactivation of NCC ( Patel-Chamberlin et al, 2016 ). Inactivation or inhibition of pendrin provokes robust diuresis by HCTZ, a potent thiazide analog ( Soleimani et al, 2012 ; Zahedi et al, 2013 ; Alshahrani et al, 2017 ).…”

“…The diuretic effect of HCTZ, a specific inhibitor of NCC, is significantly blunted consequent to compensatory salt absorption by pendrin ( Zahedi et al, 2013 ; Patel-Chamberlin et al, 2016 ). Based on the inhibitory effect of probenecid on pendrin ( Rillema and Hill, 2003 ), we tested the hypothesis that pre-treatment with probenecid will downregulate or inactivate pendrin; therefore, leaving NCC as the main salt absorbing transporter in the distal nephron, thus significantly enhancing the diuretic effect of HCTZ.…”

Slc4a8 in the Kidney: Expression, Subcellular Localization and Role in Salt Reabsorption

“…Five (5) animals placed in metabolic cages for the same duration were used as baseline. Urine pH and electrolyte excretion rate as well as serum electrolytes and acid base status were determined as described [37]. For dehydration, mice were subjected to water deprivation for 24 hrs with free access to food.…”

“…Plasma membrane protein were isolated from kidneys of WT and Slc4a8 KO mice, size-fractionated by SDS/PAGE (90 μg/lane), and blotted against pendrin or NCC antibodies according to established protocols [36,37]. Pendrin antibodies for Western blotting were generous gifts from Dr. Peter Aronson (Yale University).…”