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.

The 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.WT 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.Amiloride injection significantly increased the urine output (UO) in NCC KO mice (from 1.3 ml/day before to 2.5 ml/day after amiloride, p0.05). 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, p80% 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.1. 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

Downregulation of the Cl-/HCO3-Exchanger Pendrin in Kidneys of Mice with Cystic Fibrosis: Role in the Pathogenesis of Metabolic Alkalosis

Background/Aims: Patients with cystic fibrosis (CF) are prone to the development of metabolic alkalosis; however, the pathogenesis of this life threatening derangement remains unknown. We hypothesized that altered acid base transport machinery in the kidney collecting duct underlies the mechanism of impaired bicarbonate elimination in the CF kidney. Methods: Balance studies in metabolic cages were performed in WT and CFTR knockout (CF) mice with the intestinal rescue in response to bicarbonate loading or salt restriction, and the expression levels and cellular distribution of acid base and electrolyte transporters in the proximal tubule, collecting duct and small intestine were examined by western blots, northern blots and/or immunofluorescence labeling. Results: Baseline parameters, including acid-base and systemic vascular volume status were comparable in WT and CF mice, as determined by blood gas, kidney renin expression and urine chloride excretion. Compared with WT animals, CF mice demonstrated a significantly higher serum HCO3- concentration (22.63 in WT vs. 26.83 mEq/l in CF mice; n=4, p=0.013) and serum pH (7.33 in WT vs. 7.42 in CF mice; n=4, p=0.00792) and exhibited impaired kidney HCO3- excretion (urine pH 8.10 in WT vs. 7.35 in CF mice; n=7, p=0.00990) following a 3-day oral bicarbonate load. When subjected to salt restriction, CF mice developed a significantly higher serum HCO3- concentration vs. WT animals (29.26 mEq/L in CF mice vs. 26.72 in WT; n=5, p=0.0291). Immunofluorescence labeling demonstrated a profound reduction in the apical expression of the Cl-/HCO3- exchanger pendrin in cortical collecting duct cells and western and northern blots indicated diminished plasma membrane abundance and mRNA expression of pendrin in CF kidneys. Conclusions: We propose that patients with cystic fibrosis are prone to the development of metabolic alkalosis secondary to the inactivation of the bicarbonate secreting transporter pendrin, specifically during volume depletion, which is a common occurrence in CF patients

Supplementary Material for: Downregulation of the Cl^-/HCO₃^-Exchanger Pendrin in Kidneys of Mice with Cystic Fibrosis: Role in the Pathogenesis of Metabolic Alkalosis

<b><i>Background/Aims:</i></b> Patients with cystic fibrosis (CF) are prone to the development of metabolic alkalosis; however, the pathogenesis of this life threatening derangement remains unknown. We hypothesized that altered acid base transport machinery in the kidney collecting duct underlies the mechanism of impaired bicarbonate elimination in the CF kidney. <b><i>Methods:</i></b> Balance studies in metabolic cages were performed in WT and CFTR knockout (CF) mice with the intestinal rescue in response to bicarbonate loading or salt restriction, and the expression levels and cellular distribution of acid base and electrolyte transporters in the proximal tubule, collecting duct and small intestine were examined by western blots, northern blots and/or immunofluorescence labeling. <b><i>Results:</i></b> Baseline parameters, including acid-base and systemic vascular volume status were comparable in WT and CF mice, as determined by blood gas, kidney renin expression and urine chloride excretion. Compared with WT animals, CF mice demonstrated a significantly higher serum HCO₃^- concentration (22.63 in WT vs. 26.83 mEq/l in CF mice; n=4, p=0.013) and serum pH (7.33 in WT vs. 7.42 in CF mice; n=4, p=0.00792) and exhibited impaired kidney HCO₃^- excretion (urine pH 8.10 in WT vs. 7.35 in CF mice; n=7, p=0.00990) following a 3-day oral bicarbonate load. When subjected to salt restriction, CF mice developed a significantly higher serum HCO₃^- concentration vs. WT animals (29.26 mEq/L in CF mice vs. 26.72 in WT; n=5, p=0.0291). Immunofluorescence labeling demonstrated a profound reduction in the apical expression of the Cl^-/HCO₃^- exchanger pendrin in cortical collecting duct cells and western and northern blots indicated diminished plasma membrane abundance and mRNA expression of pendrin in CF kidneys. <b><i>Conclusions:</i></b> We propose that patients with cystic fibrosis are prone to the development of metabolic alkalosis secondary to the inactivation of the bicarbonate secreting transporter pendrin, specifically during volume depletion, which is a common occurrence in CF patients

Effect of Acetazolamide on pendrin expression, urine output and water intake in WT and NCC KO mice.

<p>Fig 5a shows the expression of pendrin in WT and NCC KO mice treated with acetazolamide for 2 or 6 days (left and right panels). Fig 5b and 5c depicts the urine output (i) and water intake (ii) in WT and NCC KO mice treated with ACTZ. The increase in urine output by ACTZ in NCC KO mice correlated with a reduction in pendrin expression. Fig 5d demonstrates enhanced sodium excretion by ACTZ in NCC KO mice vs. WT animals.</p

Expression of pendrin in kidneys of WT and NCC KO mice.

<p>Expression of pendrin by (a) Northern hybridization and (b) Immunofluorescence labeling in kidneys of WT and NCC KO mice. Northern hybridizations showed significant increase in pendrin expression in NCC KO mice vs. WT littermates (with expression of pendrin increasing by 130% vs. wt littermates, p<0.03). Immunofluorescence labeling showed significant increase in the number of pendrin positive cells in kidneys of NCC KO mice vs. WT littermates (p<0.01 vs. WT).</p

Expression of ENaC in kidneys of WT and NCC KO mice.

<p>Expression of ENaC subunits by (a) Northern hybridization and (b). Western blot in kidneys of WT and NCC KO mice. (a) Expression levels of all 3 subunits increased by Northern hybridization, with α, β and γ subunits increasing by 110, 80 and 70%, respectively, vs. wt littermates. Western blots show significant increase in the cleaved ENaC γ subunit in NCC KO mice, consistent with published reports (29). (b) The abundance of cleaved ENaC γ subunit form increased by ~420% in NCC KO mice. The full length bands for α, β and γ subunits are shown, which did not show significant changes vs. WT mice. c. Expression of NKCC2 isotypes in WT and NCC KO mice.</p

Effect of Acetazolamide on ENaC expression: Impact of amiloride on salt excretion in WT and NCC KO mice.

<p>Fig 6a shows the expression of ENaC in kidney medulla and cortex of WT and NCC KO mice treated with acetazolamide for 6 days (left and right panels). Fig 6b depicts the effect of amiloride treatment on salt excretion in WT and NCC KO mice pretreated with ACTZ for 6 days.</p

Effect of Amiloride and Acetazolamide on water and salt excretion in WT and pendrin KO mice.

<p>Fig 7a depicts urine output in pendrin KO mice compared to WT mice, before and after amiloride injection. Fig 7b shows water intake in the two genotypes at baseline and after amiloride injection. Fig 7c and 7d compares urine output and water intake in WT vs pendrin KO mice post ACTZ injection.</p