Impaired Renal Base Excretion in Secretin Receptor Knock‐Out Mice During Prolonged Base‐Loading

Figures

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  FIGURE 1. SCTR KO mice respond with diminished urine alkalization upon prolonged base‐loading. Urine (A) pH, (B) NH4 + excretion, (C) titratable acid (TA) excretion and (D) NH4 + + TA excretion in metabolic cages with control drinking water and after 24 and 48 h of base‐loading with NaHCO3‐enriched drinking water in SCTR WT mice (112 mM: n = 9, black and 160 mM: n = 11, blue) and SCTR KO mice (112 mM: n = 19, red). Statistical differences were assessed by two‐way ANOVA followed by multiple comparisons. NH4 + excretion was log‐transformed before statistical analysis. $: p < 0.05 for SCTR KO vs. 112 mM NaHCO3 WTs. *, **, and ***: p < 0.05, < 0.01, and < 0.001 for SCTR KO vs. 160 mM NaHCO3 WTs, respectively. Error‐bars indicate SEM.

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  FIGURE 2. 24 h of base‐loading increases venous HCO3 − more in SCTR KO mice compared to SCTR WT mice. (A) Venous standard (std.) HCO3 − at baseline and after 24 h of base‐loading with NaHCO3‐enriched drinking water and (B) the change in venous HCO3 − after 24 h of base‐loading in SCTR WT mice (112 mM: n = 21, black and 160 mM: n = 15, blue) and SCTR KO mice (112 mM: n = 23, red). Statistical differences were assessed by one‐way ANOVA followed by multiple comparisons (B). Error‐bars indicate SEM.

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  FIGURE 3. SCTR KO mice exhibit increased base retention during prolonged base‐loading. Venous (A) Std. HCO3 −, (B) pH, (C) Cl− and (D) pCO2 at baseline and after 1, 4 and 8 days of base‐loading with NaHCO3‐enriched drinking water in SCTR WT mice (160 mM: n = 15–17, blue) and SCTR KO mice (112 mM, n = 11–23, red). Statistical differences between the two groups were assessed by two‐way ANOVA followed by multiple comparisons. Error‐bars indicate SEM.

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  FIGURE 4. SCTR KO mice reach a negative acid excretion at higher venous HCO3 − concentrations. Urinary acid (NH4 + + titratable acid) excretion as a function of venous standard (std.) HCO3 − in SCTR WT and SCTR KO mice. Data are combined from Figures 1 and 2. Note that a negative total NH4 + and TA excretion equates to a positive net excretion of base. Error bars indicate SEM.

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  FIGURE 5. SCTR KO mice show normal pendrin protein abundance regulation during prolonged base‐loading. (A) original blot showing protein abundance of pendrin in WT and KO mice under resting conditions and after 24 h of base‐loading with NaHCO3‐enriched drinking water (112 mM). Uncropped full‐length blots are shown in Figure S5. (B) quantification of pendrin protein abundance in the SCTR WT (n = 10–11) and KO mice (n = 13–14) during resting conditions and after 24 h of base‐loading with NaHCO3‐enriched drinking water (112 mM). Pendrin protein abundance was normalized to total loaded protein (using Coomassie staining) [34] and expressed relative to SCTR WT mice during control conditions. Statistical differences were assessed by two‐way ANOVA followed by multiple comparisons (panel B). Error bars indicate SEM.

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  FIGURE 6. SCTR KO mice exhibit impaired pendrin function regulation during prolonged base‐loading. (A + B) original traces depicting intracellular pH as a function of time in isolated perfused cortical collecting ducts of SCTR WT mice (A) and SCTR KO mice (B). (C) Summary of pendrin function in isolated perfused cortical collecting ducts from SCTR WT and KO mice during control conditions and after 24 h of base‐loading with NaHCO3‐enriched drinking water (112 mM). (D) Baseline (i.e., before luminal Cl− removal) intracellular pH in SCTR WT and KO pendrin positive cells in control and base‐loaded animals. (E) The association between baseline intracellular pH and the acute intracellular alkalization rate upon luminal chloride removal in base‐loaded SCTR WT and KO mice. Pendrin function was assessed as the initial rate change of intracellular pH after luminal chloride removal (graphically illustrated by the blue line in panels A + B). Statistical differences between pendrin function and baseline intracellular pH assessed by two‐way ANOVA followed by multiple comparisons (panel C + D), slope differences (panel E) was assessed by testing if there was a significant interaction between genotype and…

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  FIGURE 7. Plasma secretin and renal SCTR expression levels are modulated by acid/base‐loading. (A) Plasma secretin (pmol/L) in C57BL/6 mice under control conditions (n = 7, black dots), after 24 h of acid‐loading (200 mM NH4Cl, n = 7; red dots) and after 24 h of base‐loading (200 mM NaHCO3, n = 9, blue dots). (B) Plasma secretin (pmol/L) as a function of arterial HCO3 − (mmol/L) across the same three groups (n = 23). (C) SCTR mRNA expression in kidney tissue from the same three groups (control: n = 10, 200 mM NH4Cl: n = 8, 200 mM NaHCO3: n = 9). SCTR mRNA expression was normalized to the mean of the control group. (D) Relative SCTR expression as a function of arterial HCO3 − (mmol/L) across the same three groups (n = 27). SCTR expression was normalized to that of two housekeeping genes (mPPIA and mHPRT) and expressed relative to that of control mice. Statistical differences were assessed using one‐way ANOVA followed by multiple comparisons (panel A + C) or linear regression (panel B + D). In one‐way ANOVA analyses, a test for trend, that is, linear trends in plasma secretin/kidney SCTR levels from NH4Cl‐loaded to control‐loaded to NaHCO3‐loaded animals. Plasma secretin and kidney SCTR…