Impaired Renal HCO3- Excretion in Cystic Fibrosis

Abstract

Background: Patients with cystic fibrosis (CF) do not respond with increased urinary HCO3- excretion after stimulation with secretin and often present with metabolic alkalosis.

Methods: By combining RT-PCR, immunohistochemistry, isolated tubule perfusion, in vitro cell studies, and in vivo studies in different mouse models, we elucidated the mechanism of secretin-induced urinary HCO3- excretion. For CF patients and CF mice, we developed a HCO3- drinking test to assess the role of the cystic fibrosis transmembrane conductance regulator (CFTR) in urinary HCO3-excretion and applied it in the patients before and after treatment with the novel CFTR modulator drug, lumacaftor-ivacaftor.

Results: β-Intercalated cells express basolateral secretin receptors and apical CFTR and pendrin. In vivo application of secretin induced a marked urinary alkalization, an effect absent in mice lacking pendrin or CFTR. In perfused cortical collecting ducts, secretin stimulated pendrin-dependent Cl-/HCO3- exchange. In collecting ducts in CFTR knockout mice, baseline pendrin activity was significantly lower and not responsive to secretin. Notably, patients with CF (F508del/F508del) and CF mice showed a greatly attenuated or absent urinary HCO3--excreting ability. In patients, treatment with the CFTR modulator drug lumacaftor-ivacaftor increased the renal ability to excrete HCO3-.

Conclusions: These results define the mechanism of secretin-induced urinary HCO3- excretion, explain metabolic alkalosis in patients with CF, and suggest feasibility of an in vivo human CF urine test to validate drug efficacy.

Keywords: cystic fibrosis; ion transport; kidney tubule; renal tubular acidosis.

Copyright © 2020 by the American Society of Nephrology.

Figures

Graphical abstract

Figure 1.

The secretin receptor and CFTR is present in pendrin-positive cells in the cortex of WT mice. (A) Immunohistochemical localization of pendrin (red, upper panel), the SCTR (green, middle panel), and double staining (lower panel) in the cortex of WT mice. (B) Immunohistochemical localization of pendrin (red, upper panel), CFTR (green, middle panel), and double staining (lower panel) in the cortex of WT mice. (C) Higher magnification of the indicated area in (B) showing apical colocalization of CFTR and pendrin in the apical membrane. DAPI, 4′,6-diamidino-2-phenylindole.

Figure 2.

Secretin stimulates acute urine alkalization in mice. (A) Photographic image of the in vivo experimental setup. (B) Two original pHu traces are shown, indicating a prompt urine alkalization after application of 400 pg/g body wt (i.p.) secretin (red) and no effect in the control injected mouse (black). (C) Dose response relationship of the secretin effect on pHu, (n=4–7).

Figure 3.

The secretin-stimulated urine alkalization is absent in mice lacking pendrin and dramatically reduced in global and tubule-specific CFTR KO mice. (A–C) Summary pHu curves of control (black) and secretin-injected mice (400 pg/g body wt, i.p.; red) in (A) pendrin WT and KO mice, (B) global CFTR (CFTRG) WT and KO mice, and (C) CFTRTS WT and KO mice. n=6–7 in all experimental series. Peak difference between vehicle- and secretin-treated mice was tested with t test. (D–F) Urine [HCO3−] is significantly higher in WT mice after stimulation with secretin as compared with controls. This effect is absent in KO mice. Urine [HCO3−] in control mice (black) and secretin-treated mice (red) in (D) pendrin WT and KO mice, (E) CFTRG WT and KO mice, and (F) CFTRTS WT and KO mice. n=6–7 in all experimental series. Differences between vehicle- and secretin-treated mice were tested by two-way ANOVA with Bonferroni multiple comparison test. (G–I) Baseline urine [HCO3−] is higher in CFTR WT mice. Baseline pHU and urine [HCO3−] in (G) pendrin WT and KO mice, (H) CFTRG WT and KO mice, and (I) CFTRTS WT and KO mice. n=12–14 in all experimental series. Difference in pHU and urine [HCO3−] between genotypes was tested with t tests. *P<0.05, **P<0.01, ***P<0.001.

Figure 4.

Secretin stimulates pendrin-mediated HCO3− transport in isolated mouse CCD. (A) Fluorescence image of an isolated perfused CCD loaded with luminal BCECF-AM. Multiple intercalated cells protruding into the lumen can be identified. Most of the intercalated cells in the CCD show functional pendrin positivity (prompt intracellular alkalization after luminal chloride removal, data not shown). (B) A time control experiment showing an original pHi recording of a single pendrin-positive cell after a first removal of luminal chloride followed by a second removal of luminal chloride 10 minutes later. (C) A secretin stimulation experiment showing an original pHi recording of a single pendrin-positive cell. Luminal chloride was removed twice, first without stimulation and second after stimulation with 10 nM basolateral secretin for 10 minutes. Note the markedly faster alkalization effect in this experiment after secretin stimulation. (D and E) Summary of the entire experimental series. Secretin stimulated a significantly faster intracellular alkalization rate as compared with time controls, indicating activation of pendrin-mediated HCO3− secretin (n=5 tubules, n=23–28 cells, t test). (F) Note the significantly lower baseline functional pendrin activity in CFTRG KO CCDs (n=10 tubules, n=51–53 cells, t test). **P<0.01, ***P<0.001.

Figure 5.

cAMP-dependent activation of pendrin requires functional CFTR in the FRT cell model. (A) mRNA expression of pendrin and glyceraldehyde 3-phosphate dehydrogenase (GADPH) in FRT cells transfected with WT CFTR+scr, WT CFTR+siRNA, or dFCFTR+scr. (B) Protein abundance of pendrin and β-actin in FRT cells transfected with WT CFTR+scr, WT CFTR+siRNA, or dFCFTR+scr. (C) Pendrin mRNA and protein abundance is greatly increased in FRT cells transfected with WT CFTR, can be downregulated with siRNA for pendrin, and is almost absent in FRT cells transfected with dFCFTR. #P<0.001 versus WT CFTR+siRNA and dFCFTR+scr. *P<0.01 versus dFCFTR+scr. One-way ANOVA with Bonferroni multiple comparisons test. (D) Pendrin colocalizes with CFTR at the apical surface of WT CFTR-transfected FRT cells (upper panels), can be downregulated with siRNA for pendrin (middle panels), and is absent in dFCFTR-transfected FRT cells (lower panels). (E) An original experiment showing marked functional upregulation of apical Cl−/HCO3− function after IF stimulation in WT CFTR transfected FRT cells. (F) Effect of IF stimulation on apical Cl−/HCO3− exchange activity in parental-, WT CFTR-, dFCFTR-transfected FRT cells and with preincubation with CFTR inhibitor 172. Note the absence of IF-induced Cl−/HCO3− in cells with either no expression of CFTR, expression of dFCFTR, and in FRT WT CFTR cells treated with CFTR inh. 172 (20 µM, 3 minutes preincubation). #P<0.0001, IF-treated FRT WT CFTR cells were significantly upregulated compared with control and all other IF-treated groups. *P=0.007, significant difference between control FRT WT CFTR cells with and without CFTR inhibitor 172. One-way ANOVA with Bonferroni multiple comparisons test (n=3–4 experiments; on average each experiment included measurement of 150 single cells). (G) An original experiment and summary data showing significantly downregulation of IF-stimulated Cl−/HCO3−-exchange in WT CFTR transfected FRT cells by siRNA knockdown of pendrin. *P<0.01, t test. dFCFTR, ΔF508 CFTR; IF, 100 µM IBMX and 2 µM forskolin; scr, scrambled siRNA; siRNA, siRNA for pendrin; ΔpHi/s, the intracelluar pH increase per second.

Figure 6.

An acute metabolic alkalosis increases plasma secretin. CFTR KO mice are unable to increase their urine HCO3− excretion after an acute oral challenge. (A) Original image illustrating the oral fluid loading procedure. (B) Plasma [HCO3−] in mice loaded with either a control or a HCO3−-containing gastric gavage after 30 minutes. Note the significant elevation of plasma [HCO3−] ([HCO3−]p) in the base loaded mice, t test (n=8–9). (C) Plasma secretin concentration in mice loaded with either a control or a HCO3−-containing gastric gavage. Note the significant elevation in the plasma concentration of secretin ([secretin]p) in the base loaded mice, t test (n=8–9). (D–F) pHu, [HCO3−], and total HCO3− excretion in urine collected over a 3-hour period from awake mice subjected either to a control gavage or a gavage containing 2.24 mmol NaHCO3/kg body wt. Note the absent HCO3− excretion in CFTR KO mice. One-way ANOVA (n=6–9). *P<0.05, ***P<0.001.

Figure 7.

Patients with CF (ΔF508/ΔF508) have reduced urine [HCO3−] and a markedly reduced ability to excrete an oral HCO3− load. (A and B) Baseline urine pH and [HCO3−] in adult patients with CF (n=9) and healthy controls (n=10–11). Note the decreased urine [HCO3−] in patients with CF, t test. *P<0.05. (C, D, and F) Urine pH, urine [HCO3−], and cumulated urinary HCO3− excretion as response to a NaHCO3 drinking test (0.94 mmol NaHCO3/kg body wt) in fasting patients with CF (red, n=9), healthy controls (blue, n=11), and a water drinking test in one fasting patient with CF (bright blue) and fasting healthy controls (black, n=5). Note the greatly reduced urinary [HCO3−] and HCO3− excretion in patients with CF as compared with healthy controls. *P<0.05, **P<0.01 difference between healthy controls and patients with CF subjected to the oral NaHCO3 challenge, two-way ANOVA with Bonferroni multiple comparisons test. (E) Area under the curve (AUC) of urinary [HCO3−] in controls (n=11) and patients with CF (n=9). **P<0.01, t test.

Figure 8.

Treatment with lumacaftor-ivacaftor improves the HCO3− excretion deficit. (A–C) pHu, urine [HCO3−], and accumulated urinary HCO3− excretion as response to a NaHCO3 drinking test in patients with CF before (red, n=3) and after 4 weeks of lumacaftor-ivacaftor treatment (black, n=3), *P<0.05, paired t test. (D–F) Individual traces of the three lumacaftor-ivacaftor–treated participants before and after treatment. Note the approximately 100% increase in urine [HCO3−] in each participant. (G) Area under the curve (AUC) of urinary [HCO3−] in patients with CF before treatment (red, n=3) and after 4 weeks of lumacaftor-ivacaftor treatment (black, n=3). *P<0.05, difference between patients with CF before and after treatment, paired t test.

Figure 9.

Secretin-release is stimulated by alkalosis and triggers HCO3- secretion in the CCD. Schematic illustration of the proposed mechanism of secretin-induced urinary HCO3− excretion. Ingestion of a meal will cause a transient alkalosis. Ingestion of a meal itself and/or metabolic alkalosis stimulates secretin release. This will increase plasma concentration of secretin ([secretin]p). An increased plasma concentration of secretin will activate HCO3− secretion by β-ICs in the CCD. This will increase urine pH and [HCO3−] excretion and thereby help compensate metabolic alkalosis. This image was created with BioRender.com.