Bicarbonate is the primary inducer of KCC3a expression in renal cortical B-type intercalated cells

Abstract

A primary function of intercalated cells in the distal tubule of the kidney is to maintain pH homeostasis. For example, type B intercalated cells secrete bicarbonate largely through the action of the apical Cl^-/HCO₃^- exchanger, pendrin, which helps correct metabolic alkalosis. Since both the K-Cl cotransporter, KCC3a and pendrin colocalize to the apical region of type B and non-A, non-B intercalated cells and since both are upregulated in models of metabolic alkalosis, such as with dietary NaHCO₃ loading, we raised the possibility that apical KCC3a facilitates pendrin-mediated bicarbonate secretion, such as through apical Cl^- recycling. The purpose of this study was to determine if KCC3a abundance changes through intake of bicarbonate alone or through bicarbonate plus its accompanying cation, and if it requires a direct interaction with pendrin or the renin-angiotensin-aldosterone system. We observed that KCC3a protein abundance, but not mRNA, increases in a mouse model of metabolic alkalosis, achieved with dietary NaHCO₃ or KHCO₃ intake. Bicarbonate ion increases KCC3a abundance, both in vivo and in vitro, independently of the accompanying cation. Moreover, bicarbonate intake upregulates KCC3a independently of aldosterone or angiotensin II. Since NaHCO₃ intake increased KCC3a abundance in wild-type as well as in pendrin knockout mice, this KCC3a upregulation by bicarbonate does not depend on a direct interaction with pendrin. We conclude that increased extracellular bicarbonate, as observed in models of metabolic alkalosis, directly raises KCC3a abundance independently of angiotensin II, aldosterone, or changes in KCC3a transcription and does not involve a direct interaction with pendrin.NEW & NOTEWORTHY KCC3a expression is stimulated in alkalemia. This paper shows that bicarbonate itself is mediating this effect through a posttranscriptional mechanism. The paper also shows that this phenomenon is not mediated by aldosterone or angiotensin II.

Keywords: K-Cl cotransport; alkalemia; bicarbonate; intercalated cells; pendrin.

Figure 1.

Changes in KCC3a and pendrin abundance in response to long-term NaHCO₃. A and B: in contrast to NaCl treatment, a 7-day NaHCO₃ treatment increased KCC3a and pendrin abundances. For blot quantification, densitometric values were normalized to β-actin. Values are represented as means ± SE (two-tailed unpaired t test, *P < 0.05; ***P < 0.001). Individual data points in any given group represent one kidney or one mouse. C and D: Localization of KCC3a following the 7-day NaCl or NaHCO₃ treatment, respectively. NaHCO₃ causes an increase in KCC3a apical localization and intensity compared with NaCl. Scale bar = 20 µm.

Figure 2.

Short-term NH₄Cl and K-citrate treatments. A and B: a 24-h NH₄Cl treatment had no effect on expression of KCC3a and pendrin (two-tailed unpaired t test, ns, nonsignificant or P > 0.05). C: KCC3a abundance increased on K-citrate treatment compared with control (Ctrl) or KCl treatment (one-way ANOVA followed by Tukey’s multiple-comparison test, *P < 0.05). For blot quantification, densitometric values were normalized to β-actin. Values are represented as means ± SE. Individual data points in the given group represent one kidney or one mouse.

Figure 3.

The main signaling molecule responsible for the increase in KCC3a abundance is bicarbonate. A and B: KCC3a and pendrin abundance were elevated by both a normal salt (NS) and high sodium (HS) KHCO₃ diet in comparison with a high-salt diet (HS) alone (one-way ANOVA followed by Tukey’s multiple-comparison test). C and D: KCC3a abundance on a normal or Na⁺-deficient diet was unaffected by losartan [angiotensin-II receptor type 1 (AT1) antagonist]. For blot quantification, densitometric values were normalized to β-actin. Values are represented as means ± SE. *P < 0.05; **P < 0.01. Individual data points in the given group represent one kidney or one mouse. ns, nonsignificant or P > 0.05.

Figure 4.

KCC3a abundance is independent of pendrin, not upregulated at the mRNA level, and not sensitive to pH. A: compared with the normal diet group, KCC3a abundance increased in pendrin knockout (KO) mice maintained on a high KHCO₃ diet for 4 days (two-tailed unpaired t test). For blot quantification, densitometric values were normalized to β-actin. Individual data points in the given group represent one kidney or one mouse. B: KCC3a transcript levels were not affected by bicarbonate treatment. Curves are for GAPDH (green), KCC3-1a-2 (red), and KCC31a-3 (blue) indicating relative abundance of the transcripts. The real-time PCR Systems software computed a relative quantification (RQ) factor, based on the delta-delta Ct (2^−ΔΔCt). The control group (Ctrl) was then set to 100% and the fold change in gene expression in the NaHCO3-treated group was calculated. Data were normalized to the expression of GAPDH. Values are represented as means ± SE. *P < 0.05. Individual data points in the given group represent one kidney or one mouse. C: expression of KCC3a in M-1 cells, wild-type (WT), and KCC3 knockout (KO) kidney samples. D: 24-h exposure of M-1 cells to acidic to alkaline pHs does not affect KCC3a expression. E: 24-h exposure of M-1 cells to increasing amounts of bicarbonate stimulates KCC3 expression. ns, nonsignificant or P > 0.05.