NBCe1 expression is required for normal renal ammonia metabolism

Abstract

The mechanisms regulating proximal tubule ammonia metabolism are incompletely understood. The present study addressed the role of the proximal tubule basolateral electrogenic Na(+)-coupled bicarbonate cotransporter (NBCe1; Slc4a4) in renal ammonia metabolism. We used mice with heterozygous and homozygous NBCe1 gene deletion and compared these mice with their wild-type littermates. Because homozygous NBCe1 gene deletion causes 100% mortality before day 25, we studied mice at day 8 (±1 day). Both heterozygous and homozygous gene deletion caused a gene dose-related decrease in serum bicarbonate. The ability to lower urinary pH was intact, and even accentuated, with NBCe1 deletion. However, in contrast to the well-known effect of metabolic acidosis to increase urinary ammonia excretion, NBCe1 deletion caused a gene dose-related decrease in ammonia excretion. There was no identifiable change in proximal tubule structure by light microscopy. Examination of proteins involved in renal ammonia metabolism showed decreased expression of phosphate-dependent glutaminase and phosphoenolpyruvate carboxykinase, key enzymes in proximal tubule ammonia generation, and increased expression of glutamine synthetase, which recycles intrarenal ammonia and regenerates glutamine. Expression of key proteins involved in ammonia transport outside of the proximal tubule (rhesus B glycoprotein and rhesus C glycoprotein) was not significantly changed by NBCe1 deletion. We conclude from these findings that NBCe1 expression is necessary for normal proximal tubule ammonia metabolism.

Keywords: acid-base; ammonia; electrogenic sodium-coupled bicarbonate cotransporter 1; proximal tubule.

Fig. 1.

Effect of electrogenic Na⁺-coupled bicarbonate (HCO₃⁻) cotransporter (NBCe1) deletion on serum electrolytes. Left: effects on serum HCO₃⁻. NBCe1 deletion induced a gene dose-dependent decrease in serum HCO₃⁻. n = 7, 7, and 6 for wild-type (Wt), heterozygous (Het), and homozygous NBCe1 deletion [knockout (KO)], respectively. Middle and right: effects on serum Na⁺ (middle) and K⁺ (right). NBCe1 deletion did not significantly alter either serum Na⁺ or K⁺. n = 5, 5, and 4 for wild-type, heterozygous, and homozygous NBCe1 deletion, respectively, for Na⁺ and n = 7, 7, and 5 for wild-type, heterozygous, and homozygous NBCe1 deletion, respectively, for K⁺.

Fig. 2.

Effect of NBCe1 deletion on urinary parameters. Top: effects on urine ammonia. Despite the associated metabolic acidosis with NBCe1 gene deletion, ammonia excretion decreased with heterozygous gene deletion and was suppressed significantly further with homozygous deletion. n = 26, 44, and 16 for wild-type, heterozygous, and homozygous NBCe1 deletion, respectively. Bottom: effects on urine pH. Urine pH was significantly more acidic (lower) in mice with heterozygous NBCe1 deletion than in wild-type mice and was decreased significantly further by homozygous NBCe1 deletion. n = 18, 29, and 11 for wild-type, heterozygous, and homozygous NBCe1 deletion, respectively.

Fig. 3.

Effect of NBCe1 deletion on renal morphology. Low-power micrographs (top) and high-power micrographs (bottom) of hematoxylin and eosin-stained kidney sections from wild-type and homozygous NBCe1 deletion (KO) mice. There was no detectable difference in proximal tubule morphology as a consequence of NBCe1 deletion. *Proximal tubule segments.

Fig. 4.

Effect of NBCe1 deletion on phosphate-dependent glutaminase (PDG) expression. Top: effects of NBCe1 deletion on PDG expression. Middle: expression of the “housekeeping” protein β-actin in the same blot showing that changes in PDG expression were not due to changes in protein loading or transfer. Bottom: quantitative analysis of PDG expression relative to β-actin expression. NBCe1 deletion resulted in significant inhibition of PDG expression. n = 7 for each genotype. *P < 0.05.

Fig. 5.

Effect of NBCe1 deletion on phosphoenolpyruvate carboxykinase (PEPCK) expression. Top: effects of NBCe1 deletion on PEPCK expression by immunoblot analysis. Heterozygous NBCe1 deletion significantly decreased PEPCK expression despite the associated metabolic acidosis. Homozygous NBCe1 deletion resulted in more marked loss of PEPCK expression. n = 7 for each genotype. Bottom: effects of NBCe1 deletion on PEPCK expression by immunohistochemistry. Compared with PEPCK immunolabel in wild-type mice, there was very weak PEPCK immunolabel in the proximal tubule of homozygous NBCe1 deletion (KO) mice.

Fig. 6.

Effect of NBCe1 deletion of glutamine synthetase expression. Top: effects of NBCe1 on glutamine synthetase expression by immunoblot analysis. Heterozygous NBCe1 deletion significantly increased glutamine synthetase expression, and homozygous deletion (KO) increased expression significantly further. This was in contrast to the expected inhibition of glutamine synthetase expression in response to metabolic acidosis. n = 7 for each genotype. Bottom: representative immunohistochemistry of glutamine synthetase expression in the cortex. Homozygous deletion (KO) increased glutamine synthetase immunolabel in proximal tubule segments.

Fig. 7.

Na⁺/H⁺ exchanger 3 (NHE3) expression and the effect of NBCe1 deletion. Top: immunoblot analysis of NHE3 expression in kidneys from mice with intact (Wt), heterozygous NBCe1 deletion (Het), and homozygous NBCe1 deletion (KO). The apparent molecular mass for mouse NHE3 of ∼75 kDa was similar to that which we and others have previously reported (33, 36, 40). Middle: expression of the housekeeping protein β-actin in the same blot showing that differences in NHE3 expression were not due to differences in protein loading or transfer. Bottom: quantitative analysis. NBCe1 deletion caused a trend for a gene dose-related decrease of NHE3 expression, but the differences were not statistically significant (NS).

Fig. 8.

Effect of NBCe1 deletion on proteins involved in collecting duct ammonia secretion. Top: rhesus B glycoprotein (Rhbg) localization in the cortex and medulla of mice with intact NBCe1 expression and with NBCe1 deletion. There was no detectable difference in Rhbg immunolabel in either the cortex or medulla as a result of NBCe1 deletion. Bottom: rhesus C glycoprotein (Rhcg) localization in the cortex and medulla of mice with intact NBCe1 expression and with NBCe1 deletion. There was no detectable difference in Rhcg immunolabel in the cortex or medulla as result of NBCe1 deletion.