Handlogten, Mary E.

The proximal tubule electrogenic, sodium‐coupled bicarbonate transporter, NBCe1, has a critical role in two proximal tubule acid‐base functions, bicarbonate reabsorption and ammonia metabolism. A third component of acid‐base homeostasis involves organic anion metabolism. The purpose of the current study was to determine NBCe1's role in organic anion metabolism by comparing mice with NBCe1 deletion to wild‐type (WT) littermates. Because NBCe1 KO causes early post‐natal mortality, we studied mice at day 8±1. Also, because NBCe1 deletion causes metabolic acidosis, we compared effects of NBCe1 deletion to that of experimental metabolic acidosis in adult WT mice. Both NBCe1 knockout (KO) and acidosis decreased citrate excretion, but their effect on proximal tubule citrate transporters differed. NaDC‐1 is the primary apical citrate transporter, and NBCe1 KO decreased NaDC‐1 mRNA expression and it decreased proximal tubule apical immunolabel. In contrast, acidosis in WT mice increased NaDC‐1 mRNA increased and proximal tubule apical immunolabel. The basolateral citrate transporter is NaDC‐3. NBCe1 deletion decreased NaDC‐3 expression whereas metabolic acidosis increased expression. A second key urinary organic anion is 2‐oxoglutarate. NBCe1 KO increased 2‐oxoglutarate excretion ~15‐fold, whereas metabolic acidosis decreased excretion by ~85%. NBCe1 deletion did not alter expression of the enzyme involved in 2‐oxoglutarate generation (Glud1), whereas metabolic acidosis increased it. There were no substantial changes in expression of the 2‐oxoglutarate dehydrogenase complex components, Ogdh, Dlst or Dld, with either NBCe1 KO or acidosis. Expression of the putative apical 2‐oxoglutarate transporter, OAT10, was decreased similarly by NBCe1 KO and by acidosis. Thus, effects of NBCe1 KO on 2‐oxoglutarate excretion did not correlate with changes in proteins involved in production, metabolism, basolateral transport (NaDC‐3) or its putative apical transporter (OAT10). 2‐oxoglutarate is proposed to stimulate urine alkalinization by activating the Oxgr1 receptor in pendrin‐positive intercalated cells. However, NBCe1 KO causes a urine pH of ~4.2 despite increased 2‐oxoglutarate excretion. Pendrin, the primary mechanism of collecting duct bicarbonate secretion, exhibited significantly decreased expression with NBCe1 KO. Expression of the 2‐oxoglutarate receptor, Oxgr1, was unaffected by NBCe1 KO, whereas it was decreased by metabolic acidosis. In summary: 1) NBCe1 is necessary for normal expression of the proximal tubule citrate transporters, NaDC‐1 and NaDC‐3; 2) NBCe1 is necessary for normal 2‐oxoglutarate metabolism through mechanisms independent of expression of known transport and metabolic pathways; and, 3) the lack of 2‐oxoglutarate urinary alkalinization in NBCe1 KO mice likely results from decreased expression of its target transporter, pendrin. Thus, NBCe1 expression is necessary for normal renal metabolism of the key organic acids, citrate and 2‐oxoglutarate. We conclude that NBCe1 expression is necessary for normal regulation of each of the three major components of proximal tubule acid‐base homeostasis, filtered bicarbonate reabsorption, ammonia metabolism and organic anion transport. Support or Funding Information Funds from the NIH (R01–DK045788 to IDW, R01‐EY017732 to MFR and R01‐DK095879 to KHS) and from the Department of Veterans Affairs (1I01BX000818 to IDW) supported these studies.

Renal ammonia metabolism is the primary mechanism through which the kidneys maintain acid-base homeostasis, but the molecular mechanisms regulating renal ammonia generation are unclear. In these studies, we evaluated the role of the proximal tubule basolateral plasma membrane electrogenic sodium bicarbonate cotransporter 1 variant A (NBCe1-A) in this process. Deletion of the NBCe1-A gene caused severe spontaneous metabolic acidosis in mice. Despite this metabolic acidosis, which normally causes a dramatic increase in ammonia excretion, absolute urinary ammonia concentration was unaltered. Additionally, NBCe1-A deletion almost completely blocked the ability to increase ammonia excretion after exogenous acid loading. Under basal conditions and during acid loading, urine pH was more acidic in mice with NBCe1-A deletion than in wild-type controls, indicating that the abnormal ammonia excretion was not caused by a primary failure of urine acidification. Instead, NBCe1-A deletion altered the expression levels of multiple enzymes involved in proximal tubule ammonia generation, including phosphate-dependent glutaminase, phosphoenolpyruvate carboxykinase, and glutamine synthetase, under basal conditions and after exogenous acid loading. Deletion of NBCe1-A did not impair expression of key proteins involved in collecting duct ammonia secretion. These studies demonstrate that the integral membrane protein NBCe1-A has a critical role in basal and acidosis-stimulated ammonia metabolism through the regulation of proximal tubule ammonia-metabolizing enzymes.

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.