Lee, Hyun‐Wook

The A‐splice variant of Na + ‐dependent, electrogenic bicarbonate transporter‐1 (SLC4A4, NBCe1‐A) is generally believed to be the only SLC4A4 isoform expressed in the mammalian proximal tubule, and to have critical roles in filtered bicarbonate reabsorption, proximal tubule ammonia metabolism and proximal tubule organic anion metabolism. The purpose of the current study was to examine expression of other splice variants in the mouse kidney. We used recently generated mice with TALEN‐generated deletion of A and D NBCe1 splice‐variants, but with intact expression of the B, C and E isoforms. RT‐PCR using primers specific to the B, C and E isoforms showed mRNA of non‐A,D splice variants was expressed in the kidney of mice with intact NBCe1‐A expression and with NBCe1‐A‐specific deletion. We then used an antibody that recognizes portions of NBCe1 present in all splice isoforms and examined immunolabel in the kidney of NBCe1‐A‐deletion mice. There was basolateral immunolabel in the proximal convoluted tubule in the cortex, and lesser intensity basolateral immunolabel in the proximal tubule straight tubule in both the cortex and outer medulla. Specificity of this antibody was demonstrated by identifying that mice with deletion of all splice variants had no detectable immunolabel. An antibody specific to the A‐splice variant showed basolateral immunolabel throughout the entire proximal tubule of mice with intact NBCe1‐A expression, but no detectable immunolabel in mice with NBCe1‐A‐specific deletion. Thus, an NBCe1 splice variant other than NBCe1‐A appears to be expressed in the mouse proximal tubule. In acid‐loaded NBCe1‐A‐deletion mice, immunolabel observed using the pan‐specific anti‐NBCe1 antibody showed increased immunolabel in the proximal straight tubule, particularly in the outer medulla. These studies show that splice variants of NBCe1 other than NBCe1‐A/D are present in the mouse kidney and that their expression appears to be increased with acid‐loading. We conclude that these splice variants may have important roles in proximal tubule filtered bicarbonate reabsorption, ammonia metabolism and organic anion metabolism. Support or Funding Information The studies were supported by funds from the NIH (R01–DK045788 to IDW, R01‐DK107798 to IDW and JWV, and R01‐EY017732 to MFR) and the Department of Veterans Affairs (1I01BX000818 to IDW).

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.

Hypokalemia increases ammonia excretion and decreases K+excretion. The present study examined the role of the proximal tubule protein NBCe1-A in these responses. We studied mice with Na+-bicarbonate cotransporter electrogenic, isoform 1, splice variant A (NBCe1-A) deletion [knockout (KO) mice] and their wild-type (WT) littermates were provided either K+control or K+-free diet. We also used tissue sections to determine the effect of extracellular ammonia on NaCl cotransporter (NCC) phosphorylation. The K+-free diet significantly increased proximal tubule NBCe1-A and ammonia excretion in WT mice, and NBCe1-A deletion blunted the ammonia excretion response. NBCe1-A deletion inhibited the ammoniagenic/ammonia recycling enzyme response in the cortical proximal tubule (PT), where NBCe1-A is present in WT mice. In the outer medulla, where NBCe1-A is not present, the PT ammonia metabolism response was accentuated by NBCe1-A deletion. KO mice developed more severe hypokalemia and had greater urinary K+excretion during the K+-free diet than did WT mice. This was associated with blunting of the hypokalemia-induced change in NCC phosphorylation. NBCe1-A KO mice have systemic metabolic acidosis, but experimentally induced metabolic acidosis did not alter NCC phosphorylation. Although KO mice have impaired ammonia metabolism, experiments in tissue sections showed that lack of ammonia does impair NCC phosphorylation. Finally, urinary aldosterone was greater in KO mice than in WT mice, but neither expression of epithelial Na+channel α-, β-, and γ-subunits nor of H+-K+-ATPase α1- or α2-subunits correlated with changes in urinary K+. We conclude that NBCe1-A is critical for the effect of diet-induced hypokalemia to increase cortical proximal tubule ammonia generation and for the expected decrease in urinary K+excretion.

Citrate is critical for acid-base homeostasis and to prevent calcium nephrolithiasis. Both metabolic acidosis and hypokalemia decrease citrate excretion and increase expression of Na+-dicarboxylate cotransporter 1 (NaDC1; SLC13A2), the primary protein involved in citrate reabsorption. However, the mechanisms transducing extracellular signals and mediating these responses are incompletely understood. The purpose of the present study was to determine the role of the Na+-coupled electrogenic bicarbonate cotransporter (NBCe1) A variant (NBCe1-A) in citrate metabolism under basal conditions and in response to acid loading and hypokalemia. NBCe1-A deletion increased citrate excretion and decreased NaDC1 expression in the proximal convoluted tubules (PCT) and proximal straight tubules (PST) in the medullary ray (PST-MR) but not in the PST in the outer medulla (PST-OM). Acid loading wild-type (WT) mice decreased citrate excretion. NaDC1 expression increased only in the PCT and PST-MR and not in the PST-MR. In NBCe1-A knockout (KO) mice, the acid loading change in citrate excretion was unaffected, changes in PCT NaDC1 expression were blocked, and there was an adaptive increase in PST-MR. Hypokalemia in WT mice decreased citrate excretion; NaDC1 expression increased only in the PCT and PST-MR. NBCe1-A KO blocked both the citrate and NaDC1 changes. We conclude that 1) adaptive changes in NaDC1 expression in response to metabolic acidosis and hypokalemia occur specifically in the PCT and PST-MR, i.e., in cortical proximal tubule segments; 2) NBCe1-A is necessary for normal basal, metabolic acidosis and hypokalemia-stimulated citrate metabolism and does so by regulating NaDC1 expression in cortical proximal tubule segments; and 3) adaptive increases in PST-OM NaDC1 expression occur in NBCe1-A KO mice in response to acid loading that do not occur in WT mice.

Sodium-coupled bicarbonate transporters are critical for renal electrolyte transport. The electrogenic, sodium-coupled bicarbonate cotransporter, isoform 1 (NBCe1), encoded by the SLC4A4 geneencoded by the SLC4A4 gene has five multiple splice variants; the A splice variant, NBCe1-A, is the primary basolateral bicarbonate transporter in the proximal convoluted tubule. This study’s purpose was to determine if there is expression of additional NBCe1 splice variants in the mouse kidney, their cellular distribution, and their regulation by metabolic acidosis. In wild-type mice, an antibody reactive only to NBCe1-A showed basolateral immunolabel only in cortical proximal tubule (PT) segments, whereas an antibody reactive to all NBCe1 splice variants (pan-NBCe1) showed basolateral immunolabel in PT segments in both the cortex and outer medulla. In mice with NBCe1-A deletion, the pan-NBCe1 antibody showed basolateral PT immunolabel in both the renal cortex and outer stripe of the outer medulla, and immunoblot analysis showed expression of a ~121-kDa protein. RT-PCR of mRNA from NBCe1-A knockout mice directed at splice variant-specific regions showed expression of only NBCe1-B mRNA. In wild-type kidney, RT-PCR confirmed expression of mRNA for the NBCe1-B splice variant and absence of mRNA for the C, D, and E splice variants. Finally, exogenous acid loading increased expression in the proximal straight tubule in the outer stripe of the outer medulla. These studies demonstrate that the NBCe1-B splice variant is present in the PT, and its expression increases in response to exogenous acid loading, suggesting it participates in the PT contribution to acid-base homeostasis.

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.