Effect of NBCe1 deletion on renal citrate and 2-oxoglutarate handling

Abstract

The bicarbonate transporter, NBCe1 (SLC4A4), is necessary for at least two components of the proximal tubule contribution to acid-base homeostasis, filtered bicarbonate reabsorption, and ammonia metabolism. This study's purpose was to determine NBCe1's role in a third component of acid-base homeostasis, organic anion metabolism, by studying mice with NBCe1 deletion. Because NBCe1 deletion causes metabolic acidosis, we also examined acid-loaded wild-type adult mice to determine if the effects of NBCe1 deletion were specific to NBCe1 deletion or were a non-specific effect of the associated metabolic acidosis. Both NBCe1 KO and acid-loading decreased citrate excretion, but in contrast to metabolic acidosis alone, NBCe1 KO decreased expression of the apical citrate transporter, NaDC-1. Thus, NBCe1 expression is necessary for normal NaDC-1 expression, and NBCe1 deletion induces a novel citrate reabsorptive pathway. Second, NBCe1 KO increased 2-oxoglutarate excretion. This could not be attributed to the metabolic acidosis as experimental acidosis decreased excretion. Increased 2-oxoglutarate excretion could not be explained by changes in plasma 2-oxoglutarate levels, the glutaminase I or the glutaminase II generation pathways, 2-oxoglutarate metabolism, its putative apical 2-oxoglutarate transporter, OAT10, or its basolateral transporter, NaDC-3.

In summary: (1) NBCe1 is necessary for normal proximal tubule NaDC-1 expression; (2) NBCe1 deletion results in stimulation of a novel citrate reabsorptive pathway; and (3) NBCe1 is necessary for normal 2-oxoglutarate metabolism through mechanisms independent of expression of known transport and metabolic pathways.

Keywords: 2‐oxoglutarate; NBCe1; NaDC‐1; acidosis; citrate; proximal tubule.

Figure 1

Effect of NBCe1 deletion and of experimental metabolic acidosis on urinary citrate and 2‐oxoglutarate excretion. Top left panel shows effects of NBCe1 deletion on citrate excretion. NBCe1 KO resulted in no detectable (ND) urinary citrate excretion. Top right panel shows effects of NBCe1 deletion on 2‐oxoglutarate excretion. Mice with NBCe1 deletion excreted significantly more 2‐oxoglutarate than did wild‐type littermates. Bottom left panel shows effects of experimental metabolic acidosis on urinary citrate excretion in adult wild‐type mice. Metabolic acidosis in adult wild‐type mice resulted in complete suppression of urinary citrate excretion. Bottom right panel shows effects of experimental metabolic acidosis on 2‐oxoglutarate excretion in adult wild‐type mice. Metabolic acidosis significantly decreased 2‐oxoglutarate excretion. N = 4 in each group in each experiment. Note that the units are different in the measurements performed in pup kidneys, μg per mg creatinine, from those in the experimental metabolic acidosis studies, μg per 24 h. ND, not detectable.

Figure 2

Effect of NBCe1 deletion on NaDC‐1 expression. Left side of figure shows that NBCe1 deletion decreased NaDC‐1 mRNA expression significantly. Right side shows that experimental metabolic acidosis, examined in adult wild‐type mice, in contrast increased NaDC‐1 mRNA expression significantly. Because NBCe1 deletion causes spontaneous metabolic acidosis (Gawenis et al. 2007; Handlogten et al. 2015), these results indicate that NBCe1 expression is necessary for the normal regulation of NaDC‐1 expression. NaDC‐1 mRNA expression was determined using Taqman real‐time RT‐PCR, and normalized to GAPDH expression. N = 4 in all samples. Results are normalized such that wild‐type expression, for pups, and control diet expression, for adult mice, is 100.0%. Because this approach is used in all experiments quantifying mRNA expression, this information is not repeated in subsequent figure legends.

Figure 3

NaDC‐1 immunolabel in wild‐type and NBCe1 knock‐out pup kidneys. Top panels show characterization of NaDC‐1 antibody used in these studies. Panel A shows that apical immunolabel is present in proximal tubule segments (arrows) of wild‐type C57Bl/6 mice. Panel B shows that no detectable immunolabel is present in proximal tubule segments (identified by apical brush border) (black arrows) of mice with NaDC‐1 deletion. Results are representative of findings in four wild‐type and four NaDC‐1 KO kidneys. In middle panels, Panel C shows NaDC‐1 immunolabel in the cortex of wild‐type (WT) pups and Panel D shows NaDC‐1 immunolabel in the cortex of NBCe1 knockout (KO) kidney. Apical NaDC‐1 immunolabel intensity is decreased in proximal tubule cells (arrows) of NBCe1 KO as compared to wild‐type mice. In the bottom panels, Panel E shows NaDC‐1 immunolabel in the cortex of control mice and Panel F shows NaDC‐1 immunolabel in the cortex of acid‐loaded mice. Apical NaDC‐1 immunolabel is present in renal proximal tubule cells (“*”) and expression is increased in response to acid‐loading. Results are representative of findings in four mice in each condition.

Figure 4

NBCe1 deletion and 2‐oxoglutarate metabolism. Effects of NBCe1 deletion on expression of key enzymes involved in 2‐oxoglutarate generation (Glud1) and metabolism (Ogdh, Dlst and Dld). Because NBCe1 causes spontaneous metabolic acidosis, adult wild‐type mice with experimentally induced metabolic acidosis were also examined to allow separate assessment of effects of NBCe1 deletion and metabolic acidosis. NBCe1 KO did not alter Glud1 expression significantly, whereas experimental metabolic acidosis increased it significantly. Because NBCe1 KO mice have metabolic acidosis, this indicates that NBCe1 expression is necessary for normal Glud1 response to metabolic acidosis. NBCe1 KO decreased Dlst expression slightly, but significantly. There were no effects of either NBCe1 KO or experimental metabolic acidosis on the other components of the 2‐oxoglutarate dehydrogenase complex, Ogdh or Dld. N = 4 in all samples.

Figure 5

Effect of NBCe1 deletion on glutaminase II pathway. Top left panel shows effect of NBCe1 deletion on glutamine transaminase K (GTK) expression and top right panel shows effect on ω‐amidase expression using immunoblot analysis. NBCe1 deletion does not alter expression of either protein significantly. Bottom left panel shows effect of experimental metabolic acidosis on GTK expression and bottom right panel shows the effect of experimental metabolic acidosis on ω‐amidase expression. There was no significant effect of experimental metabolic acidosis on expression of either GTK or ω‐amidase.

Figure 6

Effect of NBCe1 deletion on 2‐oxoglutarate transport mechanisms. Left panel shows expression of the basolateral organic anion transporter, NaDC‐3. NBCe1 KO decreased NaDC‐3 expression significantly. Experimental metabolic acidosis, in contrast, increased NaDC‐3 expression significantly. Right panel shows expression of the putative apical 2‐oxoglutarate transporter, OAT10. NBCe1 KO decreased OAT10 expression slightly, but significantly. Experimental metabolic acidosis induced a quantitatively similar, but not statistically significant, decrease. N = 4 in all samples.

Figure 7

Effects of NBCe1 deletion and experimental metabolic acidosis on Oxgr1 mRNA expression. Left bars show effect of NBCe1 deletion on Oxgr1 expression. NBCe1 KO did not alter Oxgr1 expression significantly. Right bars show that experimental metabolic acidosis decreased Oxgr1 expression significantly. Results are normalized to WT expression, for pups, and to control diet, for adult mice. N = 4 in all samples.

Figure 8

NBCe1 deletion alters pendrin protein expression. Top panel shows immunoblot assay for pendrin in wild‐type and NBCe1 KO pup kidneys. Bottom panel shows quantification of immunoblot analysis. NBCe1 deletion decreased pendrin protein expression significantly.