Regulation of renal NaDC1 expression and citrate excretion by NBCe1-A

Abstract

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.

Keywords: Na+-dicarboxylate cotransporter 1; citrate; proximal tubule.

Fig. 1.

Na⁺-dicarboxylate cotransporter 1 (NaDC1) immunolabel in the normal mouse kidney. Top left: low-power micrograph of NaDC1 immunolabel in the normal mouse kidney. Top right: high-power micrograph of the cortical labyrinth. Apical immunolabel was present only in proximal convoluted tubule (PCT) segments. Bottom left: high-power micrograph in the medullary ray in the cortex. Apical immunolabel was present only in proximal straight tubules in the medullary ray (PST-MR). Bottom right: high-power micrograph in the outer stripe of the outer medulla. Apical immunolabel was present only in proximal straight tubules in the outer medulla (PST-OM). In no region was significant NaDC1 immunolabel evident in nonproximal tubule cells. *Proximal tubule lumen. Results are representative of findings in 10 mice.

Fig. 2.

Na⁺-dicarboxylate cotransporter 1 (NaDC1) immunolabel in acid-loaded normal mouse kidney. Top left: low-power micrograph of NaDC1 immunolabel. Increased immunolabel intensity in the cortex compared with the nonacid-loaded wild-type mouse was apparent. Top right: NaDC1 immunolabel involving the brush border in proximal convoluted tubule (PCT) segments in the cortical labyrinth. Moderate to more intense apical NaDC1 immunolabel was evident. Bottom left: apical NaDC1 immunolabel involving the brush border in proximal straight tubules in the medullary ray (PST-MR). Immunolabel intensity in both PCT and PST-MR appeared greater than in nonacid-loaded mouse kidneys (see Fig. 1). Bottom right: apical NaDC1 immunolabel involving the brush border in proximal straight tubules in the outer medulla (PST-OM). Immunolabel intensity appeared unchanged compared with nonacid-loaded mouse kidneys (see Fig. 1). Results are representative of findings in 12 mice. *Proximal tubule segments. In no region was significant NaDC1 immunolabel evident in nonproximal tubule cells.

Fig. 3.

Quantitative assessment of Na⁺-dicarboxylate cotransporter 1 (NaDC1) expression in the proximal tubule of normal animals comparing basal conditions with an acid-loading diet. Cell-specific NaDC1 immunolabel intensity was determined in the proximal convoluted tubules (PCT), proximal straight tubules in the medullary ray (PST-MR), and proximal straight tubules in the outer medulla (PST-OM) of normal mice that received either control or acid-loading diet (representative immunohistochemistry shown in Figs. 1 and 2). Acid loading increased expression significantly in the PCT and PST-MR but not in the PST-OM. n = 10 normal diet-fed mice and 12 acid diet-fed mice. NS, not significant.

Fig. 4.

Na⁺-dicarboxylate cotransporter 1 (NaDC1) immunolabel expression in kidneys from Na⁺-coupled electrogenic bicarbonate cotransporter A variant (NBCe1-A) knockout (KO) mice on a normal diet. Top left: low-power micrograph of NaDC1 immunolabel in a NBCe1-A KO kidney. Relatively low cortical immunolabel, but intact outer medullary NaDC1 immunolabel, was evident. Top right: high-power micrograph of NaDC1 immunolabel in proximal convoluted tubules (PCT) illustrating low-intensity apical NaDC1 immunolabel. Bottom left: low-intensity apical NaDC1 immunolabel involving the brush border in proximal straight tubules in the medullary ray (PST-MR). Bottom right: modestly more intense apical NaDC1 immunolabel involving the brush border in proximal straight tubules in the outer medulla (PST-OM). Results are representative of findings in 11 separate mice. *Proximal tubule segments. In no region was significant NaDC1 immunolabel evident in nonproximal tubule cells.

Fig. 5.

Na⁺-dicarboxylate cotransporter 1 (NaDC1) immunolabel in acid-loaded Na⁺-coupled electrogenic bicarbonate cotransporter A variant (NBCe1-A) knockout (KO) mouse kidneys. Top left: low-power micrograph of NaDC1 immunolabel in an acid-loaded NBCe1-A KO kidney. Low-intensity NaDC1 immunolabel was present in the cortical labyrinth, where intensity appeared more intense compared with that observed in nonacid-loaded KO mice (see Fig. 4) in proximal straight tubule segments in both the medullary ray (PST-MR) and outer medulla (PST-OM). Top right: higher magnification of low-intensity apical NaDC1 immunolabel in proximal convoluted tubules (PCT). Bottom left: substantially more intense apical NaDC1 immunolabel involving the brush border in the PST-MR compared with that observed in nonacid-loaded KO mice (see Fig. 4). Bottom right: intense apical NaDC1 immunolabel involving the brush border in the PST-OM. Results are representative of findings in 11 separate mice. *Proximal tubule segments. In no region was significant NaDC1 immunolabel evident in nonproximal tubule cells.

Fig. 6.

Effect of Na⁺-coupled electrogenic bicarbonate cotransporter A variant (NBCe1-A) deletion on the Na⁺-dicarboxylate cotransporter 1 (NaDC1) response to acid loading in proximal convoluted tubules (PCT). Left: quantitative analysis of NaDC1 immunolabel in the PCT of wild-type (WT) and NBCe1-A knockout (KO) mice under control conditions and after acid loading for 7 days. NBCe1-A deletion decreased NaDC1 immunolabel intensity significantly both under control conditions and after acid loading. Acid loading increased NaDC1 expression significantly in both genotypes. Right: the increase in NaDC1 immunolabel intensity in response to acid loading was significantly less in NBCe1-A KO mice than in WT mice. n = 8 for WT mice on normal and acid-loading diets and n = 11 for KO mice on normal and acid-loading diets.

Fig. 7.

Effect of Na⁺-coupled electrogenic bicarbonate cotransporter A variant (NBCe1-A) deletion on the Na⁺-dicarboxylate cotransporter 1 (NaDC1) response to acid loading in proximal straight tubules in the medullary ray (PST-MR). Left: quantitative analysis of NaDC1 expression in wild-type (WT) and NBCe1-A knockout (KO) mice under control conditions and after acid loading. NBCe1-A deletion decreased NaDC1 expression under control conditions and after acid loading. Acid loading increased NaDC1 expression in both WT and NBCe1-A KO mice. Right: the increase in response to acid loading did not differ significantly between WT and NBCe1-A KO mice. n = 7 for WT mice on normal and acid-loading diets and n = 11 for KO mice on normal and acid-loading diets. NS, not significant.

Fig. 8.

Effect of Na⁺-coupled electrogenic bicarbonate cotransporter A variant (NBCe1-A) deletion on the Na⁺-dicarboxylate cotransporter 1 (NaDC1) response to acid loading in proximal straight tubules in the outer medulla (PST-OM). Left: quantitative analysis of NaDC1 expression in the PST-OM in WT and NBCe1-A KO mice under control conditions and after acid loading. NBCe1-A deletion did not alter NaDC1 expression under control conditions significantly. Acid loading increased NaDC1 expression significantly in NBCe1-A KO mice but not in WT mice. Right: the NaDC1 response in the PST-OM to acid loading was significantly greater in NBCe1-A KO mice than in WT mice. n = 8 for WT mice on normal and acid-loading diets, n = 11 for KO mice under control conditions, and n = 12 for NBCe1-A KO mice after acid loading. NS, not significant.

Fig. 9.

Effect of acid loading and Na⁺-coupled electrogenic bicarbonate cotransporter A variant (NBCe1-A) deletion on urinary citrate excretion. Urinary citrate excretion was determined by NMR spectroscopy of 24-h urine collections in wild-type (WT) and NBCe1-A knockout (KO) mice before and after acid loading for 7 days. Urinary citrate excretion was significantly greater in NBCe1-A KO mice than in WT mice fed a normal diet. Acid loading significantly suppressed citrate excretion in both WT and NBCe1-A KO mice. After acid loading, there was no significant difference in citrate excretion between WT and NBCe1-A KO mice. n = 11 mice for each genotype. NS, not significant.

Fig. 10.

Effect of hypokalemia on Na⁺-dicarboxylate cotransporter 1 (NaDC1) expression in wild-type (WT) mice. Top and middle: NaDC1 immunohistochemistry in WT mice on the K⁺ control diet (top) and after the K⁺-free diet (middle) for 4 days. Bottom: results of quantitative immunohistochemistry for NaDC1 immunolabel. Results are normalized to mean proximal convoluted tubule (PCT) expression in WT mice on the K⁺ control diet equal to 100.0. K⁺-free diet increased NaDC1 expression significantly in PCT and proximal straight tubules in the medullary ray (PST-MR) but not in proximal straight tubules in the in the outer medulla (PST-OM). Results are from 5 mice on the K⁺ control diet and 6 mice on the K⁺-free diet. *Proximal tubule.

Fig. 11.

Na⁺-dicarboxylate cotransporter 1 (NaDC1) immunohistochemistry in response to hypokalemia in Na⁺-coupled electrogenic bicarbonate cotransporter A variant (NBCe1-A) knockout (KO) mice. Top and middle: NaDC1 immunohistochemistry in NBCe1-A KO mice on the K⁺control diet (top) and after the K⁺-free diet (middle) for 4 days. Bottom: results of quantitative immunohistochemistry for NaDC1 immunolabel. Results are normalized to mean proximal convoluted tubule (PCT) expression in wild-type (WT) mice on the K⁺ control diet equal to 100.0. The K⁺-free diet did not alter NaDC1 expression significantly in either the PCT, proximal straight tubules in the medullary ray (PST-MR), or proximal straight tubules in the in the outer medulla (PST-OM). Results are from 5 mice on the K⁺ control diet and 6 mice on the K⁺-free diet. *Proximal tubule.

Fig. 12.

Effect of Na⁺-coupled electrogenic bicarbonate cotransporter A variant (NBCe1-A) deletion on the urinary citrate response to a K⁺-free diet. Left: urine citrate excretion on the last day of the K⁺-control diet and then on day 4 of the K⁺-free diet in wild-type (WT) and NBCe1-A knockout (KO) mice. NBCe1-A deletion increased urine citrate excretion significantly in mice fed either the K⁺ control diet or K⁺-free diet. Right: the percent decrease in citrate excretion in response to the K⁺-free diet was significantly less in NBCe1-A KO mice than in WT mice. Results are from 6 WT mice and 6 KO mice.