Dicarboxylate Cotransporter Research Articles

SLC35G1 is a highly chloride-sensitive transporter responsible for the basolateral membrane transport in intestinal citrate absorption.

The intestinal absorption of essential nutrients, especially those not readily biosynthesized, is a critical physiological process for maintaining homeostasis. Numerous studies have indicated that intestinal absorption is mediated by various membrane transporters. Citrate, a crucial bioactive compound produced as an intermediate in the Krebs cycle, is absorbed in the small intestine through carrier-mediated systems because of its high hydrophilicity. While the luminal absorption of citrate is mediated by Na+-dicarboxylate cotransporter 1 (NaDC1/SLC13A2), the mechanism governing the release of the transported citrate into the bloodstream remains unknown. Here, we explored the transporters responsible for intestinal citrate absorption at the basolateral membrane, focusing on highly expressed orphan transporters in the small intestine as candidates. Consequently, SLC35G1, originally identified as a partner of stromal interaction molecule 1, a cell surface transmembrane glycoprotein, was found to play a role in the intestinal absorption of citrate at the basolateral membrane. Furthermore, our results revealed that SLC35G1-mediated citrate transport was diminished by chloride ions at physiologically relevant extracellular concentrations. This suggests that SLC35G1, to our best knowledge, is the first transporter identified to be extremely sensitive to chloride ions among those functioning on the basolateral membrane of intestinal epithelial cells. This study provides valuable insights into the intestinal absorption of citrate and significantly contributes to elucidating the poorly understood molecular basis of the intestinal absorption system.

Structural and functional implications of SLC13A3 and SLC9A6 mutations: an in silico approach to understanding intellectual disability

BackgroundIntellectual disability (ID) is a condition that varies widely in both its clinical presentation and its genetic underpinnings. It significantly impacts patients’ learning capacities and lowers their IQ below 70. The solute carrier (SLC) family is the most abundant class of transmembrane transporters and is responsible for the translocation of various substances across cell membranes, including nutrients, ions, metabolites, and medicines. The SLC13A3 gene encodes a plasma membrane-localized Na+/dicarboxylate cotransporter 3 (NaDC3) primarily expressed in the kidney, astrocytes, and the choroid plexus. In addition to three Na + ions, it brings four to six carbon dicarboxylates into the cytosol. Recently, it was discovered that patients with acute reversible leukoencephalopathy and a-ketoglutarate accumulation (ARLIAK) carry pathogenic mutations in the SLC13A3 gene, and the X-linked neurodevelopmental condition Christianson Syndrome is caused by mutations in the SLC9A6 gene, which encodes the recycling endosomal alkali cation/proton exchanger NHE6, also called sodium-hydrogen exchanger-6. As a result, there are severe impairments in the patient’s mental capacity, physical skills, and adaptive behavior.Methods and resultsTwo Pakistani families (A and B) with autosomal recessive and X-linked intellectual disorders were clinically evaluated, and two novel disease-causing variants in the SLC13A3 gene (NM 022829.5) and the SLC9A6 gene (NM 001042537.2) were identified using whole exome sequencing. Family-A segregated a novel homozygous missense variant (c.1478 C > T; p. Pro493Leu) in the exon-11 of the SLC13A3 gene. At the same time, family-B segregated a novel missense variant (c.1342G > A; p.Gly448Arg) in the exon-10 of the SLC9A6 gene. By integrating computational approaches, our findings provided insights into the molecular mechanisms underlying the development of ID in individuals with SLC13A3 and SLC9A6 mutations.ConclusionWe have utilized in-silico tools in the current study to examine the deleterious effects of the identified variants, which carry the potential to understand the genotype-phenotype relationships in neurodevelopmental disorders.

Case Report: Compound Heterozygous Variants of SLC13A3 Identified in a Chinese Patient With Acute Reversible Leukoencephalopathy and α-Ketoglutarate Accumulation

Background: SLC13A3 gene encodes the Na+/dicarboxylate cotransporter 3 (NaDC3), which locates on the plasma membrane and is mainly expressed in kidney, astrocytes and the choroid plexus. It imports four to six carbon dicarboxylates together with three Na+ ions into the cytosol. Nowadays, pathogenic variants of SLC13A3 gene were found to cause acute reversible leukoencephalopathy and α-ketoglutarate accumulation (ARLIAK) in patients. Here, we report two novel SLC13A3 variants c.185C>T (p.T62M) and c.331C>T (p.R111*) identified in a Chinese patient with ARLIAK.Case Presentation: The patient was a Chinese girl aged 13 years and 7 months old, who had acute, recurrent neurological deterioration during two febrile episodes. She presented with reversible leukoencephalopathy and increased urinary excretion of α-ketoglutarate. Genetic studies revealed compound heterozygous variants (c.185C>T, p.T62M, and c.331C>T, p.R111*) in SLC13A3, which had not been reported previously.Conclusions: These findings expand the variant spectrum of SLC13A3, providing the basis for the further study of this rare disease.

Abstract P015: Factors Altering Luminal Succinate Effect Blood Pressure

Luminal succinate (Suc) has been reported to activate its receptor Sucnr1, stimulate renin release and increase BP in certain contexts. NaDC1 (Na + dicarboxylate cotransporter), located only on apical membrane in the proximal tubule of the kidney, reabsorbs filtered citrate and Suc, and is upregulated in acidosis. NaDC1 regulation is key in preventing stones and in maintaining acid-base homeostasis; but its role in BP regulation is not known. We postulate that luminal Suc alters BP. Our purpose was to examine the role of NaDC1, luminal Suc, and acidosis in BP regulation. To address these issues, we used NaDC1 KO (knock out) and WT (wild type) mice on normal diet or 72 hr acid diet (AD). Acidosis should lower luminal Suc due to upregulation of NaDC1 in WT mice. AD was associated with statistically significant BP decreases in both male and female WT but not in NaDC1 KO. Clearance studies compared Suc infused (SI) with non-infused NaDC1 KO and WT. ANOVA showed borderline increases between some groups: WT males on AD, BP increased from 71.48 ± 2.30 to 81.63 ± 2.16 with SI; and NaDC1 KO females on normal diet BP increased from 86.20 ± 3.98 to 96.48 ± 3.39 with SI. KO was only associated with increased BP in males on AD (p=0.05). Recently Khamaysi et al (JASN 2019) found only activity-dependent BP was altered by Suc. So, in additional studies we used telemetry (BP/T) for 24/7 monitoring of NaDC1 KO vs WT. On normal diets, mean arterial pressure (MAP) was significantly higher in KO than in WT. MAP at 9 pm: KO 122.44 ± 1.85 vs WT 110.40 ± 2.59, p <0.002, 5 am: KO 118.81 ± 2.64 vs WT 103.18 ± 4.00, p <0.01 and 8 am: KO 106.86 ± 1.28 vs 99.22 ± 2.64, p <0.02. Acidosis appeared to lessen these BP/T differences. Thus, increased luminal delivery of succinate due to KO of NaDC1 yields significant MAP increases during active periods. We examined levels of Sucnr1 in kidney cortex using droplet digital PCR (data are expressed as copy numbers of target gene in 1 ng PCR reaction). Sucnr1 expression was higher in NaDC1 KO (1343.6 ± 51) vs WT (1052.8 ± 50, p< 0.007) mice on normal diet. When fed acid diet Sucnr1 expression in KO fell (1039.6 ± 33) but increased in WT (1148 ± 50, p <0.006). In sum luminal Suc predominantly via NaDC1 reabsorption, does influence BP but this is modified by a variety of factors such as acidosis and activity.

Short bowel upregulates oxalate and citrate transporters and increases CaOx crystal deposition in the rat hyperoxaluric kidney

Short bowel (SB) increases the risk of kidney stones. However, the underlying mechanism related to intrarenal changes is unclear. Here we examine SB may affect oxalate and citrate handlings on calcium oxalate (CaOx) crystal formation in the hyperoxaluric kidney. SB was induced by small intestine resection in male Wistar rats. Sham‐operation was performed the same surgery without resection. After 7 days of recovery, rats were then fed with 0.75% ethylene glycol (EG and SB+EG groups) for 28 days as a hyperoxaluric induction. The corresponding controls were fed with distilled water (control and SB groups). Blood and 24 h of urine were collected for analysis. Renal CaOx deposition was examined by Pizzolato’s staining. Oxalate transporters Slc26a2 and Slc26a6, and citrate transporter sodium‐dependent dicarboxylate cotransporter NaDC‐1 at their mRNA levels were examined (n=6 in each group). After 7 days of SB, hypocalciuria and hypocitraturia were found when compared with those in sham‐operated control rats. These were associated with hypocalcemia and low renal NaDC‐1 expression after 28 days. SB aggravated EG‐mediated CaOx crystal nephropathy by fostering hyperoxaluria, degree of supersaturation, and CaOx crystal deposition. Renal expression of Slc26a6 but not Slc26a2 was increased in SB+EG group when compared to those in EG or SB group. SB lowered NaDC‐1 expression, but it was reversed in SB+EG group. Intestinal expression of these transporters was similar among groups. We conclude that SB enhances CaOx crystal formation in the hyperoxaluric kidney. More oxalate and less citrate secretion due to Slc26a6 upregulation and insufficient NaDC‐1 expression, respectively, possibly contribute to the aggravated effect of SB on crystal formation.Support or Funding InformationThis research was supported by grants of the Far Eastern Memorial Hospital (FEMH‐2018‐C‐046, Tseng YS and Ma MC) and the Ministry of Science and Technology (MOST108‐2314‐B‐030‐004‐MY3, Ma MC), Taiwan.Hyperoxaluria and hypocitraturia in the short bowl (SB)‐treated hyperoxlauric rats.More prominent hyperoxaluria (A) and oxalate transporter Slc26a6 upregulation (B) was seen in the SB+EG group. Hypocitraturia were found in the SB group (C), this associated with citrate transporter NaDC‐1 downregulation (D). SB increased CaOx formation in the hyperoxaluric kidney (E). N=6 in each group. *P<0.05, EG vs. C group; #P<0.05, SB+EG vs. EG group; @P<0.05, SB+EG vs. SB groupFigure 1

Luminal Succinate and Acidosis: Effects on Blood Pressure

Succinate (Suc) is well known as a Krebs cycle intermediate but extracellular Suc has been associated with a variety of pathologic processes. A distinct GPCR receptor for Suc (Sucnr1) was identified in the kidney and some (but not all) studies have documented that Suc infusions increase blood pressure (BP) via paracrine signaling. Luminal Suc has been reported to activate Sucnr1, stimulate renin release and alter BP in certain circumstances.In the nephron, apical NaDC1 (Na+ dicarboxylate cotransporter) is located only in the proximal tubule, reabsorbs filtered citrate and Suc, and is upregulated in acidosis. Thus NaDC1’s regulation is important in preventing calcium stones and promoting acid‐base homeostasis via effects of citrate reabsorption but its role in BP regulation is not known. Our purpose was to explore the role of NaDC1, luminal Suc, and acidosis in BP regulation.To address these issues, we used NaDC1 KO and WT mice on normal diet or 72 hr acid diet (0.4 M HCl). In initial clearance (CL) studies, KO mice had increased urinary Suc and citrate but no statistical difference in BP even with the infusion of 1 mmol/kg Suc. Suc infused WT did not have increased BP in CL. Acid diet reduced urinary Suc in WT, but had no consistent effect on BP in either KO or WT in CL.Since recent reports suggested that Suc may affect BP exclusively during more active periods (nocturnal in mice), we additionally examined BP using telemetry (BP/T) for 24/7 monitoring. During 8 PM‐5 AM on a normal diet mean arterial pressure (MAP) was significantly higher in KO than in WT. Acidosis appeared to lessen these BP/T differences. As an example of this, Figure 1 shows MAP at selected time points in a 24 hour cycle, 8 AM, 9 PM, 5AM and 8AM.To determine whether Sucnr1 gene expression alterations could influence these results, droplet digital PCR (Bio Rad) was performed. Data are expressed as copy numbers of target gene in 1 ng PCR reaction. Thus with ddPCR we determined on normal diet, Sucnr1 expression levels in KO (1343.6 ± 51) are significantly increased from that in WT (1052.8 ± 50, p< 0.007). Furthermore, on 72 hr acid diet the expression of Sucnr1 in KO (1039.6 ± 33) fell while in WT increased to 1148 ± 50, p <0.006).In sum, on normal diet, Sucnr1 expression is significantly greater in NaDC1 KO vs WT and in chronic acidosis expression decreases in KO but increases in WT. On normal diet MAP in KO is significantly higher than in WT while acidosis diminishes these differences (by slightly lowering KO MAP but raising slightly WT MAP).These diverse findings illustrate that NaDC1 (and presumably luminal Suc) can regulate BP but that this regulation varies with time of day (or associated factors such as activity) and acidosis.Support or Funding InformationNIH, DCI PTRF, LA CaTSMAP differences in NaDC1 KO mice vs WT mice on a normal dietFigure 1

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

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.

Renal expression and urinary excretion of Na+/dicarboxylate cotransporter 1 (NaDC1) in obstructive nephropathy: a candidate biomarker for this pathology.

Obstructive nephropathy is characterized by alterations in renal function that depends on the degree and type of obstruction. To increase the knowledge about the physiopathological mechanisms involved in the renal damage associated with bilateral ureteral obstruction (BUO), we studied the renal expression and function (as urinary citrate excretion) of sodium-dependent dicarboxylate cotransporter (NaDC1) in rats. In addition, we evaluated the urinary excretion of NaDC1 as a candidate biomarker forthis pathology. Male Wistar rats underwent bilateral ureteral obstruction for 1 (BUO1), 2 (BUO2), 5 (BUO5), and 24 (BUO24) h or sham operation. After 24h of ureteral releasing, traditional parameters of renal function and citrate levels were determined, and NaDC1 levels were evaluated in total renal homogenates, apical plasma membranes, and urine by electrophoresis and Western blotting. Traditional parameters of renal function were only modified in BUO5 and BUO24. The renal expression of NaDC1 was decreased in BUO5 and BUO24, with a concomitant increase in urinary excretion of citrate. Moreover, the urinary excretion of NaDC1 increased after short times of ureteral obstruction (BUO1 and BUO2) and was positively correlated with the time elapsed after obstruction. The results obtained from the renal expression of NaDC1 could explain an adaptive mechanism to prevent the formation of kidney stones by increasing the levels of citrate, a calcium chelator. The urinary excretion of NaDC1 could be postulated as an early biomarker of obstructive nephropathy that also gives information about the duration of the obstruction.

Acid Stimulation of the Citrate Transporter NaDC-1 Requires Pyk2 and ERK1/2 Signaling Pathways.

Background Urine citrate is reabsorbed exclusively along the renal proximal tubule via the apical Na+-dicarboxylate cotransporter NaDC-1. We previously showed that an acid load in vivo and media acidification in vitro increase NaDC-1 activity through endothelin-1 (ET-1)/endothelin B (ETB) signaling. Here, we further examined the signaling pathway mediating acid-induced NaDC-1 activity.Methods We transiently transfected cultured opossum kidney cells, a model of the proximal tubule, with NaDC-1 and ETB and measured [14C]-citrate uptake after media acidification under various experimental conditions, including inactivation of Pyk2 and c-Src, which were previously shown to be activated by media acidification. Wild-type (Pyk2+/+) and Pyk2-null (Pyk2-/-) mice were exposed to NH4Cl loading and euthanized after various end points, at which time we harvested the kidneys for immunoblotting and brush border membrane NaDC-1 activity studies.Results Inhibition of Pyk2 or c-Src prevented acid stimulation but not ET-1 stimulation of NaDC-1 in vitro Consistent with these results, NH4Cl loading stimulated NaDC-1 activity in kidneys of wild-type but not Pyk2-/- mice. In cultured cells and in mice, ERK1/2 was rapidly phosphorylated by acid loading, even after Pyk2 knockdown, and it was required for acid but not ET-1/ETB stimulation of NaDC-1 in vitro Media acidification also induced the phosphorylation of Raf1 and p90RSK, components of the ERK1/2 pathway, and inhibition of these proteins blocked acid stimulation of NaDC-1 activity.Conclusions Acid stimulation of NaDC-1 activity involves Pyk2/c-Src and Raf1-ERK1/2-p90RSK signaling pathways, but these pathways are not downstream of ET-1/ETB in this process.

Sensors of Succinate: Neural Stem Cell Grafts Fight Neuroinflammation.

In this issue of Cell Stem Cell, Peruzzotti-Jametti etal. (2018) demonstrate how neural stem cells, transplanted in a mouse model of multiple sclerosis, respond to extracellular succinate and modulate neuroinflammation by releasing anti-inflammatory prostaglandin E2 and scavenging succinate. This mechanism reduces CNS damage and ameliorates motor impairment.

Mapping Functionally Important Residues in the Na+/Dicarboxylate Cotransporter, NaDC1.

Transporters from the SLC13 family couple the transport of two to four Na+ ions with a di- or tricarboxylate, such as succinate or citrate. We have previously modeled mammalian members of the SLC13 family, including the Na+/dicarboxylate cotransporter NaDC1 (SLC13A2), based on a structure of the bacterial homologue VcINDY in an inward-facing conformation with one sodium ion bound at the Na1 site. In the study presented here, we modeled the outward-facing conformation of rabbit and human NaDC1 (rbNaDC1 and hNaDC1, respectively) using an outward-facing model of VcINDY as a template and identified residues in or near the putative Na2 and Na3 cation binding sites. Guided by the structural models in both conformations, we performed site-directed mutagenesis in rbNaDC1 for residues proposed to be in the Na+ or substrate binding sites. Cysteine substitution of T474 in the predicted Na2 binding site results in an inactive protein. The M539C mutant has a low apparent affinity for both sodium and lithium cations, suggesting that M539 may form part of the putative Na3 binding site. The Y432C and T86C mutants have increased Km values for succinate, supporting their proposed location in the outward-facing substrate binding site. In addition, cysteine labeling by MTSEA-biotin shows that Y432C is accessible from the outside of the cell, and the accessibility changes in the presence or absence of Na+. The results of this study improve our understanding of substrate and ion recognition in the mammalian members of the SLC13 family and provide a framework for developing conformationally specific inhibitors against these transporters.

Evidence for epistatic interaction between VDR and SLC13A2 genes in the pathogenesis of hypocitraturia in recurrent calcium oxalate stone formers.

Genetic factors play a key role in the pathogenesis of hypocitraturia, a common risk factor for nephrolithiasis. The Na+-dicarboxylate cotransporter NaDC1, encoded by the sodium-dicarboxylate cotransporter (SLC13A2) gene, is a major determinant of urinary citrate excretion and its biological functions are regulated also by the vitamin D/Vitamin D receptor (VDR) biological system. The aim of this case-control study was to evaluate the possible epistatic interaction between VDR rs731236and SLC13A2 rs11567842 allelic variants in the pathogenesis of hypocitraturia. Recurrent calcium-oxalate stone formers (SF) with or without hypocitraturia and healthy controls (C) were genotyped. Gene-gene interactions were estimated by the 1.0 software package of multifactor dimensionality reduction (MDR). The prevalence of VDR TT and SLC13A2 GG genotypes was higher in hypocitraturic SF compared to C (odds ratio [OR] 3.24, 95 % confidence interval [CI] 1.38-7.60 for VDR TT vs. VDR tt and OR 4.06, 95 % CI 1.75-9.42 for SLC13A2 GG vs. SLC13A2 AA ). MDR analysis indicated a significant interaction between VDR TT and SLC13A2 GG in hypocitraturic SF compared to C [OR 3.81 (2.11-6.88)]. These data are compatible with an epistatic interaction between the VDR TT and SLC13A2 GG genotypes with a significant impact on the magnitude of the effect (suppressive effect). These results point to an epistatic interaction between the VDR and the SLC13A2 alleles in the pathogenesis of idiopathic hypocitraturia in calcium-oxalate SF.

Distribution of organic anion transporters NaDC3 and OAT1-3 along the human nephron.

The initial step in renal secretion of organic anions (OAs) is mediated by transporters in the basolateral membrane (BLM). Contributors to this process are primary active Na(+)-K(+)-ATPase (EC 3.6.3.9), secondary active Na(+)-dicarboxylate cotransporter 3 (NaDC3/SLC13A3), and tertiary active OA transporters (OATs) OAT1/SLC22A6, OAT2/SLC22A7, and OAT3/SLC22A8. In human kidneys, we analyzed the localization of these transporters by immunochemical methods in tissue cryosections and isolated membranes. The specificity of antibodies was validated with human embryonic kidney-293 cells stably transfected with functional OATs. Na(+)-K(+)-ATPase was immunolocalized to the BLM along the entire human nephron. NaDC3-related immunostaining was detected in the BLM of proximal tubules and in the BLM and/or luminal membrane of principal cells in connecting segments and collecting ducts. The thin and thick ascending limbs, macula densa, and distal tubules exhibited no reactivity with the anti-NaDC3 antibody. OAT1-OAT3-related immunostaining in human kidneys was detected only in the BLM of cortical proximal tubules; all three OATs were stained more intensely in S1/S2 segments compared with S3 segment in medullary rays, whereas the S3 segment in the outer stripe remained unstained. Expression of NaDC3, OAT1, OAT2, and OAT3 proteins exhibited considerable interindividual variability in both male and female kidneys, and sex differences in their expression could not be detected. Our experiments provide a side-by-side comparison of basolateral transporters cooperating in renal OA secretion in the human kidney.

Structural Characterization of Substrate Transport Selectivity of the SLC13 Family of Na+/Dicarboxylate Cotransporters

Members of the sodium dependent SLC13 transporter family mediate the transport of di and tricarboxylates of the citric acid cycle into cells. SLC13 transporters such as NaCT (SLC13A5) have been implicated in lifespan extension and resistance to high fat diets and are therefore emerging drug targets for aging and metabolic disorders. Here, we combine various computational and experimental methods to improve our understanding of the structural components that are important for substrate specificity in this physiologically important family. We first used homology modeling, site-directed mutagenesis and transport assays to characterize the human NaDC3 (SLC13A3) and identify key residues involved in substrate and Na+ binding. We then expanded our study and generated homology models for various SLC13 transporters from the human and mouse. Virtual screening of various small molecule libraries followed by experimental testing revealed nine previously unknown inhibitors of multiple members of the familly. Ligand optimization showed that differential ligand binding specificity among SLC13 family members is largely determined by the length of the carbon chain and the size of the ligands. Additionally, we identified two key positions that define a unique binding site for each SLC13 member. Our findings rationalize substrate specificity in the SLC13 family and provide a framework for the development of more potent inhibitors.

Structure-Based Identification of Inhibitors for the SLC13 Family of Na(+)/Dicarboxylate Cotransporters.

In mammals, citric acid cycle intermediates play a key role in regulating various metabolic processes, such as fatty acid synthesis and glycolysis. Members of the sodium-dependent SLC13 transporter family mediate the transport of di- and tricarboxylates into cells. SLC13 family members have been implicated in lifespan extension and resistance to high-fat diets; thus, they are emerging drug targets for aging and metabolic disorders. We previously characterized key structural determinants of substrate and cation binding for the human NaDC3/SLC13A3 transporter using a homology model. Here, we combine computational modeling and virtual screening with functional and biochemical testing, to identify nine previously unknown inhibitors for multiple members of the SLC13 family from human and mouse. Our results reveal previously unknown substrate selectivity determinants for the SLC13 family, including key residues that mediate ligand binding and transport, as well as promiscuous and specific SLC13 small molecule ligands. The newly discovered ligands can serve as chemical tools for further characterization of the SLC13 family or as lead molecules for the future development of potent inhibitors for the treatment of metabolic diseases and aging. Our results improve our understanding of the structural components that are important for substrate specificity in this physiologically important family as well as in other structurally related transport systems.

Citrate Transport in NaDC1 Knockout Mice

Urinary citrate is the major endogenous inhibitor of calcium nephrolithiasis. Regulation of urinary citrate has been presumed to occur solely by the sodium dependent dicarboxylate cotransporter (NaDC1) located on the apical membrane of the proximal tubule. Using NaDC1 knockout (KO) and heterozygous (Het) mice (originally generated by Ho et al Kidney Int 2007) along with their wild type litter (WT) mates, we studied citrate and other Krebs cycle intermediates in plasma and urine with mice on either a normal diet (Nl) or on a 72 hour acid loading protocol (AL). Plasma citrate concentrations did not differ substantially in any of the conditions. On Nl diets, fractional excretion (FE) of citrate was higher in KO animals (0.24±0.02) than in WT (0.13±0.05); Hets were intermediate (0.18±0.04). Therefore, although KO of NaDC1 increases citrate excretion, substantial reabsorption of citrate still occurs in these animals (fractional reabsorption = 1-FE). With acidosis FE in WT was nearly abolished (0.003±0.001) but did not change in the KO animals (0.24± 0.02 Nl vs 0.25±0.0.02 AL diet). Surprisingly Het animals responded somewhat to acidosis but not completely, FE of 0.10±0.06 with AL and 0.18±0.04 with Nl diet. Urinary excretion of other dicarboxylates paralleled that of citrate except that urinary excretion of each decreased significantly in KO animals with acidosis. Thus, in examining FE in NaDC1 KO mice we found that most of renal reabsorption of citrate is not via NaDC1; although NaDC1 appears to account for the response to acidosis for citrate. Interestingly, the reabsorption of other Krebs cycle intermediates did respond significantly to acidosis. Also and surprisingly, Het animals do not have normal citrate reabsorption indicating that two normal alleles are important for normal citrate transport. NIH/NIDDK

NaDC3 Induces Premature Cellular Senescence by Promoting Transport of Krebs Cycle Intermediates, Increasing NADH, and Exacerbating Oxidative Damage.

High-affinity sodium-dependent dicarboxylate cotransporter 3 (NaDC3) is a key metabolism-regulating membrane protein responsible for transport of Krebs cycle intermediates. NaDC3 is upregulated as organs age, but knowledge regarding the underlying mechanisms by which NaDC3 modulates mammalian aging is limited. In this study, we showed that NaDC3 overexpression accelerated cellular senescence in young human diploid cells (MRC-5 and WI-38) and primary renal tubular cells, leading to cell cycle arrest in G1 phase and increased expression of senescent biomarkers, senescence-associated β-galactosidase and p16. Intracellular levels of reactive oxygen species, 8-hydroxy-2'-deoxyguanosine, malondialdehyde, and carbonyl were significantly enhanced, and activities of respiratory complexes I and III and ATP level were significantly decreased in NaDC3-infected cells. Stressful premature senescent phenotypes induced by NaDC3 were markedly ameliorated via treatment with the antioxidants Tiron and Tempol. High expression of NaDC3 caused a prominent increase in intracellular levels of Krebs cycle intermediates and NADH. Exogenous NADH and NAD(+) may aggravate and attenuate the aging phenotypes induced by NaDC3, respectively. These results suggest that NaDC3 can induce premature cellular senescence by promoting the transport of Krebs cycle intermediates, increasing generation of NADH and reactive oxygen species and leading to oxidative damage. Our results clarify the aging signaling pathway regulated by NaDC3.

Transporters involved in renal excretion of N-carbamoylglutamate, an orphan drug to treat inborn n-acetylglutamate synthase deficiency.

Inborn defects in N-acetylglutamate (NAG) synthase (NAGS) cause a reduction of NAG, an essential cofactor for the initiation of the urea cycle. As a consequence, blood ammonium concentrations are elevated, leading to severe neurological disorders. The orphan drug N-carbamoylglutamate (NCG; Carbaglu), efficiently overcomes NAGS deficiency. However, not much is known about the transporters involved in the uptake, distribution, and elimination of the divalent organic anion NCG. Organic anion-transporting polypeptides (OATPs) as well as organic anion transporters (OATs) working in cooperation with sodium dicarboxylate cotransporter 3 (NaDC3) accept a wide variety of structurally unrelated drugs. To test for possible interactions with OATPs and OATs, the impact of NCG on these transporters in stably transfected human embryonic kidney-293 cells was measured. The two-electrode voltage-clamp technique was used to monitor NCG-mediated currents in Xenopus laevis oocytes that expressed NaDC3. Neither OATPs nor OAT2 and OAT3 interacted with NCG, but OAT1 transported NCG. In addition, NCG was identified as a high-affinity substrate of NaDC3. Preincubation of OAT4-transfected human embryonic kidney-293 cells with NCG showed an increased uptake of estrone sulfate, the reference substrate of OAT4, indicating efflux of NCG by OAT4. In summary, NaDC3 and, to a lesser extent, OAT1 are likely to be responsible for the uptake of NCG from the blood. Efflux of NCG across the luminal membrane into the tubular lumen probably occurs by OAT4 completing renal secretion of this drug.

A Na+-coupled C4-dicarboxylate transporter (Asuc_0304) and aerobic growth of Actinobacillus succinogenes on C4-dicarboxylates.

Actinobacillus succinogenes, which is known to produce large amounts of succinate during fermentation of hexoses, was able to grow on C4-dicarboxylates such as fumarate under aerobic and anaerobic conditions. Anaerobic growth on fumarate was stimulated by glycerol and the major product was succinate, indicating the involvement of fumarate respiration similar to succinate production from glucose. The aerobic growth on C4-dicarboxylates and the transport proteins involved were studied. Fumarate was oxidized to acetate. The genome of A. succinogenes encodes six proteins with similarity to secondary C4-dicarboxylate transporters, including transporters of the Dcu (C4-dicarboxylate uptake), DcuC (C4-dicarboxylate uptake C), DASS (divalent anion : sodium symporter) and TDT (tellurite resistance dicarboxylate transporter) family. From the cloned genes, Asuc_0304 of the DASS family protein was able to restore aerobic growth on C4-dicarboxylates in a C4-dicarboxylate-transport-negative Escherichia coli strain. The strain regained succinate or fumarate uptake, which was dependent on the electrochemical proton potential and the presence of Na(+). The transport had an optimum pH ~7, indicating transport of the dianionic C4-dicarboxylates. Transport competition experiments suggested substrate specificity for fumarate and succinate. The transport characteristics for C4-dicarboxylate uptake by cells of aerobically grown A. succinogenes were similar to those of Asuc_0304 expressed in E. coli, suggesting that Asuc_0304 has an important role in aerobic fumarate uptake in A. succinogenes. Asuc_0304 has sequence similarity to bacterial Na(+)-dicarboxylate cotransporters and contains the carboxylate-binding signature. Asuc_0304 was named SdcA (sodium-coupled C4-dicarboxylate transporter from A. succinogenes).

Determinants of Substrate and Cation Transport in the Human Na+/Dicarboxylate Cotransporter NaDC3

Metabolic intermediates, such as succinate and citrate, regulate important processes ranging from energy metabolism to fatty acid synthesis. Cytosolic concentrations of these metabolites are controlled, in part, by members of the SLC13 gene family. The molecular mechanism underlying Na(+)-coupled di- and tricarboxylate transport by this family is understood poorly. The human Na(+)/dicarboxylate cotransporter NaDC3 (SLC13A3) is found in various tissues, including the kidney, liver, and brain. In addition to citric acid cycle intermediates such as α-ketoglutarate and succinate, NaDC3 transports other compounds into cells, including N-acetyl aspartate, mercaptosuccinate, and glutathione, in keeping with its dual roles in cell nutrition and detoxification. In this study, we construct a homology structural model of NaDC3 on the basis of the structure of the Vibrio cholerae homolog vcINDY. Our computations are followed by experimental testing of the predicted NaDC3 structure and mode of interaction with various substrates. The results of this study show that the substrate and cation binding domains of NaDC3 are composed of residues in the opposing hairpin loops and unwound portions of adjacent helices. Furthermore, these results provide a possible explanation for the differential substrate specificity among dicarboxylate transporters that underpin their diverse biological roles in metabolism and detoxification. The structural model of NaDC3 provides a framework for understanding substrate selectivity and the Na(+)-coupled anion transport mechanism by the human SLC13 family and other key solute carrier transporters.