Sat1 is dispensable for active oxalate secretion in mouse duodenum

Mice deficient for the apical membrane oxalate transporter SLC26A6 develop hyperoxalemia, hyperoxaluria, and calcium oxalate stones due to a defect in intestinal oxalate secretion. However, the nature of the basolateral membrane oxalate transport process that operates in series with SLC26A6 to mediate active oxalate secretion in the intestine remains unknown. Sulfate anion transporter-1 (Sat1 or SLC26A1) is a basolateral membrane anion exchanger that mediates intestinal oxalate transport. Moreover, Sat1-deficient mice also have a phenotype of hyperoxalemia, hyperoxaluria, and calcium oxalate stones. We, therefore, tested the role of Sat1 in mouse duodenum, a tissue with Sat1 expression and SLC26A6-dependent oxalate secretion. Although the active secretory flux of oxalate across mouse duodenum was strongly inhibited (>90%) by addition of the disulfonic stilbene DIDS to the basolateral solution, secretion was unaffected by changes in medium concentrations of sulfate and bicarbonate, key substrates for Sat1-mediated anion exchange. Inhibition of intracellular bicarbonate production by acetazolamide and complete removal of bicarbonate from the buffer also produced no change in oxalate secretion. Finally, active oxalate secretion was not reduced in Sat1-null mice. We conclude that a DIDS-sensitive basolateral transporter is involved in mediating oxalate secretion across mouse duodenum, but Sat1 itself is dispensable for this process.     

Regulation of the sodium/sulfate co-transporter by farnesoid X receptor alpha

Fxralpha is known to regulate a variety of metabolic processes, including bile acid, cholesterol, and carbohydrate metabolism. In this study, we show direct evidence that Fxralpha is a key player in maintaining sulfate homeostasis. We identified and characterized the sodium/sulfate co-transporter (NaS-1; Slc13a1) as an Fxralpha target gene expressed in the kidney and intestine. Electromobility shift assays, chromatin immunoprecipitation, and promoter reporter studies identified a single functional Fxralpha response element in the second intron of the mouse Slc13a1 gene. Treatment of wild-type mice with GW4064, a synthetic Fxralpha agonist, induced Slc13a1 mRNA in the intestine and kidney. Slc13a1 mRNA was also induced in the kidney and intestine of wild-type, but not Fxralpha-/- mice, after treatment with the hepatotoxin alpha-naphthylisothiocyanate, which is known to result in elevated blood bile acid levels. Finally, we observed a decrease in Slc13a1 mRNA in the kidney and intestine of Fxralpha-/- mice and a corresponding increase in urinary excretion of free sulfates as compared with wild-type mice. These results demonstrate that mouse Slc13a1 is a novel Fxralpha target gene expressed in the kidney and intestine and that in the absence of Fxralpha, mice waste sulfate into the urine. Thus, Fxralpha is necessary for normal sulfate homeostasis in vivo.     

Plasma and Urinary Sulfate Determination in a Cohort with Autism

Sulfate is important for mammalian development but is not routinely measured in clinical settings. The renal NaS1 sulfate transporter maintains circulating sulfate levels and is linked to renal sulfate wasting in mice. Some autistic individuals exhibit renal sulfate wasting, but the etiology is yet unknown. We measured plasma and urinary sulfate levels, calculated the fractional excretion index (FEI) of sulfate, and screened for two loss-of-function NaS1 sequence variants (R12X and N174S) in 23 autistic individuals. The FEI sulfate values ranged from 0.13 to 0.50. NaS1 variants were detected in 18 of the 23 individuals (11 heterozygous N174S, four homozygous N174S, two heterozygous R12X, and one individual carried both R12X and N174S). Those individuals with neither sequence variant had FEI sulfate ≤ 0.34, whereas FEI sulfate ≥ 0.35 was found in about 60 % (11 of 18) of individuals that had R12X and/or N174S. This study links renal sulfate wasting with loss-of-function NaS1 sequence variants in humans.     

Renal and intestinal transport defects in Slc26a6-null mice

SLC26A6 (PAT1, CFEX) is an anion exchanger that is expressed on the apical membrane of the kidney proximal tubule and the small intestine. Modes of transport mediated by SLC26A6 include Cl-/formate exchange, Cl-/HCO3- exchange, and Cl-/oxalate exchange. To study its role in kidney and intestinal physiology, gene targeting was used to prepare mice lacking Slc26a6. Homozygous mutant Slc26a6-/- mice appeared healthy and exhibited a normal blood pressure, kidney function, and plasma electrolyte profile. In proximal tubules microperfused with a low-HCO3-/high-Cl- solution, the baseline rate of fluid absorption (Jv), an index of NaCl transport under these conditions, was the same in wild-type and null mice. However, the stimulation of Jv by oxalate observed in wild-type mice was completely abolished in Slc26a6-null mice (P<0.05). Formate stimulation of Jv was partially reduced in null mice, but the difference from the response in wild-type mice did not reach statistical significance. Apical membrane Cl-/base exchange activity, assayed with the pH-sensitive dye BCPCF in microperfused proximal tubules, was decreased by 58% in Slc26a6-/- animals (P<0.001 vs. wild types). In the duodenum, the baseline rate of HCO3- secretion measured in mucosal tissue mounted in Ussing chambers was decreased by approximately 30% (P<0.03), whereas the forskolin-stimulated component of HCO3- secretion was the same in wild-type and Slc26a6-/- mice. We conclude that Slc26a6 mediates oxalate-stimulated NaCl absorption, contributes to apical membrane Cl-/base exchange in the kidney proximal tubule, and also plays an important role in HCO3- secretion in the duodenum.     

Urinary Metabolic Phenotyping the slc26a6 (Chloride-Oxalate Exchanger) Null Mouse Model

The prevalence of renal stone disease is increasing, although it remains higher in men than in women when matched for age. While still somewhat controversial, several studies have reported an association between renal stone disease and hypertension, but this may be confounded by a shared link with obesity. However, independent of obesity, hyperoxaluria has been shown to be associated with hypertension in stone-formers, and the most common type of renal stone is composed of calcium oxalate. The chloride-oxalate exchanger slc26a6 (also known as CFEX or PAT-1), located in the renal proximal tubule, was originally thought to have an important role in sodium homeostasis and thereby blood pressure control, but it has recently been shown to have a key function in oxalate balance by mediating oxalate secretion in the gut. We have applied two orthogonal analytical platforms (NMR spectroscopy and capillary electrophoresis with UV detection) in parallel to characterize the urinary metabolic signatures related to the loss of the renal chloride-oxalate exchanger in slc26a6 null mice. Clear metabolic differentiation between the urinary profiles of the slc26a6 null and the wild type mice were observed using both methods, with the combination of NMR and CE-UV providing extensive coverage of the urinary metabolome. Key discriminating metabolites included oxalate, m-hydroxyphenylpropionylsulfate (m-HPPS), trimethylamine-N-oxide, glycolate and scyllo-inositol (higher in slc26a6 null mice) and hippurate, taurine, trimethylamine, and citrate (lower in slc26a6 null mice). In addition to the reduced efficiency of anion transport, several of these metabolites (hippurate, m-HPPS, methylamines) reflect alteration in gut microbial cometabolic activities. Gender-related metabotypes were also observed in both wild type and slc26a6 null groups. Urinary metabolites that showed a sex-specific pattern included trimethylamine, trimethylamine-N-oxide, citrate, spermidine, guanidinoacetate, and 2-oxoisocaproate. The gender-dependent metabolic expression of the consequences of slc26a6 deletion might have relevance to the difference in prevalence of renal stone formation in men and women. The different composition of microbial metabolites in the slc26a6 null mice is consistent with the fact that the slc26a6 transporter is found in a range of tissues, including the kidney and intestine, and provides further evidence for the "long reach" of the microbiota in physiological and pathological processes.     

Ileal oxalate absorption and urinary oxalate excretion are enhanced in Slc26a6 null mice

Intestinal oxalate transport, mediated by anion exchange proteins, is important to oxalate homeostasis and consequently to calcium oxalate stone diseases. To assess the contribution of the putative anion transporter (PAT)1 (Slc26a6) to transepithelial oxalate transport, we compared the unidirectional and net fluxes of oxalate across isolated, short-circuited segments of the distal ileum of wild-type (WT) mice and Slc26a6 null mice [knockout (KO)]. Additionally, urinary oxalate excretion was measured in both groups. In WT mouse ileum, there was a small net secretion of oxalate (J(net)(Ox) = -5.0 +/-5.0 pmol.cm(-2).h(-1)), whereas in KO mice J(net)(Ox) was significantly absorptive (75 +/- 10 pmol.cm(-2)h.h(-1)), which was the result of a smaller serosal-to-mucosal oxalate flux (J(sm)(Ox)) and a larger mucosal-to-serosal oxalate flux (J(ms)(Ox)). Mucosal DIDS (200 microM) reduced J(sm)(Ox) in WT mice, leading to reversal of the direction of net oxalate transport from secretion to absorption (J(net)(Ox) = 15.0 +/- 5.0 pmol.cm(-2).h(-1)) , but DIDS had no significant effect on KO ileum. In WT mice in the absence of mucosal Cl(-), there were small increases in J(ms)(Ox) and decreases in J(sm)(Ox) that led to a small net oxalate absorption. In KO mice, J(net)(Ox) was 1.5-fold greater in the absence of mucosal Cl(-), due solely to an increase in J(ms)(Ox). Urinary oxalate excretion was about fourfold greater in KO mice compared with WT littermates. We conclude that PAT1 is DIDS sensitive and mediates a significant fraction of oxalate efflux across the apical membrane in exchange for Cl(-); as such, PAT1 represents a major apical membrane pathway mediating J(sm)(Ox).     

Specificity of anion exchange mediated by mouse Slc26a6

Recently, CFEX, the mouse orthologue of human SLC26A6, was localized to the brush border membrane of proximal tubule cells and was demonstrated to mediate Cl(-)-formate exchange when expressed in Xenopus oocytes. The purpose of the present study was to examine whether mouse Slc26a6 can mediate one or more of the additional anion exchange processes observed to take place across the apical membrane of proximal tubule cells. Influx of [(14)C]formate into Slc26a6-expressing oocytes was inhibited by sulfate, oxalate, and p-aminohippurate (PAH), indicating affinity for these anions. Measurements of uptake of [(14)C]oxalate, [(14)C]PAH, and [(35)S]sulfate indicated that Slc26a6 can mediate transport of oxalate and sulfate but not PAH. Studies of the effect of external anions on [(14)C]oxalate efflux demonstrated Slc26a6-mediated Cl(-)-oxalate, oxalate-formate, oxalate-oxalate, and oxalate-sulfate exchange. Two-electrode voltage clamp measurements indicated that Slc26a6-mediated Cl(-)-oxalate exchange is electrogenic. Intracellular pH recordings demonstrated that Slc26a6 can mediate Cl(-)-HCO(3)(-) exchange, but Cl(-)-OH(-) exchange was not detected. The presence of 100 microm oxalate inhibited the rate of Cl(-)-HCO(3)(-) exchange by 60%. We conclude that mouse Slc26a6 has affinity for oxalate, sulfate, and HCO(3)(-) in addition to Cl(-) and formate and can function in multiple exchange modes involving pairs of these anions. In the presence of high oxalate concentrations as found in renal tubular fluid and urine, Slc26a6 may largely function as an electrogenic Cl(-)-oxalate exchanger.     

Roles of Slc13a1 and Slc26a1 sulfate transporters of eel kidney in sulfate homeostasis and osmoregulation in freshwater

Sulfate is required for proper cell growth and development of all organisms. We have shown that the renal sulfate transport system has dual roles in euryhaline eel, namely, maintenance of sulfate homeostasis and osmoregulation of body fluids. To clarify the physiological roles of sulfate transporters in teleost fish, we cloned orthologs of the mammalian renal sulfate transporters Slc13a1 (NaSi-1) and Slc26a1 (Sat-1) from eel (Anguilla japonica) and assessed their functional characteristics, tissue localization, and regulated expression. Full-length cDNAs coding for ajSlc13a1 and ajSlc26a1 were isolated from a freshwater eel kidney cDNA library. Functional expression in Xenopus oocytes revealed the expected sulfate transport characteristics; furthermore, both transporters were inhibited by mercuric chloride. Northern blot analysis, in situ hybridization, and immunohistochemistry demonstrated robust apical and basolateral expression of ajSlc13a1 and ajSlc26a1, respectively, within the proximal tubule of freshwater eel kidney. Expression was dramatically reduced after the transfer of eels from freshwater to seawater; the circulating sulfate concentration in eels was in turn markedly elevated in freshwater compared with seawater conditions (19 mM vs. 1 mM). The reabsorption of sulfate via the apical ajSlc13a1 and basolateral ajSlc26a1 transporters may thus contribute to freshwater osmoregulation in euryhaline eels, via the regulation of circulating sulfate concentration.     

Prevention of the Disrupted Enamel Phenotype in Slc4a4-Null Mice Using Explant Organ Culture Maintained in a Living Host Kidney Capsule

Slc4a4-null mice are a model of proximal renal tubular acidosis (pRTA). Slc4a4 encodes the electrogenic sodium base transporter NBCe1 that is involved in transcellular base transport and pH regulation during amelogenesis. Patients with mutations in the SLC4A4 gene and Slc4a4-null mice present with dysplastic enamel, amongst other pathologies. Loss of NBCe1 function leads to local abnormalities in enamel matrix pH regulation. Loss of NBCe1 function also results in systemic acidemic blood pH. Whether local changes in enamel pH and/or a decrease in systemic pH are the cause of the abnormal enamel phenotype is currently unknown. In the present study we addressed this question by explanting fetal wild-type and Slc4a4-null mandibles into healthy host kidney capsules to study enamel formation in the absence of systemic acidemia. Mandibular E11.5 explants from NBCe1^−/− mice, maintained in host kidney capsules for 70 days, resulted in teeth with enamel and dentin with morphological and mineralization properties similar to cultured NBCe1^+/+ mandibles grown under identical conditions. Ameloblasts express a number of proteins involved in dynamic changes in H⁺/base transport during amelogenesis. Despite the capacity of ameloblasts to dynamically modulate the local pH of the enamel matrix, at least in the NBCe1^−/− mice, the systemic pH also appears to contribute to the enamel phenotype. Extrapolating these data to humans, our findings suggest that in patients with NBCe1 mutations, correction of the systemic metabolic acidosis at a sufficiently early time point may lead to amelioration of enamel abnormalities.     

Defective intestinal amino acid absorption in Ace2 null mice

Mutations in the main intestinal and kidney luminal neutral amino acid transporter B(0)AT1 (Slc6a19) lead to Hartnup disorder, a condition that is characterized by neutral aminoaciduria and in some cases pellagra-like symptoms. These latter symptoms caused by low-niacin are thought to result from defective intestinal absorption of its precursor l-tryptophan. Since Ace2 is necessary for intestinal B(0)AT1 expression, we tested the impact of intestinal B(0)AT1 absence in ace2 null mice. Their weight gain following weaning was decreased, and Na(+)-dependent uptake of B(0)AT1 substrates measured in everted intestinal rings was defective. Additionally, high-affinity Na(+)-dependent transport of l-proline, presumably via SIT1 (Slc6a20), was absent, whereas glucose uptake via SGLT1 (Slc5a1) was not affected. Measurements of small intestine luminal amino acid content following gavage showed that more l-tryptophan than other B(0)AT1 substrates reach the ileum in wild-type mice, which is in line with its known lower apparent affinity. In ace2 null mice, the absorption defect was confirmed by a severalfold increase of l-tryptophan and of other neutral amino acids reaching the ileum lumen. Furthermore, plasma and muscle levels of glycine and l-tryptophan were significantly decreased in ace2 null mice, with other neutral amino acids displaying a similar trend. A low-protein/low-niacin diet challenge led to differential changes in plasma amino acid levels in both wild-type and ace2 null mice, but only in ace2 null mice to a stop in weight gain. Despite the combination of low-niacin with a low-protein diet, plasma niacin concentrations remained normal in ace2 null mice and no pellagra symptoms, such as photosensitive skin rash or ataxia, were observed. In summary, mice lacking Ace2-dependent intestinal amino acid transport display no total niacin deficiency nor clear pellagra symptoms, even under a low-protein and low-niacin diet, despite gross amino acid homeostasis alterations.     

Ascorbic-acid transporter Slc23a1 is essential for vitamin C transport into the brain and for perinatal survival

The only proven requirement for ascorbic acid (vitamin C) is in preventing scurvy, presumably because it is a cofactor for hydroxylases required for post-translational modifications that stabilize collagen. We have created mice deficient in the mouse ortholog (solute carrier family 23 member 1 or Slc23a1) of a rat ascorbic-acid transporter, Svct2 (ref. 4). Cultured embryonic fibroblasts from homozygous Slc23a1(-/-) mice had less than 5% of normal ascorbic-acid uptake. Ascorbic-acid levels were undetectable or markedly reduced in the blood and tissues of Slc23a1(-/-) mice. Prenatal supplementation of pregnant females did not elevate blood ascorbic acid in Slc23a1(-/-) fetuses, suggesting Slc23a1 is important in placental ascorbic-acid transport. Slc23a1(-/-) mice died within a few minutes of birth with respiratory failure and intraparenchymal brain hemorrhage. Lungs showed no postnatal expansion but had normal surfactant protein B levels. Brain hemorrhage was unlikely to be simply a form of scurvy since Slc23a1(-/-) mice showed no hemorrhage in any other tissues and their skin had normal skin 4-hydroxyproline levels despite low ascorbic-acid content. We conclude that Slc23a1 is required for transport of ascorbic acid into many tissues and across the placenta. Deficiency of the transporter is lethal in newborn mice, thereby revealing a previously unrecognized requirement for ascorbic acid in the perinatal period.     

Targeted disruption of Slc2a8 (GLUT8) reduces motility and mitochondrial potential of spermatozoa

GLUT8 is a class 3 sugar transport facilitator which is predominantly expressed in testis and also detected in brain, heart, skeletal muscle, adipose tissue, adrenal gland, and liver. Since its physiological function in these tissues is unknown, we generated a Slc2a8 null mouse and characterized its phenotype. Slc2a8 knockout mice appeared healthy and exhibited normal growth, body weight development and glycemic control, indicating that GLUT8 does not play a significant role for maintenance of whole body glucose homeostasis. However, analysis of the offspring distribution of heterozygous mating indicated a lower number of Slc2a8 knockout offspring (30.5:47.3:22.1%, Slc2a8(+/+), Slc2a8(+/-), and Slc2a8(-/-) mice, respectively) resulting in a deviation (p=0.0024) from the expected Mendelian distribution. This difference was associated with lower ATP levels, a reduced mitochondrial membrane potential and a significant reduction of sperm motility of the Slc2a8 knockout in comparison to wild-type spermatozoa. In contrast, number and survival rate of spermatozoa were not altered. These data indicate that GLUT8 plays an important role in the energy metabolism of sperm cells.     

PSGL-1 regulates the migration and proliferation of CD8(+) T cells under homeostatic conditions

P-selectin glycoprotein ligand-1 (PSGL-1), a heavily glycosylated sialomucin expressed on most leukocytes, has dual function as a selectin ligand for leukocyte rolling on vascular selectins expressed in inflammation and as a facilitator of resting T cell homing into lymphoid organs. In this article, we document disturbances in T cell homeostasis present in PSGL-1(null) mice. Naive CD4(+) and CD8(+) T cell frequencies were profoundly reduced in blood, whereas T cell numbers in lymph nodes and spleen were at or near normal levels. Although PSGL-1(null) T cells were less efficient at entering lymph nodes, they also remained in lymph nodes longer than PSGL-1(+/+) T cells, suggesting that PSGL-1 supports T cell egress. In addition, PSGL-1(null) CD8(+) T cell proliferation was observed under steady-state conditions and PSGL-1(null) CD8(+) T cells were found to be hyperresponsive to homeostatic cytokines IL-2, IL-4, and IL-15. Despite these disturbances in T cell homeostasis, PSGL-1(null) mice exhibited a normal acute response (day 8) to lymphocytic choriomeningitis virus infection but generated an increased frequency of memory T cells (day 40). Our observations demonstrate a novel pleiotropic influence of PSGL-1 deficiency on several aspects of T cell homeostasis that would not have been anticipated based on the mild phenotype of PSGL-1(null) mice. These potentially offsetting effects presumably account for the near-normal cellularity seen in lymph nodes of PSGL-1(null) mice.     

Critical role of vitamin D in sulfate homeostasis: regulation of the sodium-sulfate cotransporter by 1,25-dihydroxyvitamin D3

As the fourth most abundant anion in the body, sulfate plays an essential role in numerous physiological processes. One key protein involved in transcellular transport of sulfate is the sodium-sulfate cotransporter NaSi-1, and previous studies suggest that vitamin D modulates sulfate homeostasis by regulating NaSi-1 expression. In the present study, we found that, in mice lacking the vitamin D receptor (VDR), NaSi-1 expression in the kidney was reduced by 72% but intestinal NaSi-1 levels remained unchanged. In connection with these findings, urinary sulfate excretion was increased by 42% whereas serum sulfate concentration was reduced by 50% in VDR knockout mice. Moreover, levels of hepatic glutathione and skeletal sulfated proteoglycans were also reduced by 18 and 45%, respectively, in the mutant mice. Similar results were observed in VDR knockout mice after their blood ionized calcium levels and rachitic bone phenotype were normalized by dietary means, indicating that vitamin D regulation of NaSi-1 expression and sulfate metabolism is independent of its role in calcium metabolism. Treatment of wild-type mice with 1,25-dihydroxyvitamin D3 or vitamin D analog markedly stimulated renal NaSi-1 mRNA expression. These data provide strong in vivo evidence that vitamin D plays a critical role in sulfate homeostasis. However, the observation that serum sulfate and skeletal proteoglycan levels in normocalcemic VDR knockout mice remained low in the absence of rickets and osteomalacia suggests that the contribution of sulfate deficiency to development of rickets and osteomalacia is minimal.     

Targeted disruption of Slc2a8 (GLUT8) reduces motility and mitochondrial potential of spermatozoa

GLUT8 is a class 3 sugar transport facilitator which is predominantly expressed in testis and also detected in brain, heart, skeletal muscle, adipose tissue, adrenal gland, and liver. Since its physiological function in these tissues is unknown, we generated a Slc2a8 null mouse and characterized its phenotype. Slc2a8 knockout mice appeared healthy and exhibited normal growth, body weight development and glycemic control, indicating that GLUT8 does not play a significant role for maintenance of whole body glucose homeostasis. However, analysis of the offspring distribution of heterozygous mating indicated a lower number of Slc2a8 knockout offspring (30.5:47.3:22.1%, Slc2a8^+/+, Slc2a8^+/?, and Slc2a8^?/? mice, respectively) resulting in a deviation (p=0.0024) from the expected Mendelian distribution. This difference was associated with lower ATP levels, a reduced mitochondrial membrane potential and a significant reduction of sperm motility of the Slc2a8 knockout in comparison to wild-type spermatozoa. In contrast, number and survival rate of spermatozoa were not altered. These data indicate that GLUT8 plays an important role in the energy metabolism of sperm cells.