Oxalate Exchange Research Articles

Characterization of renal NaCl and oxalate transport in Slc26a6-/- mice.

The apical membrane Cl-/oxalate exchanger SLC26A6 has been demonstrated to play a role in proximal tubule NaCl transport based on studies in microperfused tubules. The present study is directed at characterizing the role of SLC26A6 in NaCl homeostasis in vivo under physiological conditions. Free-flow micropuncture studies revealed that volume and Cl- absorption were similar in surface proximal tubules of wild-type and Slc26a6-/- mice. Moreover, the increments in urine flow rate and sodium excretion following thiazide and furosemide infusion were identical in wild-type and Slc26a6-/- mice, indicating no difference in NaCl delivery out of the proximal tubule. The absence of an effect of deletion of SLC26A6 on NaCl homeostasis was further supported by the absence of lower blood pressure in Slc26a6-/- compared with wild-type mice on normal or low-salt diets. Moreover, raising plasma and urine oxalate by feeding mice a diet enriched in soluble oxalate did not affect mean blood pressure. In contrast to the lack of effect of SLC26A6 deletion on NaCl homeostasis, fractional excretion of oxalate was reduced from 1.6 in wild-type mice to 0.7 in Slc26a6-/- mice. We conclude that, although SLC26A6 is dispensable for renal NaCl homeostasis, it is required for net renal secretion of oxalate.

The apical anion exchanger Slc26a6 promotes oxalate secretion by murine submandibular gland acinar cells

The solute carrier family 26 (SLC26) gene family encodes at least 10 different anion exchangers. SLC26 member 6 (SLC26A6 or CFEX/PAT-1) and the cystic fibrosis transmembrane conductance regulator (CFTR) co-localize to the apical membrane of pancreatic duct cells, where they act in concert to drive HCO3- and fluid secretion. In contrast, in the small intestine, SLC26A6 serves as the major pathway for oxalate secretion. However, little is known about the function of Slc26a6 in murine salivary glands. Here, RNA sequencing-based transcriptional profiling and Western blots revealed that Slc26a6 is highly expressed in mouse submandibular and sublingual salivary glands. Slc26a6 localized to the apical membrane of salivary gland acinar cells with no detectable immunostaining in the ducts. CHO-K1 cells transfected with mouse Slc26a6 exchanged Cl- for oxalate and HCO3-, whereas two other anion exchangers known to be expressed in salivary gland acinar cells, Slc4a4 and Slc4a9, mediated little, if any, Cl-/oxalate exchange. Of note, both Cl-/oxalate exchange and Cl-/HCO3- exchange were significantly reduced in acinar cells isolated from the submandibular glands of Slc26a6-/- mice. Oxalate secretion in submandibular saliva also decreased significantly in Slc26a6-/- mice, but HCO3- secretion was unaffected. Taken together, our findings indicate that Slc26a6 is located at the apical membrane of salivary gland acinar cells, where it mediates Cl-/oxalate exchange and plays a critical role in the secretion of oxalate into saliva.

Показники уратно-оксалатного обміну в осіб з цукровим діабетом, що зазнали малоінвазивного лікування з приводу уретеролітіазу

The aim of the study: was to study the state of urate oxalate metabolism in patients with ureterolithiasis and concomitant diabetes mellitus, who underwent various methods of minimally invasive surgical treatment of urinary stones. Materials and methods. The study involved 204 people, of which men were 130 (63.7%), women – 74 (36.3%), the average age was 34.6±5.5 years. The study groups were divided into VI clinical groups: I – individuals with diabetes and ureterolithiasis, who underwent TUCL and cytrate per os (n=57); II – persons with DM and ureterolithiasis, who were TUCL (n=44); III – persons with DM and , who were administered by the ESWL (n=32); IV – persons with ureterolithiasis without DM, who underwent TUCL (n=41); V (control 1) – of the person with diabetes, who had an independent passage of stones from the ureter (n=18); VI (control 2) – healthy volunteers, with no signs of pathology (n=12). The diagnosis of the ureterolithiasis was verified using clinico-anamnestic, laboratory and instrumental research methods according to the methabolic homeostasis protocols, before and after the disintegration of the stones by the method of transurethral contact lithotripsy (TUСL) and ESWL. Results. Analysis of the above data showed that, in the presence of ureterolithiasis, there were significant changes in diurnal pH values in the form of monotony of the curve of indicators, with a significantly high shift towards acidification. Persistently acidic urine reflects the peculiarities of the urate urolythiasis in conditions of NAM, indicates the absence of an isolated violation by acute calculous obstruction and defines this state as a bilateral process. Hypocytraturia was established in all cases of ureterolithiasis, but in the urine of people with diabetes excretion rates of citrate were as low as possible. The conclusion. In patients with diabetes mellitus and concomitant ureterolithiasis there are significant disruptions in the exchange of uric acid and oxalate, with the possibility of correction and complete restoration of urodynamics with the help of the TUСL procedure. Key words: ureterolithiasis, diabetes mellitus, urates, oxalates, minimally invasive methods of lithotripsy.

N-glycosylation critically regulates function of oxalate transporter SLC26A6.

The brush border Cl--oxalate exchanger SLC26A6 plays an essential role in mediating intestinal secretion of oxalate and is crucial for the maintenance of oxalate homeostasis and the prevention of hyperoxaluria and calcium oxalate nephrolithiasis. Previous in vitro studies have suggested that SLC26A6 is heavily N-glycosylated. N-linked glycosylation is known to critically affect folding, trafficking, and function in a wide variety of integral membrane proteins and could therefore potentially have a critical impact on SLC26A6 function and subsequent oxalate homeostasis. Through a series of enzymatic deglycosylation studies we confirmed that endogenously expressed mouse and human SLC26A6 are indeed glycosylated, that the oligosaccharides are principally attached via N-glycosidic linkage, and that there are tissue-specific differences in glycosylation. In vitro cell culture experiments were then used to elucidate the functional significance of the addition of the carbohydrate moieties. Biotinylation studies of SLC26A6 glycosylation mutants indicated that glycosylation is not essential for cell surface delivery of SLC26A6 but suggested that it may affect the efficacy with which it is trafficked and maintained in the plasma membrane. Functional studies of transfected SLC26A6 demonstrated that glycosylation at two sites in the putative second extracellular loop of SLC26A6 is critically important for chloride-dependent oxalate transport and that enzymatic deglycosylation of SLC26A6 expressed on the plasma membrane of intact cells strongly reduced oxalate transport activity. Taken together, these studies indicated that oxalate transport function of SLC26A6 is critically dependent on glycosylation and that exoglycosidase-mediated deglycosylation of SLC26A6 has the capacity to profoundly modulate SLC26A6 function.

Loss of Cystic Fibrosis Transmembrane Regulator Impairs Intestinal Oxalate Secretion.

Patients with cystic fibrosis have an increased incidence of hyperoxaluria and calcium oxalate nephrolithiasis. Net intestinal absorption of dietary oxalate results from passive paracellular oxalate absorption as modified by oxalate back secretion mediated by the SLC26A6 oxalate transporter. We used mice deficient in the cystic fibrosis transmembrane conductance regulator gene (Cftr) to test the hypothesis that SLC26A6-mediated oxalate secretion is defective in cystic fibrosis. We mounted isolated intestinal tissue from C57BL/6 (wild-type) and Cftr-/- mice in Ussing chambers and measured transcellular secretion of [14C]oxalate. Intestinal tissue isolated from Cftr-/- mice exhibited significantly less transcellular oxalate secretion than intestinal tissue of wild-type mice. However, glucose absorption, another representative intestinal transport process, did not differ in Cftr-/- tissue. Compared with wild-type mice, Cftr-/- mice showed reduced expression of SLC26A6 in duodenum by immunofluorescence and Western blot analysis. Furthermore, coexpression of CFTR stimulated SLC26A6-mediated Cl--oxalate exchange in Xenopus oocytes. In association with the profound defect in intestinal oxalate secretion, Cftr-/- mice had serum and urine oxalate levels 2.5-fold greater than those of wild-type mice. We conclude that defective intestinal oxalate secretion mediated by SLC26A6 may contribute to the hyperoxaluria observed in this mouse model of cystic fibrosis. Future studies are needed to address whether similar mechanisms contribute to the increased risk for calcium oxalate stone formation observed in patients with cystic fibrosis.

Functional Significance of Glycosylation of Cl‐/oxalate exchanger Slc26a6

The brush border Cl‐/oxalate exchanger, Slc26a6, plays a crucial role in enteric oxalate secretion. Mice devoid of Slc26a6 develop profound hyperoxalemia, hyperoxaluria, and calcium oxalate urolithiasis. Modifications such as glycosylation that alter Slc26a6 trafficking or function may therefore be important determinants of oxalate homeostasis. The study aims are to confirm Slc26a6 glycosylation and to characterize its functional significance. By use of glycosidic enzyme digestions and site‐specific mutagenesis of transfected human Slc26a6 expressed in OKP cells, we demonstrate that Slc26a6 is glycosylated and that carbohydrate residues are added via N‐glycosidic linkage at two distinct sites in the putative second extracellular loop. Biotinylation studies suggest that glycosylation is not absolutely essential for surface delivery but does dramatically affect the efficiency by which Slc26a6 is processed and delivered to the cell surface. Moreover, cell‐surface biotinylation trafficking studies indicate that rates of endocytic retrieval of enzymatically deglycosylated Slc26a6 are 2‐3 fold higher than that observed with fully glycosylated Slc26a6, further supporting a role for glycosylation in Slc26a6 trafficking. Finally, investigation of the intrinsic transport function of Slc26a6, assayed as Cl‐ gradient‐dependent uptake of [14C]oxalate normalized to cell‐surface expression, demonstrate that deglycosylation achieved through either mutation or enzymatic digest consistently reduces transport rates by up to 75%. In conclusion, we confirm that Slc26a6 is glycosylated and demonstrate that the presence of glycan residues at two glycosylation sites is critically important for both protein trafficking and oxalate transport function. NIH DK33793.

Extracellular nucleotides inhibit oxalate transport by human intestinal Caco-2-BBe cells through PKC-δ activation

Nephrolithiasis remains a major health problem in Western countries. Seventy to 80% of kidney stones are composed of calcium oxalate, and small changes in urinary oxalate affect risk of kidney stone formation. Intestinal oxalate secretion mediated by the anion exchanger SLC26A6 plays an essential role in preventing hyperoxaluria and calcium oxalate nephrolithiasis, indicating that understanding the mechanisms regulating intestinal oxalate transport is critical for management of hyperoxaluria. Purinergic signaling modulates several intestinal processes through pathways including PKC activation, which we previously found to inhibit Slc26a6 activity in mouse duodenal tissue. We therefore examined whether purinergic stimulation with ATP and UTP affects oxalate transport by human intestinal Caco-2-BBe (C2) cells. We measured [¹⁴C]oxalate uptake in the presence of an outward Cl⁻ gradient as an assay of Cl⁻/oxalate exchange activity, ≥50% of which is mediated by SLC26A6. We found that ATP and UTP significantly inhibited oxalate transport by C2 cells, an effect blocked by the PKC inhibitor Gö-6983. Utilizing pharmacological agonists and antagonists, as well as PKC-δ knockdown studies, we observed that ATP inhibits oxalate transport through the P2Y₂ receptor, PLC, and PKC-δ. Biotinylation studies showed that ATP inhibits oxalate transport by lowering SLC26A6 surface expression. These findings are of potential relevance to pathophysiology of inflammatory bowel disease-associated hyperoxaluria, where supraphysiological levels of ATP/UTP are expected and overexpression of the P2Y₂ receptor has been reported. We conclude that ATP and UTP inhibit oxalate transport by lowering SLC26A6 surface expression in C2 cells through signaling pathways including the P2Y₂ purinergic receptor, PLC, and PKC-δ.

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.

Hepatic and renal sat‐1 and CFEX in ethylene glycol‐induced oxalate nephrolithiasis in rats

The epidemiological incidence of oxalate (OX) nephrolithiasis is higher in men than in women. More than 85% of the body OX is produced in liver and then released into the systemic circulation by the action of sulfate‐OX exchanger sat1 in the hepatocyte sinusoidal membrane. In kidneys, sat1 in the proximal tubule basolateral membrane mediates OX uptake, whereas the chloride‐formate‐ oxalate exchanger CFEX in the brush‐border membrane finally extrudes OX into the urine. Here we used the model of ethylene glycol (EG)‐induced OX nephrolithiasis in adult male (M) and female (F) rats to study sat1, CFEX, and rate‐limiting enzymes of OX synthesis, alcohol dehydrogenase (Adh1) and hydroxy acid oxidase (Hao1). Compared to controls, EG‐treated animals exhibited higher concentrations of OX in plasma and urine and higher abundance of OX crystals in urine, with a M dominance. Sat1 protein in liver and kidneys, which was M‐dominant in control animals, in EG‐treated animals increased only in F rats, while sat1 mRNA stayed unchanged. CFEX mRNA in both organs was sex‐independent and unaffected by EG treatment. Adh1 and Hao1 mRNA in both organs remained also unchanged upon EG treatment. In conclusion, despite hyperoxaluria in EG‐treated animals, the expression of sat1 in M and of CFEX in both sexes was sufficient to handle the EG‐induced production and secretion of OX.

Parsing apical oxalate exchange in Caco-2BBe1 monolayers: siRNA knockdown of SLC26A6 reveals the role and properties of PAT-1

The purpose of this investigation was to quantitate the contribution of the anion exchanger PAT-1 (putative anion transporter-1), encoded by SLC26A6, to oxalate transport in a model intestinal epithelium and to discern some characteristics of this exchanger expressed in its native environment. Control (Con) Caco-2 BBe1 monolayers, 6-8 days postseeding, were compared with those transfected with a small interfering RNA targeted to SLC26A6 (A6KD). Radiotracer and Ussing chamber techniques were used to determine the transepithelial unidirectional fluxes of Ox(2-), Cl(-), and SO(4)(2-) whereas fluorometric/BCECF measurements of intracellular pH were used to assess HCO(3)(-) exchange. PAT-1 was functionally targeted to the apical membrane, and SLC26A6 knockdown reduced PAT-1 protein (>60%) and mRNA (>75%) expression in A6KD. No net flux of Ox(2-), Cl(-), or SO(4)(2-) was detected in Con or A6KD monolayers, yet the unidirectional fluxes in A6KD were reduced 50, 35, and 15%, respectively. Cl(-)-dependent HCO(3)(-) efflux from A6KD was reduced 50% compared with Con. The difference between Con and A6KD properties represents that mediated solely by PAT-1, and by this approach we found that PAT-1-mediated oxalate influx and efflux are inhibited equally by mucosal DIDS (EC(50) approximately 5 microM) and that mucosal Cl(-) inhibits oxalate uptake with an EC(50) < 20 mM. Transepithelial Cl(-) gradients supported large, DIDS-sensitive net absorptive or secretory fluxes of oxalate in a direction opposite that of the imposed Cl(-) gradient. The overall symmetry of PAT-1-mediated oxalate exchange suggests that vectorial oxalate transport observed in vivo is principally dependent on the magnitude and direction of counterion gradients.

Oxalate in renal stone disease: the terminal metabolite that just won't go away

The incidence of calcium oxalate nephrolithiasis in the US has been increasing throughout the past three decades. Biopsy studies show that both calcium oxalate nephrolithiasis and nephrocalcinosis probably occur by different mechanisms in different subsets of patients. Before more-effective medical therapies can be developed for these conditions, we must understand the mechanisms governing the transport and excretion of oxalate and the interactions of the ion in general and renal physiology. Blood oxalate derives from diet, degradation of ascorbate, and production by the liver and erythrocytes. In mammals, oxalate is a terminal metabolite that must be excreted or sequestered. The kidneys are the primary route of excretion and the site of oxalate's only known function. Oxalate stimulates the uptake of chloride, water, and sodium by the proximal tubule through the exchange of oxalate for sulfate or chloride via the solute carrier SLC26A6. Fecal excretion of oxalate is stimulated by hyperoxalemia in rodents, but no similar phenomenon has been observed in humans. Studies in which rats were treated with (14)C-oxalate have shown that less than 2% of a chronic oxalate load accumulates in the internal organs, plasma, and skeleton. These studies have also demonstrated that there is interindividual variability in the accumulation of oxalate, especially by the kidney. This Review summarizes the transport and function of oxalate in mammalian physiology and the ion's potential roles in nephrolithiasis and nephrocalcinosis.

The Roles and Mechanisms of Intestinal Oxalate Transport in Oxalate Homeostasis

The mammalian intestine has an important role in the dynamics of oxalate exchange and thereby is significant in the etiology of calcium oxalate nephrolithiasis. Here we review some of the phenomenologic observations that have led to the conclusion that anion exchangers (antiporters) are important mediators of secondarily active, net oxalate transport along the intestine (both absorptive and secretory). Understanding the mechanisms of transepithelial oxalate transport has been advanced radically in recent years by the identification of the solute-linked carrier (SLC)26 family of anion transporters, which has facilitated the identification of specific proteins mediating individual apical or basolateral oxalate transport pathways. Moreover, identification of specific exchangers has underscored their relative importance to oxalate homeostasis as revealed by using knockout mouse models and has facilitated studies of oxalate transport regulation in heterologous expression systems. Finally, the significance of oxalate degrading bacteria to oxalate homeostasis is considered from basic and applied perspectives.

The role of SLC26A6‐mediated chloride/oxalate exchange in causing susceptibility to nephrolithiasis

The role of SLC26A6‐mediated chloride/oxalate exchange in causing susceptibility to nephrolithiasis

Specificity and Regulation of Renal Sulfate Transporters

Sulfate is essential for normal cellular function. The kidney plays a major role in sulfate homeostasis. Sulfate is freely filtered and then undergoes net reabsorption in the proximal tubule. The apical membrane Na(+)/sulfate cotransporter NaS1 (SLC13A1) has a major role in mediating proximal tubule sulfate reabsorption, as demonstrated by the findings of hyposulfatemia and hypersulfaturia in Nas1-null mice. The anion exchanger SAT1 (SLC26A1), the founding member of the SLC26 sulfate transporter family, mediates sulfate exit across the basolateral membrane to complete the process of transtubular sulfate reabsorption. Another member of this family, CFEX (SLC26A6), is present at the apical membrane of proximal tubular cells. It also can transport sulfate by anion exchange, which probably mediates backflux of sulfate into the lumen. Knockout mouse studies have demonstrated a major role of CFEX as an apical membrane Cl(-)/oxalate exchanger that contributes to NaCl reabsorption in the proximal tubule. Several additional SLC26 family members mediate sulfate transport and show some level of renal expression (e.g., SLC26A2, SLC26A7, SLC26A11). Their roles in mediating renal tubular sulfate transport are presently unknown. This paper reviews current data available on the function and regulation of three sulfate transporters (NaS1, SAT1, and CFEX) and their physiological roles in the kidney.

Essential roles of CFEX-mediated Cl−–oxalate exchange in proximal tubule NaCl transport and prevention of urolithiasis

The majority of the Na(+) and Cl(-) filtered by the kidney is reabsorbed in the proximal tubule. In this nephron segment, a significant fraction of Cl(-) is transported via apical membrane Cl(-)-base exchange: Cl(-)-formate exchange, Cl(-)-oxalate exchange, Cl(-)-OH(-) exchange, and Cl(-)-HCO(3)(-) exchange. A search for the transporter responsible for apical membrane Cl(-)-formate exchange in the proximal tubule led to the identification of CFEX (SLC26A6). Functional expression studies in Xenopus oocytes demonstrated that CFEX is capable of mediating not only Cl(-)-formate exchange but also Cl(-)-oxalate exchange, Cl(-)-OH(-) exchange, and Cl(-)-HCO(3)(-) exchange. Studies in CFEX-null mice have begun to elucidate which of the anion exchange activities mediated by CFEX is important for renal physiology and pathophysiology in vivo. Measurements of transport in renal brush border vesicles isolated from CFEX-null mice demonstrated that CFEX primarily mediates Cl(-)-oxalate exchange rather than Cl(-)-formate exchange. Microperfusion studies in CFEX-null mice revealed that CFEX plays an essential role in mediating oxalate-dependent NaCl absorption in the proximal tubule. CFEX-null mice were found to have hyperoxaluria and a high incidence of calcium oxalate urolithiasis. The etiology of hyperoxaluria in CFEX-null mice was observed to be a defect in oxalate secretion in the intestine, leading to enhanced net absorption of ingested oxalate and elevation of plasma oxalate. Thus, by virtue of its function as a Cl(-)-oxalate exchanger, CFEX plays essential roles both in proximal tubule NaCl transport and in the prevention of hyperoxaluria and calcium oxalate nephrolithiasis.

Calcium oxalate urolithiasis in mice lacking anion transporter Slc26a6

Urolithiasis is one of the most common urologic diseases in industrialized societies. Calcium oxalate is the predominant component in 70-80% of kidney stones, and small changes in urinary oxalate concentration affect the risk of stone formation. SLC26A6 is an anion exchanger expressed on the apical membrane in many epithelial tissues, including kidney and intestine. Among its transport activities, SLC26A6 mediates Cl(-)-oxalate exchange. Here we show that mutant mice lacking Slc26a6 develop a high incidence of calcium oxalate urolithiasis. Slc26a6-null mice have significant hyperoxaluria and elevation in plasma oxalate concentration that is greatly attenuated by dietary oxalate restriction. In vitro flux studies indicated that mice lacking Slc26a6 have a defect in intestinal oxalate secretion resulting in enhanced net absorption of oxalate. We conclude that the anion exchanger SLC26A6 has a major constitutive role in limiting net intestinal absorption of oxalate, thereby preventing hyperoxaluria and calcium oxalate urolithiasis.

Role of PDZK1 in membrane expression of renal brush border ion exchangers.

Na-H exchanger NHE3 and Cl-anion exchanger CFEX (SLC26A6, PAT1) play principal roles in the reabsorption of Na and Cl in the proximal tubule of the mammalian kidney. The mechanisms by which NHE3 and CFEX are localized to and maintained in the brush border of the proximal tubule are largely unknown. To investigate the possible interaction of NHE3 and CFEX with the PDZ-domain-containing scaffolding protein PDZK1, we performed a series of in vitro interaction assays with GST-fusion proteins and native brush border membrane proteins. These studies demonstrated that, not only were NHE3 and CFEX capable of directly interacting with PDZK1, but that this interaction was mediated through their C-terminal PDZ-interaction sites. To determine whether PDZK1 interaction is essential for brush border localization of NHE3 and CFEX in vivo, we examined the expression of NHE3 and CFEX in kidneys of wild-type and PDZK1-null mutant mice by both Western analysis and immunocytochemistry. These studies indicated that, although brush border expression of NHE3 was unaffected by the loss of PDZK1, the expression of CFEX was markedly reduced. Finally, we assayed CFEX functional activity as Cl-oxalate exchange in brush border membrane vesicles and oxalate-stimulated volume absorption in microperfused proximal tubules. Consistent with the observed decrease in CFEX protein expression, both measures of CFEX functional activity were dramatically reduced in PDZK1-null animals. In conclusion, the scaffolding protein PDZK1 is essential for the normal expression and function of Cl-anion exchanger CFEX in the proximal tubule of the mammalian kidney.

Alterations in band 3 protein and anion exchange in red blood cells of renal failure patients

The precise nature of band 3 protein and its involvement in oxalate exchange in the red blood cells (RBCs) of renal failure patients has not been studied in detail. Therefore, here we studied the oxalate exchange and binding by band 3 protein in RBCs of humans with conditions of acute and chronic renal failure (ARF and CRF). The RBCs of ARF and CRF patients exhibited abnormal red cell morphology and an increased resistance to osmotic hemolysis. Further, an increase in the cholesterol content and decrease in the activities of Na(+)-K(+)-, Ca(2+)-, and Mg(2+)-ATPases of membranes were observed in the RBCs of ARF and CRF patients. A decrease in the oxalate flux was observed in the RBCs of ARF and CRF patients. The oxalate-binding activities of the RBC membranes were significantly lower in ARF (20 pmoles/mg protein) and CRF (5.3 pmoles/mg protein) patients as compared to that in the normal subjects (36 pmoles/mg protein). DEAE-cellulose and Sephadex G-200 column chromatography purification profiles revealed a distinctive shift in oxalate-binding activity of band 3 protein of RBCs of ARF and CRF patients as compared to that of the normal subjects. It was also observed from the binding studies with a fluorescent dye, eosin-5-maleimide, which specifically binds to band 3 protein, that the RBCs of ARF and CRF patients exhibited only 53 and 32% of abundance of band 3 protein, respectively, as compared to that in the RBCs of the normal subjects, thus revealing a decrease in the band 3 protein content in ARF and CRF patients. These results for the first time showed a decrease in the oxalate exchange in RBCs of patients with ARF and CRF, which was also concomitant with the low levels of abundance of band 3 protein.

Functional Comparison of Mouse slc26a6 Anion Exchanger with Human SLC26A6 Polypeptide Variants: DIFFERENCES IN ANION SELECTIVITY, REGULATION, AND ELECTROGENICITY

The unusually low 78% amino acid identity between the orthologous human SLC26A6 and mouse slc26a6 polypeptides prompted systematic comparison of their anion transport functions in Xenopus oocytes. Multiple human SLC26A6 variant polypeptides were also functionally compared. Transport was studied as unidirectional fluxes of (36)Cl(-), [(14)C]oxalate, and [(35)S]sulfate; as net fluxes of HCO(3)(-) by fluorescence ratio measurement of intracellular pH; as current by two-electrode voltage clamp; and as net Cl(-) flux by fluorescence intensity measurement of relative changes in extracellular and intracellular [Cl(-)]. Four human SLC26A6 polypeptide variants each exhibited rates of bidirectional [(14)C]oxalate flux, Cl(-)/HCO(3)(-) exchange, and Cl(-)/OH(-) exchange nearly equivalent to those of mouse slc26a6. Cl(-)/HCO(3)(-) exchange by both orthologs was cAMP-sensitive, further enhanced by coexpressed wild type cystic fibrosis transmembrane regulator but inhibited by cystic fibrosis transmembrane regulator DeltaF508. However, the very low rates of (36)Cl(-) and [(35)S]sulfate transport by all active human SLC26A6 isoforms contrasted with the high rates of the mouse ortholog. Human and mouse orthologs also differed in patterns of acute regulation. Studies of human-mouse chimeras revealed cosegregation of the high (36)Cl(-) transport phenotype with the transmembrane domain of mouse slc26a6. Mouse slc26a6 and human SLC26A6 each mediated electroneutral Cl(-)/HCO(3)(-) and Cl(-)/OH(-) exchange. In contrast, whereas Cl(-)/oxalate exchange by mouse slc26a6 was electrogenic, that mediated by human SLC26A6 appeared electroneutral. The increased currents observed in oocytes expressing either mouse or human ortholog were pharmacologically distinct from the accompanying monovalent anion exchange activities. The human SLC26A6 polypeptide variants SLC26A6c and SLC26A6d were inactive as transporters of oxalate, sulfate, and chloride. Thus, the orthologous mouse and human SLC26A6 proteins differ in anion selectivity, transport mechanism, and acute regulation, but both mediate electroneutral Cl(-)/HCO(3)(-) exchange.

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.