Oxalate Secretion Research Articles

Oxygen control in bioreactor drives high yield production of functional hiPSC-like hepatocytes for advanced liver disease modelling

Hepatocytes-like cells (HLC) derived from human induced pluripotent stem cells show great promise for cell-based liver therapies and disease modelling. However, their application is currently hindered by the low production yields of existing protocols. We aim to develop a bioprocess able to generate high numbers of HLC. We used stirred-tank bioreactors with a rational control of dissolved oxygen concentration (DO) for the optimization of HLC production as 3D aggregates. We evaluated the impact of controlling DO at physiological levels (4%O2) during hepatic progenitors’ stage on cell proliferation and differentiation efficiency. Whole transcriptome analysis and biochemical assays were performed to provide a detailed characterization of HLC quality attributes. When DO was controlled at 4%O2 during the hepatic progenitors’ stage, cells presented an upregulation of genes associated with hypoxia-inducible factor pathway and a downregulation of oxidative stress genes. This condition promoted higher HLC production (maximum cell concentration: 2 × 106 cell/mL) and improved differentiation efficiencies (80% Albumin-positive cells) when compared to the bioreactor operated under atmospheric oxygen levels (21%O2, 0.6 × 106 cell/mL, 43% Albumin positive cells). These HLC exhibited functional characteristics of hepatocytes: capacity to metabolize drugs, ability to synthesize hepatic metabolites, and inducible cytochrome P450 activity. Bioprocess robustness was confirmed with HLC derived from different donors, including a primary hyperoxaluria type 1 (PH1) patient. The generated PH1.HLC showed metabolic features of PH1 disease with higher secretion of oxalate compared with HLC generated from healthy individuals. This work reports a reproducible bioprocess, that shows the importance of controlling DO at physiological levels to increase HLC production, and the HLC capability to display PH1 disease features.

A Direct Link Implicating Loss of SLC26A6 to Gut Microbial Dysbiosis, Compromised Barrier Integrity, and Inflammation

BackgroundPutative anion transporter-1 (PAT1, SLC26A6) plays a key role in intestinal oxalate and bicarbonate secretion. PAT1 knockout (PKO) mice exhibit hyperoxaluria and nephrolithiasis. Notably, diseases such as inflammatory bowel diseases (IBD) are also associated with higher risk of hyperoxaluria and nephrolithiasis. However, the potential role of PAT1 deficiency in gut barrier integrity and susceptibility to colitis is currently elusive. MethodsAge-matched PKO and wild-type (WT) littermates were administered 3.5%-DSS in drinking water for 6 days. Ileum and colon of control and treated mice were harvested. mRNA and protein expression of tight junction (TJ) proteins were determined by RT-PCR and western blotting. Severity of inflammation was assessed by measuring diarrheal phenotype, cytokine expression and H&E staining. Gut microbiome and associated metabolome were analyzed by 16S rRNA sequencing and mass spectrometry, respectively. ResultsPKO mice exhibited significantly higher loss of body weight, gut permeability, colonic inflammation, and diarrhea in response to DSS treatment. Additionally, PKO mice showed microbial dysbiosis and significantly reduced levels of butyrate and butyrate-producing microbes compared to controls. Cohousing WT and PKO mice for 4 weeks resulted in PKO-like signatures on the expression of TJ proteins in the colon of WT mice. ConclusionOur data demonstrate that loss of PAT1 disrupts gut microbiome and related metabolites, decreases gut barrier integrity, and increases host susceptibility to intestinal inflammation. These findings, thus, highlight a novel role of the oxalate transporter PAT1 in promoting gut barrier integrity and its deficiency appears to contribute to the pathogenesis of IBD.

The colonization of Penicillium oxalicum SL2 on rice root surface increased Pb interception capacity of iron plaque and decreased Pb uptake by roots

The exploration of microbial resources to reduce Pb accumulation in rice attracted great attention. In this study, we found Penicillium oxalicum SL2, a Pb-tolerant strain with good capability of dissolving phosphorus and stabilizing Pb in soil, was able to colonize on the root surface of rice seedlings without additional carbon sources, and promoted the secretion of metabolites related to amino acid metabolism, organic acid metabolism, signal transduction and other pathways in rhizosphere exudates, in which the secretion of oxalate increased by 47.7 %. However, P. oxalicum SL2 increased Fe(II) proportion and Fe availability on the root surface, resulting in iron plaque content decrease. Moreover, by converting root surface Pb from Pb-Fe state to PbC2O4 and Pb-P compounds, P. oxalicum SL2 increased Pb intercept capacity of iron plaque by 118.0 %. Furthermore, P. oxalicum SL2 regulated element distribution on the root surface, and reduced the relative content of Pb on the maturation zone of root tip, which was conducive to reducing Pb uptake by apoplastic pathway and the risk of Pb accumulation in root system. Our findings further revealed the interaction between P. oxalicum SL2 and rice root, providing a theoretical basis for the development and application of microbial agents in Pb-contaminated farmland.

Sel1-like proteins and peptides are the major Oxalobacter formigenes-derived factors stimulating oxalate transport by human intestinal epithelial cells.

Kidney stones (KSs) are very common, excruciating, and associated with tremendous healthcare cost, chronic kidney disease (CKD), and kidney failure (KF). Most KSs are composed of calcium oxalate and small increases in urinary oxalate concentration significantly enhance the stone risk. Oxalate also potentially contributes to CKD progression, kidney disease-associated cardiovascular diseases, and poor renal allograft survival. This emphasizes the urgent need for plasma and urinary oxalate lowering therapies, which can be achieved by enhancing enteric oxalate secretion. We previously identified Oxalobacter formigenes (O. formigenes)-derived factors secreted in its culture-conditioned medium (CM), which stimulate oxalate transport by human intestinal Caco2-BBE (C2) cells and reduce urinary oxalate excretion in hyperoxaluric mice by enhancing colonic oxalate secretion. Given their remarkable therapeutic potential, we now identified Sel1-like proteins as the major O. formigenes-derived secreted factors using mass spectrometry and functional assays. Crystal structures for six proteins were determined to confirm structures and better understand functions. OxBSel1-14-derived small peptides P8 and P9 were identified as the major factors, with P8 + 9 closely recapitulating the CM's effects, acting through the oxalate transporters SLC26A2 and SLC26A6 and PKA activation. Besides C2 cells, P8 + 9 also stimulate oxalate transport by human ileal and colonic organoids, confirming that they work in human tissues. In conclusion, P8 and P9 peptides are identified as the major O. formigenes-derived secreted factors and they have significant therapeutic potential for hyperoxalemia, hyperoxaluria, and related disorders, impacting the outcomes of patients suffering from KSs, enteric hyperoxaluria, primary hyperoxaluria, CKD, KF, and renal transplant recipients.NEW & NOTEWORTHY We previously identified Oxalobacter formigenes-derived secreted factors stimulating oxalate transport by human intestinal epithelial cells in vitro and reducing urinary oxalate excretion in hyperoxaluric mice by enhancing colonic oxalate secretion. We now identified Sel1-like proteins and small peptides as the major secreted factors and they have significant therapeutic potential for hyperoxalemia and hyperoxaluria, impacting the outcomes of patients suffering from kidney stones, primary and secondary hyperoxaluria, chronic kidney disease, kidney failure, and renal transplant recipients.

Oxalate secretion is stimulated by a cAMP-dependent pathway in the mouse cecum.

Elevated levels of the intracellular second messenger cAMP can stimulate intestinal oxalate secretion however the membrane transporters responsible are unclear. Oxalate transport by the chloride/bicarbonate (Cl-/HCO3-) exchanger Slc26a6 or PAT-1 (Putative Anion Transporter 1), is regulated via cAMP when expressed in Xenopus oocytes and cultured cells but whether this translates to the native epithelia is unknown. This study investigated the regulation of oxalate transport by the mouse intestine focusing on transport at the apical membrane hypothesizing PAT-1 is the target of a cAMP-dependent signaling pathway. Adopting the Ussing chamber technique we measured unidirectional 14C-oxalate and 36Cl- flux ([Formula: see text] and [Formula: see text]) across distal ileum, cecum and distal colon, employing forskolin (FSK) and 3-isobutyl-1-methylxanthine (IBMX) to trigger cAMP production. FSK/IBMX initiated a robust secretory response by all segments but the stimulation of net oxalate secretion was confined to the cecum only involving activation of [Formula: see text] and distinct from net Cl- secretion produced by inhibiting [Formula: see text]. Using the PAT-1 knockout (KO) mouse we determined cAMP-stimulated [Formula: see text] was not directly dependent on PAT-1, but it was sensitive to mucosal DIDS (4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid), although unlikely to be another Cl-/HCO3- exchanger given the lack of trans-stimulation or cis-inhibition by luminal Cl- or HCO3-. The cAMP-activated oxalate efflux was reliant on CFTR (Cystic Fibrosis Transmembrane conductance Regulator) activity, but only in the presence of PAT-1, leading to speculation on the involvement of a multi-transporter regulatory complex. Further investigations at the cellular and molecular level are necessary to define the mechanism and transporter(s) responsible.

CRISPR/Cas9-mediated knock-out of AGXT1 in HepG2 cells as a new in vitro model of Primary Hyperoxaluria Type 1

AGXT1 encodes alanine:glyoxylate aminotransferase 1 (AGT1), a liver peroxisomal pyridoxal 5′-phosphate dependent-enzyme whose deficit causes Primary Hyperoxaluria Type 1 (PH1). PH1 is a rare disease characterized by overproduction of oxalate, first leading to kidney stones formation, and possibly evolving to life-threatening systemic oxalosis. A minority of PH1 patients is responsive to pyridoxine, while the option for non-responders is liver-kidney transplantation. Therefore, huge efforts are currently focused on the identification of new therapies, including the promising approaches based on RNA silencing recently approved. Many PH1-associated mutations are missense and lead to a variety of kinetic and/or folding defects on AGT1. In this context, the availability of a reliable in vitro disease model would be essential to better understand the phenotype of known or newly-identified pathogenic variants as well as to test novel drug candidates.Here, we took advantage of the CRISPR/Cas9 technology to specifically knock-out AGXT1 in HepG2 cells, a hepatoma-derived cell model exhibiting a conserved glyoxylate metabolism. AGXT1-KO HepG2 displayed null AGT1 expression and significantly reduced transaminase activity leading to an enhanced secretion of oxalate upon glycolate challenge. Known pathogenic AGT1 variants expressed in AGXT1-KO HepG2 cells showed alteration in both protein levels and specific transaminase activity, as well as a partial mitochondrial mistargeting when associated with a common polymorphism. Notably, pyridoxine treatment was able to partially rescue activity and localization of clinically-responsive variants. Overall, our data validate AGXT1-KO HepG2 cells as a novel cellular model to investigate PH1 pathophysiology, and as a platform for drug discovery and development.

Primary hyperoxaluria type 1: novel therapies at a glance.

ABSTRACTPrimary hyperoxaluria type 1 (PH1) is a rare and severe autosomal recessive disease of oxalate metabolism, resulting from a mutation in the AGXT gene that encodes the hepatic peroxisomal enzyme alanine–glyoxylate aminotransferase (AGT). Until recently, treatment of PH1 was supportive, consisting of intensive hyperhydration, use of crystallization inhibitors (citrate and neutral phosphorus), in a subset of responsive PH1 patients’ pharmacologic doses of vitamin B6 (pyridoxine), and kidney and liver transplantation when patients progressed to kidney failure. Treatment approaches have been similar for PH2 caused by mutations in hepatic glyoxylate reductase/hydroxypyruvate reductase (GR/HPR), although pyridoxine does not have any benefit in this group. PH3 is caused by mutations of mitochondrial 4-hydroxy-2-oxoglutarate aldolase (HOGA1) and was the most recently described. Kidney failure appears less common in PH3, although kidney stones occur as frequently as in PH1 and PH2. Oxalate metabolism in the liver is complex. Novel therapies based on RNA interference (RNAi) have recently emerged to modulate these pathways, designed to deplete substrate for enzymes upstream and decrease/avoid oxalate production. Two hepatic enzymes have been targeted to date in PH: glycolate oxidase (GO) with lumasiran and lactate dehydrogenase A (LDH-A) with nedosiran. Lumasiran was approved for the treatment of PH1 in 2020 by both the European Medicines Agency and the Food and Drug Administration, whilst clinical trials with nedosiran are ongoing. Results with the two RNAi therapies demonstrate a significant reduction of urinary oxalate excretion in PH1 patients, but long-term data on efficacy (preservation of kidney function, decreased stone events) and safety remain to be established. Nevertheless, the hepatically targeted RNAi approach represents a potential ‘game changer’ in the field of PH1, bringing hope to families and patients that they may be able to avoid liver and/or kidney transplantation in the future and suffer fewer stone events, perhaps with less strict therapeutic regimens. Pharmacological compounds directly inhibiting GO or LDH are also under development and could be of special interest in developing countries where RNAi therapies may not be readily available in the near future. Approaches to manipulate the intestinal microbiome with a goal to increase oxalate degradation or to stimulate secretion of oxalate into the intestine from plasma are also under development. Overall, we appear to be entering a new phase of PH treatment, with an array of promising approaches emerging that will need optimization and evaluation to establish long-term efficacy and safety.

Oxalobacter formigenes treatment confers protective effects in a rat model of primary hyperoxaluria by preventing renal calcium oxalate deposition.

In primary hyperoxaluria, increased hepatic oxalate production sometimes leads to severe nephrocalcinosis and early end-stage kidney disease. Oral administration of Oxalobacter formigenes (O. formigenes), an oxalate-degrading bacterium, is thought to derive oxalate from systemic sources by inducing net enteric oxalate secretion. Here, the impact of O. formigenes on nephrocalcinosis was investigated in an ethylene glycol rat model mimicking hepatic oxalate overproduction in primary hyperoxaluria. Eighteen rats were administered ethylene glycol (0.75% in drinking water) for 6weeks, of which 9 were treated by oral gavage with O. formigenes and 9 received vehicle. Five control rats did not receive ethylene glycol or O. formigenes. Plasma and urinary oxalate levels, calcium oxalate crystalluria, urinary volume, fluid intake, and serum creatinine were monitored during the study. On killing, nephrocalcinosis was quantified. Ethylene glycol intake induced pronounced hyperoxalemia, hyperoxaluria, calcium oxalate crystalluria and nephrocalcinosis. Concomitant O. formigenes treatment partially prevented the ethylene glycol-induced increase in plasma oxalate and completely prevented nephrocalcinosis. Urinary oxalate excretion was not reduced by O. formigenes treatment. Nevertheless, absence of crystals in renal tissue of O. formigenes-treated ethylene glycol animals indicates that the propensity for oxalate to crystallize in the kidneys was reduced compared to non-treated animals. This is supported by the lower plasma oxalate concentrations in O. formigenes-treated animals. This study shows a beneficial effect of O. formigenes treatment on ethylene glycol-induced hyperoxalemia and nephrocalcinosis, and thus supports a possible beneficial effect of O. formigenes in primary hyperoxaluria.

Baseline Sensitivity and Control Efficacy of Strobilurin Fungicide Pyraclostrobin Against Sclerotium rolfsii.

Sclerotium rolfsii is a fungi pathogen of southern blight with broad host range. The quinone outside inhibitor fungicide pyraclostrobin was officially approved for controlling many diseases in 2015. In this study, baseline sensitivity of S. rolfsii to pyraclostrobin was established by measuring the 50% effective concentration (EC50) values of 155 isolates of S. rolfsii. The EC50 values ranged from 0.0291 to 1.0871 µg/ml, with a mean EC50 of 0.4469 ± 0.2490 µg/ml (mean ± standard deviation). In a preventive fungicide invitro experiment and a field experiment, pyraclostrobin preventive efficacy reached 90% and 80%, respectively, which were much higher than that of the control agent carbendazim. Curative efficacy of pyraclostrobin was significantly lower than its preventive efficacy. Pyraclostrobin at 0.1, 0.5, and 2 μg/ml significantly reduced the number of sclerotia produced on potato dextrose agar medium, but had no significant influence on their total weight. Pyraclostrobin had no significant influence on mycelial cell membrane permeability, but it significantly reduced oxalate secretion and protein synthesis of S. rolfsii. Our findings are of great significance for resistance monitoring of S. rolfsii and also provide new insight into the action mechanism of pyraclostrobin against S. rolfsii.

The limited exclusion and efficient translocation mediated by organic acids contribute to rare earth element hyperaccumulation in Phytolacca americana

Organic acids play an important role in metal tolerance, uptake, and translocation in hyperaccumulators. Phytolacca americana is a rare earth element (REE) hyperaccumulator, but the underlying mechanisms on REE tolerance and accumulation mediated by organic acids are poorly understood. Here, we reported for the first time the strategy of P. americana to enhance REE tolerance and accumulation through organic acids from root external secretion to internal biosynthesis. Different from the exclusion of heavy metal by organic acid in the typical plants, the results showed that oxalate secretion (0.3–0.6 μmol h−1 g−1 root DW) induced by yttrium (Y) could not prevent Y from entering the roots, resulting in excess Y uptake by P. americana. Yttrium stress also stimulated the accumulation of malate and citrate by 1.4- and 2.0-folds in the root cortex. Exogenous malate and citrate promoted the redistribution of Y from the root cell walls to the shoot by 30% and 21%, respectively. Based on comparative transcriptome analysis, 6-fold up-regulation was observed in PaNIP1;2, whose homology AtNIP1;2 is responsible for the transport of Al-malate in Arabidopsis. These results suggested that the promoted formation of Y-malate complexes within the roots potentially accelerated the transport of Y from P. americana roots to shoots through PaNIP1;2. Our study revealed the potential mechanism of organic acids in the external exclusion and internal detoxification and translocation of REE in P. americana roots, which provided a basis for improving the efficiency of REE phytoextraction.

Dehydrin MsDHN1 improves aluminum tolerance of alfalfa (Medicago sativa L.) by affecting oxalate exudation from root tips.

A SK3 -type dehydrin MsDHN1 was cloned from alfalfa (Medicago sativa L.). Its function and gene regulatory pathways were studied via overexpression and suppression of MsDHN1 in alfalfa seedlings or hairy roots. The results showed that MsDHN1 is a typical intrinsically disordered protein that exists in the form of monomers and homodimers in alfalfa. The plant growth rates increased as a result of MsDHN1 overexpression (MsDHN1-OE) and decreased upon MsDHN1 suppression (MsDHN1-RNAi) in seedlings or hairy roots of alfalfa compared with the wild-type or the vector line under Al stress. MsDHN1 interacting with aquaporin (AQP) MsPIP2;1 and MsTIP1;1 positively affected oxalate secretion from root tips and Al accumulation in root tips. MsABF2 was proven to be an upstream transcription factor of MsDHN1 and activated MsDHN1 expression by binding to the ABRE element of the MsDHN1 promoter. The transcriptional regulation of MsABF2 on MsDHN1 was dependent on the abscisic acid signaling pathway. These results indicate that MsDHN1 can increase alfalfa tolerance to Al stress via increasing oxalate secretion from root tips, which may involve in the interaction of MsDHN1 with two AQP.

FC 044THE LONG-TERM EFFECTS OF ACUTE KIDNEY INJURY ON INTESTINAL OXALATE-DEGRADING BACTERIA IN RATS

Abstract Background and Aims Gut microbiota is considered an important factor affecting oxalate handling in the intestine. It has been demonstrated that intestinal oxalate secretion provides a complementary route of excretion, and it becomes more evident when kidney function declines. A diversity of gut oxalate-degrading bacteria (ODB) has been hypothesized to play a role in this process. However, there is a general lack of research on the long-term effects of acute kidney injury (AKI) on ODB and their total oxalate-degrading activity (ODA) in fecal microbiota. In this study, we evaluated whether renal dysfunction could affect intestinal ODB and their total ODA in a rat model of glycerol-induced AKI. Method The Male Wistar rats (200-300 g, n=20) on oxalate-free diet were randomly divided into 2 groups. After 24-h of water deprivation, Group 1 (n=10) received an intramuscular injection of 50% glycerol (10 ml/kg of body weight), and Group 2 (n=10) served as control. The numbers of ODB (incubated in a highly selective Oxalate Medium and determined using culture method) and total fecal ODA were measured after injection on days 7 and 70. The method of redoximetric titration with a KMnO4 solution was adopted to evaluate total ODA in fecal microbiota; the results were expressed as % of oxalate degradation per 0.01 g of feces. Renal injury was assessed by histopathological examination, serum creatinine and daily proteinuria levels after removing the animals from the experiment on day 70. Cortical interstitial fibrosis was measured by computerized image analysis on sections stained with picrosirius red. The median (Me) and the interquartile ranges (Q25; Q75) were calculated and compared using the nonparametric Mann-Whitney test. The Spearman correlation coefficient was used to evaluate association between the examined parameters. Results The obtained results demonstrated: 1) after glycerol injection on day 7, no differences were found in the numbers of ODB and total fecal ODA between the experimental and control groups: 5.9 (5.4-6.0) vs 6.0 (5.4-6.4) CFU/g, p=0.65 and 2.0 (0.1-5.0) vs 2.5 (2.0-9.0) %/0.01g, p=0.24, respectively; 2) after AKI initiation on day 70, the numbers of ODB and total fecal ODA were significantly lower in Group I compared with control Group II (Fig. 1); 3) the higher percentage of renal interstitial fibrosis was, the higher total fecal ODA occurred in the experimental rats (Fig. 2). In addition, the number of ODB in feces in Group 1 had an inverse association with serum creatinine (r=-0.52, p=0.006) and 24-h proteinuria levels (r=-0.86, p<0.0001). Conclusion AKI had the long-term negative effects on the quantitative and qualitative characteristics of ODB in fecal microbiota in rats. Moreover, the results of our study confirmed an increasing trend in total fecal ODA according to the aggravation of renal interstitial fibrosis in rats.

Role of Impaired Oxalate Homeostasis in Cardiovascular Disease in Patients With End-Stage Renal Disease: An Opinion Article.

Role of Impaired Oxalate Homeostasis in Cardiovascular Disease in Patients With End-Stage Renal Disease: An Opinion Article.

The role of NHE3 (Slc9a3) in oxalate and sodium transport by mouse intestine and regulation by cAMP.

Intestinal oxalate transport involves Cl−/HCO3 − exchangers but how this transport is regulated is not currently known. NHE3 (Slc9a3), an apical Na+/H+ exchanger, is an established target for regulation of electroneutral NaCl absorption working in concert with Cl−/HCO3 − exchangers. To test whether NHE3 could be involved in regulation of intestinal oxalate transport and renal oxalate handling we compared urinary oxalate excretion rates and intestinal transepithelial fluxes of 14C‐oxalate and 22Na+ between NHE3 KO and wild‐type (WT) mice. NHE3 KO kidneys had lower creatinine clearance suggesting reduced GFR, but urinary oxalate excretion rates (µmol/24 h) were similar compared to the WT but doubled when expressed as a ratio of creatinine. Intestinal transepithelial fluxes of 14C‐oxalate and 22Na+ were measured in the distal ileum, cecum, and distal colon. The absence of NHE3 did not affect basal net transport rates of oxalate or sodium across any intestinal section examined. Stimulation of intracellular cAMP with forskolin (FSK) and 3‐isobutyl‐1‐methylxanthine (IBMX) led to an increase in net oxalate secretion in the WT distal ileum and cecum and inhibition of sodium absorption in the cecum and distal colon. In NHE3 KO cecum, cAMP stimulation of oxalate secretion was impaired suggesting the possibility of a role for NHE3 in this process. Although, there is little evidence for a role of NHE3 in basal intestinal oxalate fluxes, NHE3 may be important for cAMP stimulation of oxalate in the cecum and for renal handling of oxalate.

Gut-kidney axis in oxalate homeostasis.

The gut-kidney axis plays a critical role in oxalate homeostasis, and better understanding of oxalate transport regulatory mechanisms is essential for developing novel therapies. Oxalate potentially contributes to chronic kidney disease (CKD) progression, CKD - and end stage renal disease (ESRD)-associated cardiovascular diseases, polycystic kidney disease (PKD) progression, and/or poor renal allograft survival, emphasizing the need for plasma and urinary oxalate lowering therapies. One promising strategy would be to enhance the bowel's ability to secrete oxalate, which might be facilitated by the following findings. Oxalobacter formigenes (O. formigenes)-derived factors recapitulate O. formigenes colonization effects by reducing urinary oxalate excretion in hyperoxaluric mice by inducing colonic oxalate secretion. Protein kinase A activation stimulates intestinal oxalate transport by enhancing the surface expression of the oxalate transporter SLC26A6 (A6). Glycosylation also stimulates A6-mediated oxalate transport. The colon adapts to chronic acidosis in rats through increased colonic oxalate secretion as previously reported in CKD rats, and A6-mediated enteric oxalate secretion is critical in reducing the body oxalate burden in CKD mice. Intestinal oxalate transport is negatively regulated by proinflammatory cytokines and cholinergic, purinergic, and adenosinergic signaling. These findings could facilitate the development of novel therapeutics for hyperoxalemia, hyperoxaluria, and related disorders if similar regulatory mechanisms are confirmed in humans.

Oxalobacter formigenes produces metabolites and lipids undetectable in oxalotrophic Bifidobacterium animalis

In the search for new potential therapies for pathologies of oxalate, such as kidney stone disease and primary hyperoxaluria, the intestinal microbiome has generated significant interest. Resident oxalate-degrading bacteria inhabit the gastrointestinal tract and reduce absorption of dietary oxalate, thereby potentially lowering the potency of oxalate as a risk factor for kidney stone formation. Although several species of bacteria have been shown to degrade oxalate, select strains of Oxalobacter formigenes (O. formigenes) have thus far demonstrated the unique ability among oxalotrophs to initiate a net intestinal oxalate secretion into the lumen from the bloodstream, allowing them to feed on both dietary and endogenous metabolic oxalate. There is significant interest in this function as a potential therapeutic application for circulating oxalate reduction, although its mechanism of action is still poorly understood. Since this species-exclusive, oxalate-regulating function is reported to be dependent on the use of a currently unidentified secreted bioactive compound, there is much interest in whether O. formigenes produces unique biochemicals that are not expressed by other oxalotrophs which lack the ability to transport oxalate. Hence, this study sought to analyze and compare the metabolomes of O. formigenes and another oxalate degrader, Bifidobacterium animalis subsp. lactis (B. animalis), to determine whether O. formigenes could produce features undetectable in another oxalotroph, thus supporting the theory of a species-exclusive secretagogue compound. A comparative metabolomic analysis of O. formigenes strain HC1 (a human isolate) versus B. animalis, another oxalate-degrading human intestinal microbe, was performed by ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). Bacteria were cultured independently in anaerobic conditions, harvested, lysed, and extracted by protein precipitation. Metabolite extracts were chromatographically separated and analyzed by UHPLC-HRMS using reverse phase gradient elution (ACE Excel 2 C18-Pentafluorophenyl column) paired with a Q Exactive™ mass spectrometer. The purpose of this study was to assess whether O. formigenes potentially produces unique biochemicals from other oxalate degraders to better understand its metabolic profile and provide support for the theoretical production of a species-exclusive secretagogue compound responsible for enhancing intestinal oxalate secretion. We report a panel of metabolites and lipids detected in the O. formigenes metabolome which were undetectable in B. animalis, several of which were identified either by mass-to-charge ratio and retention time matching to our method-specific metabolite library or MS/MS fragmentation. Furthermore, re-examination of data from our previous work showed most of these features were also undetected in the metabolomes of Lactobacillus acidophilus and Lactobacillus gasseri, two other intestinal oxalate degraders. Our observation of O. formigenes metabolites and lipids which were undetectable in other oxalotrophs suggests that this bacterium likely holds the ability to produce biochemicals not expressed by at least a selection of other oxalate degraders. These findings provide support for the hypothesized biosynthesis of a species-exclusive secretagogue responsible for the stimulation of net intestinal oxalate secretion.

The anion exchanger PAT-1 (Slc26a6) does not participate in oxalate or chloride transport by mouse large intestine.

The membrane-bound transport proteins responsible for oxalate secretion across the large intestine remain unidentified. The apical chloride/bicarbonate (Cl-/HCO3-) exchanger encoded by Slc26a6, known as PAT-1 (putative anion transporter 1), is a potential candidate. In the small intestine, PAT-1 makes a major contribution to oxalate secretion but whether this role extends into the large intestine has not been directly tested. Using the PAT-1 knockout (KO) mouse, we compared the unidirectional absorptive ([Formula: see text]) and secretory ([Formula: see text]) flux of oxalate and Cl- across cecum, proximal colon, and distal colon from wild-type (WT) and KO mice in vitro. We also utilized the non-specific inhibitor DIDS (4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid) to confirm a role for PAT-1 in WT large intestine and (in KO tissues) highlight any other apical anion exchangers involved. Under symmetrical, short-circuit conditions the cecum and proximal colon did not transport oxalate on a net basis, whereas the distal colon supported net secretion. We found no evidence for the participation of PAT-1, or indeed any other DIDS-sensitive transport mechanism, in oxalate or Cl- by the large intestine. Most unexpectedly, mucosal DIDS concurrently stimulated [Formula: see text] and [Formula: see text] by 25-68% across each segment without impacting net transport. For the colon, these changes were directly proportional to increased transepithelial conductance suggesting this response was the result of bidirectional paracellular flux. In conclusion, PAT-1 does not contribute to oxalate or Cl- transport by the large intestine, and we urge caution when using DIDS with mouse colonic epithelium.

Metabolomic Alteration in the Mouse Distal Colonic Mucosa after Oral Gavage with Oxalobacter formigenes.

Oxalobacter formigenes has been investigated for years due to its proposed ability to produce a secretagogue compound that initiates net intestinal oxalate secretion, thereby theoretically reducing circulating oxalate and risk of kidney stone formation. Strains which have been shown to exhibit this function in vivo across native tissue include the human strain, HC1, and the wild rat strain, OxWR. While previous work on these secretagogue-relevant strains has focused on profiling their metabolome and lipidome in vitro, efforts to characterize their influence on host intestinal mucosal biochemistry in vivo are yet to be reported. Much work has been done over the years with O. formigenes in relation to the secretagogue hypothesis, but it has never been clearly demonstrated that this microorganism is capable of inducing metabolic changes in native host tissue, which would be expected with the production of a transport-inducing compound. In this work, we show how the distal colonic mucosal metabolomic profile in a mouse model exhibited significant changes in the levels of a variety of metabolites as a result of oral gavage with O. formigenes HC1. Among these significant metabolites was nicotinic acid, an essential nutrient shown in past work to be produced in the gut by the native microbiome. Our finding that the in vivo biochemical state of the distal colon was altered with O. formigenes lends support to the secretagogue hypothesis and serves as a pioneering step in characterizing the biochemical interplay between O. formigenes and the mammalian host.

Enteric Oxalate Secretion Mediated by Slc26a6 Defends against Hyperoxalemia in Murine Models of Chronic Kidney Disease.

A state of oxalate homeostasis is maintained in patients with healthy kidney function. However, as GFR declines, plasma oxalate (Pox) concentrations start to rise. Several groups of researchers have described augmentation of oxalate secretion in the colon in models of CKD, but the oxalate transporters remain unidentified. The oxalate transporter Slc26a6 is a candidate for contributing to the extrarenal clearance of oxalate via the gut in CKD. Feeding a diet high in soluble oxalate or weekly injections of aristolochic acid induced CKD in age- and sex-matched wild-type and Slc26a6-/- mice. qPCR, immunohistochemistry, and western blot analysis assessed intestinal Slc26a6 expression. An oxalate oxidase assay measured fecal and Pox concentrations. Fecal oxalate excretion was enhanced in wild-type mice with CKD. This increase was abrogated in Slc26a6-/- mice associated with a significant elevation in plasma oxalate concentration. Slc26a6 mRNA and protein expression were greatly increased in the intestine of mice with CKD. Raising Pox without inducing kidney injury did not alter intestinal Slc26a6 expression, suggesting that changes associated with CKD regulate transporter expression rather than elevations in Pox. Slc26a6-mediated enteric oxalate secretion is critical in decreasing the body burden of oxalate in murine CKD models. Future studies are needed to address whether similar mechanisms contribute to intestinal oxalate elimination in humans to enhance extrarenal oxalate clearance.

Current update and future directions on gut microbiome and nephrolithiasis.

The incidence of nephrolithiasis is increasing worldwide. Understanding how gut microbiome influences oxalate homeostasis has the potential to offer new strategies to prevent nephrolithiasis. The literature was reviewed to gather the evidence on the association between gut microbiome, hyperoxaluria and nephrolithiasis, and to identify the therapeutic interventions focused on the gut microbiome that could decrease hyperoxaluria and prevent nephrolithiasis. Gut microbiome is constituted by a plethora of microbiota including Oxalobacter formigenes (Oxf) and lactobacilli. Oxf can degrade dietary oxalate and induce enteral oxalate secretion. Animal studies suggested an association between oral Oxf supplementation and a decrease in hyperoxaluria. However, human studies have showed inconsistent results. Oral supplementation of lactobacilli did not show benefit in decreasing the hyperoxaluria. Antibiotic exposure, by affecting the gut microbiome, has been associated with an increase in nephrolithiasis. In vivo studies suggest fecal transplantation as a potential treatment option for reducing nephrolithiasis, but needs further evaluation in clinical studies. The current evidence suggests an association between gut microbiome and nephrolithiasis. However, the strategies focused on modulating gut microbiome for decreasing hyperoxaluria and preventing nephrolithiasis need further research. Judicious use of antibiotics in those predisposed to nephrolithiasis offers a preventative strategy for decreasing nephrolithiasis.