RESUMO
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.
Assuntos
Ceco , Proteínas de Membrana Transportadoras , Animais , Camundongos , 1-Metil-3-Isobutilxantina/farmacologia , 1-Metil-3-Isobutilxantina/metabolismo , Transporte de Íons , Transporte Biológico , Proteínas de Membrana Transportadoras/metabolismo , Ceco/metabolismo , Cloretos/metabolismo , Oxalatos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Bicarbonatos/metabolismo , Transportadores de Sulfato/metabolismo , Antiporters/metabolismoRESUMO
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.
Assuntos
Antiporters/metabolismo , Cloretos/metabolismo , Intestino Grosso/metabolismo , Oxalatos/metabolismo , Transportadores de Sulfato/metabolismo , Animais , Antiporters/genética , Transporte Biológico , Camundongos , Camundongos Knockout , Transportadores de Sulfato/genéticaRESUMO
Oxalobacter formigenes, a unique anaerobic bacterium that relies solely on oxalate for growth, is a key oxalate-degrading bacterium in the mammalian intestinal tract. Degradation of oxalate in the gut by O. formigenes plays a critical role in preventing renal toxicity in animals that feed on oxalate-rich plants. The role of O. formigenes in reducing the risk of calcium oxalate kidney stone disease and oxalate nephropathy in humans is less clear, in part due to difficulties in culturing this organism and the lack of studies which have utilized diets in which the oxalate content is controlled. Herein, we review the literature on the 40th anniversary of the discovery of O. formigenes, with a focus on its biology, its role in gut oxalate metabolism and calcium oxalate kidney stone disease, and potential areas of future research. Results from ongoing clinical trials utilizing O. formigenes in healthy volunteers and in patients with primary hyperoxaluria type 1 (PH1), a rare but severe form of calcium oxalate kidney stone disease, are also discussed. Information has been consolidated on O. formigenes strains and best practices to culture this bacterium, which should serve as a good resource for researchers.
Assuntos
Oxalatos/metabolismo , Oxalobacter formigenes , Animais , Microbioma Gastrointestinal , Genômica , Humanos , Inativação Metabólica , Metabolômica , Nefrolitíase , Oxalatos/urina , Oxalobacter formigenes/genética , Oxalobacter formigenes/metabolismo , Oxalobacter formigenes/fisiologiaRESUMO
INTRODUCTION: 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. METHODS: 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. OBJECTIVES: 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. RESULTS: 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. CONCLUSIONS: 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.
Assuntos
Metabolismo dos Lipídeos , Metaboloma , Metabolômica , Oxalobacter formigenes/metabolismo , Bifidobacterium animalis/metabolismo , Cromatografia Líquida de Alta Pressão , Microbioma Gastrointestinal , Humanos , Oxalatos/metabolismo , Espectrometria de Massas em TandemRESUMO
The anion exchanger SAT-1 [sulfate anion transporter 1 (Slc26a1)] is considered an important regulator of oxalate and sulfate homeostasis, but the mechanistic basis of these critical roles remain undetermined. Previously, characterization of the SAT-1-knockout (KO) mouse suggested that the loss of SAT-1-mediated oxalate secretion by the intestine was responsible for the hyperoxaluria, hyperoxalemia, and calcium oxalate urolithiasis reportedly displayed by this model. To test this hypothesis, we compared the transepithelial fluxes of 14C-oxalate, 35SO42- , and 36Cl- across isolated, short-circuited segments of the distal ileum, cecum, and distal colon from wild-type (WT) and SAT-1-KO mice. The absence of SAT-1 did not impact the transport of these anions by any part of the intestine examined. Additionally, SAT-1-KO mice were neither hyperoxaluric nor hyperoxalemic. Instead, 24-h urinary oxalate excretion was almost 50% lower than in WT mice. With no contribution from the intestine, we suggest that this may reflect the loss of SAT-1-mediated oxalate efflux from the liver. SAT-1-KO mice were, however, profoundly hyposulfatemic, even though there were no changes to intestinal sulfate handling, and the renal clearances of sulfate and creatinine indicated diminished rates of sulfate reabsorption by the proximal tubule. Aside from this distinct sulfate phenotype, we were unable to reproduce the hyperoxaluria, hyperoxalemia, and urolithiasis of the original SAT-1-KO model. In conclusion, oxalate and sulfate transport by the intestine were not dependent on SAT-1, and we found no evidence supporting the long-standing hypothesis that intestinal SAT-1 contributes to oxalate and sulfate homeostasis. NEW & NOTEWORTHY SAT-1 is a membrane-bound transport protein expressed in the intestine, liver, and kidney, where it is widely considered essential for the excretion of oxalate, a potentially toxic waste metabolite. Previously, calcium oxalate kidney stone formation by the SAT-1-knockout mouse generated the hypothesis that SAT-1 has a major role in oxalate excretion via the intestine. We definitively tested this proposal and found no evidence for SAT-1 as an intestinal anion transporter contributing to oxalate homeostasis.
Assuntos
Antiporters/genética , Homeostase/fisiologia , Hiperoxalúria/metabolismo , Nefrolitíase/metabolismo , Oxalatos/metabolismo , Transportadores de Sulfato/genética , Animais , Cloretos/metabolismo , Homeostase/genética , Mucosa Intestinal/metabolismo , Intestinos/fisiologia , Transporte de Íons/fisiologia , Rim/metabolismo , Fígado/metabolismo , Camundongos Knockout , Nefrolitíase/genética , Transportadores de Sulfato/metabolismoRESUMO
NEW FINDINGS: What is the central question of this study? The tracer 36 Cl- , currently used to measure transepithelial Cl- fluxes, has become prohibitively expensive, threatening its future use. 125 Iodide, previously validated alongside 36 Cl- as a tracer of Cl- efflux by cells, has not been tested as a surrogate for 36 Cl- across epithelia. What is the main finding and its importance? We demonstrate that 125 I- can serve as an inexpensive replacement for measuring Cl- transport across mouse large intestine, tracking Cl- transport in response to cAMP stimulation (inducing Cl- secretion) in the presence and absence of the main gastrointestinal Cl- -HCO3- exchanger, DRA. ABSTRACT: Chloride transport is important for driving fluid secretion and absorption by the large intestine, with dysregulation resulting in diarrhoea-associated pathologies. The radioisotope 36 Cl- has long been used as a tracer to measure epithelial Cl- transport but is prohibitively expensive. 125 Iodide has been used as an alternative to 36 Cl- in some transport assays but has never been validated as an alternative for tracing bidirectional transepithelial Cl- fluxes. The goal of this study was to validate 125 I- as an alternative to 36 Cl- for measurement of Cl- transport by the intestine. Simultaneous fluxes of 36 Cl- and 125 I- were measured across the mouse caecum and distal colon. Net Cl- secretion was induced by the stimulation of cAMP with a cocktail of forskolin (FSK) and 3-isobutyl-1-methylxanthine (IBMX). Unidirectional fluxes of 125 I- correlated well with 36 Cl- fluxes after cAMP-induced net Cl- secretion, occurring predominantly through a reduction in the absorptive mucosal-to-serosal Cl- flux rather than by stimulation of the secretory serosal-to-mucosal Cl- flux. Correlations between 125 I- fluxes and 36 Cl- fluxes were maintained in epithelia from mice lacking DRA (Slc26a3), the main Cl- -HCO3- exchanger responsible for Cl- absorption by the large intestine. Lower rates of Cl- and I- absorption in the DRA knockout intestine suggest that DRA might have a previously unrecognized role in iodide uptake. This study validates that 125 I- traces transepithelial Cl- fluxes across the mouse large intestine, provides insights into the mechanism of net Cl- secretion and suggests that DRA might be involved in intestinal iodide absorption.
Assuntos
Cloretos/metabolismo , Colo/metabolismo , Epitélio/metabolismo , Iodetos/metabolismo , Transporte de Íons/fisiologia , Animais , Absorção Intestinal/fisiologia , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Transportadores de Sulfato/metabolismoRESUMO
Diseases of oxalate, such as nephrolithiasis and primary hyperoxaluria, affect a significant portion of the US population and have limited treatment options. Oxalobacter formigenes, an obligate oxalotrophic bacterium in the mammalian intestine, has generated great interest as a potential probiotic or therapeutic treatment for oxalate-related conditions due to its ability to degrade both exogenous (dietary) and endogenous (metabolic) oxalate, lowering the risk of hyperoxaluria/hyperoxalemia. Although all oxalotrophs degrade dietary oxalate, Oxalobacter formigenes is the only species shown to initiate intestinal oxalate secretion to draw upon endogenous, circulating oxalate for consumption. Evidence suggests that Oxalobacter regulates oxalate transport proteins in the intestinal epithelium using an unidentified secreted bioactive compound, but the mechanism of this function remains elusive. It is essential to gain an understanding of the biochemical relationship between Oxalobacter and the host intestinal epithelium for this microbe to progress as a potential remedy for oxalate diseases. This investigation includes the first profiling of the metabolome and lipidome of Oxalobacter formigenes, specifically the human strain HC1 and rat strain OxWR, the only two strains shown thus far to initiate net intestinal oxalate secretion across native gut epithelia. This study was performed using untargeted and targeted metabolomics and lipidomics methodologies utilizing ultra-high-performance liquid chromatography-mass spectrometry. We report our findings that the metabolic profiles of these strains, although largely conserved, show significant differences in their expression of many compounds. Several strain-specific features were also detected. Discussed are trends in the whole metabolic profile as well as in individual features, both identified and unidentified. Graphical abstract á .
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Lipídeos/química , Espectrometria de Massas/métodos , Metabolômica , Oxalobacter formigenes/metabolismoRESUMO
The ileum is considered the primary site of inorganic sulfate ([Formula: see text]) absorption. In the present study, we explored the contributions of the apical chloride/bicarbonate (Cl-/[Formula: see text]) exchangers downregulated in adenoma (DRA; Slc26a3), and putative anion transporter 1 (PAT1; Slc26a6), to the underlying transport mechanism. Transepithelial 35[Formula: see text] and 36Cl- fluxes were determined across isolated, short-circuited segments of the distal ileum from wild-type (WT), DRA-knockout (KO), and PAT1-KO mice, together with measurements of urine and plasma sulfate. The WT distal ileum supported net sulfate absorption [197.37 ± 13.61 (SE) nmol·cm-2·h-1], but neither DRA nor PAT1 directly contributed to the unidirectional mucosal-to-serosal flux ([Formula: see text]), which was sensitive to serosal (but not mucosal) DIDS, dependent on Cl-, and regulated by cAMP. However, the absence of DRA significantly enhanced net sulfate absorption by one-third via a simultaneous rise in [Formula: see text] and a 30% reduction to the secretory serosal-to-mucosal flux ([Formula: see text]). We propose that DRA, together with PAT1, contributes to [Formula: see text] by mediating sulfate efflux across the apical membrane. Associated with increased ileal sulfate absorption in vitro, plasma sulfate was 61% greater, and urinary sulfate excretion (USO4) 2.2-fold higher, in DRA-KO mice compared with WT controls, whereas USO4 was increased 1.8-fold in PAT1-KO mice. These alterations to sulfate homeostasis could not be accounted for by any changes to renal sulfate handling suggesting that the source of this additional sulfate was intestinal. In summary, we characterized transepithelial sulfate fluxes across the mouse distal ileum demonstrating that DRA (and to a lesser extent, PAT1) secretes sulfate with significant implications for intestinal sulfate absorption and overall homeostasis.NEW & NOTEWORTHY Sulfate is an essential anion that is actively absorbed from the small intestine involving members of the Slc26 gene family. Here, we show that the main intestinal chloride transporter Slc26a3, known as downregulated in adenoma (DRA), also handles sulfate and contributes to its secretion into the lumen. In the absence of functional DRA (as in the disease congenital chloride diarrhea), net intestinal sulfate absorption was significantly enhanced resulting in substantial alterations to overall sulfate homeostasis.
Assuntos
Antiporters/metabolismo , Homeostase/fisiologia , Íleo/fisiologia , Sulfatos/metabolismo , Animais , Antiporters/genética , Bicarbonatos/metabolismo , Transporte Biológico , Cloretos/metabolismo , Condutividade Elétrica , Regulação da Expressão Gênica/fisiologia , Camundongos , Camundongos Knockout , Transportadores de SulfatoRESUMO
Studies have shown that compensatory adaptations in gastrointestinal oxalate transport can impact the amount of oxalate excreted by the kidney. Hyperoxaluria is a major risk factor in the formation of kidney stones, and oxalate is derived from both the diet and the liver metabolism of glyoxylate. Although the intestine generally absorbs oxalate from dietary sources and can contribute as much as 50% of urinary oxalate, enteric oxalate elimination plays a significant role when renal function is compromised. While the mechanistic basis for these changes in the direction of intestinal oxalate movements in chronic renal failure involves an upregulation of angiotensin II receptors in the large intestine, enteric secretion/excretion of oxalate can also occur by mechanisms that are independent of angiotensin II. Most notably, the commensal bacterium Oxalobacter sp. interacts with the host enterocyte and promotes the movement of oxalate from the blood into the lumen, resulting in the beneficial effect of significantly lowering urinary oxalate excretion. Changes in the passive permeability of the intestine, such as in steatorrhoea and following gastric bypass, also promote oxalate absorption and hyperoxaluria. In summary, this report highlights the two-way physiological signalling between the gut and the kidney, which may help to alleviate the consequences of certain kidney diseases.
Assuntos
Derivação Gástrica , Mucosa Intestinal/metabolismo , Rim/metabolismo , Ácido Oxálico/metabolismo , Insuficiência Renal Crônica/metabolismo , Adaptação Fisiológica , Animais , Humanos , Hiperoxalúria/metabolismo , Hiperoxalúria/fisiopatologia , Absorção Intestinal , Eliminação Intestinal , Intestinos/microbiologia , Intestinos/fisiopatologia , Rim/fisiopatologia , Cálculos Renais/metabolismo , Cálculos Renais/fisiopatologia , Ácido Oxálico/urina , Permeabilidade , Eliminação Renal , Insuficiência Renal Crônica/fisiopatologia , Transdução de SinaisRESUMO
Active transcellular oxalate transport in the mammalian intestine contributes to the homeostasis of this important lithogenic anion. Several members of the Slc26a gene family of anion exchangers have a measurable oxalate affinity and are expressed along the gut, apically and basolaterally. Mouse Slc26a6 (PAT1) targets to the apical membrane of enterocytes in the small intestine, and its deletion results in net oxalate absorption and hyperoxaluria. Apical exchangers of the Slc26a family that mediate oxalate absorption have not been established, yet the Slc26a3 [downregulated in adenoma (DRA)] protein is a candidate mediator of oxalate uptake. We evaluated the role of DRA in intestinal oxalate and Cl(-) transport by comparing unidirectional and net ion fluxes across short-circuited segments of small (ileum) and large (cecum and distal colon) intestine from wild-type (WT) and DRA knockout (KO) mice. In WT mice, all segments demonstrated net oxalate and Cl(-) absorption to varying degrees. In KO mice, however, all segments exhibited net anion secretion, which was consistently, and solely, due to a significant reduction in the absorptive unidirectional fluxes. In KO mice, daily urinary oxalate excretion was reduced 66% compared with that in WT mice, while urinary creatinine excretion was unchanged. We conclude that DRA mediates a predominance of the apical uptake of oxalate and Cl(-) absorbed in the small and large intestine of mice under short-circuit conditions. The large reductions in urinary oxalate excretion underscore the importance of transcellular intestinal oxalate absorption, in general, and, more specifically, the importance of the DRA exchanger in oxalate homeostasis.
Assuntos
Antiporters/metabolismo , Cloretos/metabolismo , Oxalatos/metabolismo , Absorção , Animais , Antiporters/genética , Transporte Biológico/fisiologia , Ceco/metabolismo , Fenômenos Eletrofisiológicos , Regulação da Expressão Gênica/fisiologia , Homeostase , Íleo/metabolismo , Camundongos , Camundongos Knockout , Transportadores de SulfatoRESUMO
Inorganic sulfate (SO4²â») is essential for a multitude of physiological processes. The specific molecular pathway has been identified for uptake from the small intestine but is virtually unknown for the large bowel, although there is evidence for absorption involving Naâº-independent anion exchange. A leading candidate is the apical chloride/bicarbonate (Clâ»/HCO3â») exchanger DRA (down-regulated in adenoma; Slc26a3), primarily linked to the Clâ» transporting defect in congenital chloride diarrhea. The present study set out to characterize transepithelial ³5SO4²â» and ³6Clâ» fluxes across the isolated, short-circuited cecum from wild-type (WT) and knockout (KO) mice and subsequently to define the contribution of DRA. The cecum demonstrated simultaneous net SO4²â» secretion (-8.39 ± 0.88 nmol·cm⻲·h⻹) and Clâ» absorption (10.85 ± 1.41 µmol·cm⻲·h⻹). In DRA-KO mice, SO4²â» secretion was reversed to net absorption via a 60% reduction in serosal to mucosal SO4²â» flux. Similarly, net Clâ» absorption was abolished and replaced by secretion, indicating that DRA represents a major pathway for transcellular SO4²â» secretion and Clâ» absorption. Further experiments including the application of DIDS (500 µM), bumetanide (100 µM), and substitutions of extracellular Clâ» or HCO3â»/CO2 helped to identify specific ion dependencies and driving forces and suggested that additional anion exchangers were operating at both apical and basolateral membranes supporting SO4²â» transport. In conclusion, DRA contributes to SO4²â» secretion via DIDS-sensitive HCO3â»/SO4²â» exchange, in addition to being the principal DIDS-resistant Clâ»/HCO3â» exchanger. With DRA linked to the pathogenesis of other gastrointestinal diseases extending its functional characterization offers a more complete picture of its role in the intestine.
Assuntos
Antiporters/metabolismo , Ceco/metabolismo , Cloretos/metabolismo , Sulfatos/metabolismo , Absorção , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Animais , Antiporters/genética , Transporte Biológico , Bumetanida/farmacologia , Ceco/efeitos dos fármacos , Diuréticos/farmacologia , Camundongos , Camundongos Knockout , Transportadores de Sulfato , Simportadores/genética , Simportadores/metabolismoRESUMO
PURPOSE: We determined the effect of dietary fat and oxalate on fecal fat excretion and urine parameters in a rat model of Roux-en-Y gastric bypass surgery. MATERIALS AND METHODS: Diet induced obese Sprague-Dawley® rats underwent sham surgery as controls (16), or Roux-en-Y gastric bypass surgery (19). After recovery, rats had free access to a normal calcium, high fat (40%) diet with or without 1.5% potassium oxalate for 5 weeks and then a normal (10%) fat diet for 2 weeks. Stool and urine were collected after each period. Fecal fat was determined by gas chromatography and urine metabolites were evaluated by assay spectrophotometry. RESULTS: Daily fecal fat excretion remained low in controls on either diet. However, Roux-en-Y gastric bypass rats ingested a food quantity similar to that of controls but had eightfold higher fecal fat excretion (p <0.001) and heavier stools (p = 0.02). Compared to controls, gastric bypass rats on the high fat diet with potassium oxalate had a fivefold increase in urine oxalate excretion (p <0.001), while gastric bypass rats without potassium oxalate had a twofold increase in urine calcium (p <0.01). Lowering dietary fat in gastric bypass rats with potassium oxalate led to a 50% decrease in oxalate excretion (p <0.01), a 30% decrease in urine calcium and a 0.3 U increase in urine pH (p <0.001). CONCLUSIONS: In this Roux-en-Y gastric bypass model high fat feeding resulted in steatorrhea, hyperoxaluria and low urine pH, which were partially reversible by lowering the dietary fat and oxalate content. Roux-en-Y gastric bypass rats on normal fat and no oxalate diets excreted twice as much oxalate as age matched, sham operated controls. Although Roux-en-Y gastric bypass hyperoxaluria appears primarily mediated by gut and diet, secondary causes of oxalogenesis from liver or other mechanisms deserve further exploration.
Assuntos
Gorduras na Dieta/metabolismo , Derivação Gástrica/efeitos adversos , Hiperoxalúria/etiologia , Obesidade/cirurgia , Oxalatos/metabolismo , Esteatorreia/etiologia , Animais , Modelos Animais de Doenças , Fezes/química , Derivação Gástrica/métodos , Hiperoxalúria/fisiopatologia , Masculino , Obesidade/complicações , Complicações Pós-Operatórias/epidemiologia , Complicações Pós-Operatórias/fisiopatologia , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Valores de Referência , Medição de Risco , Esteatorreia/fisiopatologia , Resultado do Tratamento , UrináliseRESUMO
Little is known about oxalate transport in renal epithelia under basal conditions, let alone in hyperoxaluria when the capacity for renal oxalate excretion is increased. Sulfate anion transporter 1 (SAT1, Slc26a1) is considered to be a major basolateral anion-oxalate exchanger in the proximal tubule and we hypothesized its expression may correlate with urinary oxalate excretion. We quantified changes in the renal expression of SAT1 mRNA and protein in two rat models, one with hyperoxaluria (HYP) and one with renal insufficiency (HRF) induced by hyperoxaluria. The hyperoxaluria observed in the HYP group could not simply be ascribed to changes in SAT1 mRNA or protein abundance. However, when hyperoxaluria was accompanied by renal insufficiency, significant reductions in SAT1 mRNA and protein were detected in medullary and papillary tissue. Together, the results indicate that transcriptional modulation of the SAT1 gene is not a significant component of the hyperoxaluria observed in these rat models.
Assuntos
Acetiltransferases/genética , Acetiltransferases/metabolismo , Regulação da Expressão Gênica , Hiperoxalúria/genética , Hiperoxalúria/metabolismo , Rim/metabolismo , RNA Mensageiro/metabolismo , Animais , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Oxalobacter colonization of rat intestine was previously shown to promote enteric oxalate secretion and elimination, leading to significant reductions in urinary oxalate excretion (Hatch et al. Kidney Int 69: 691-698, 2006). The main goal of the present study, using a mouse model of primary hyperoxaluria type 1 (PH1), was to test the hypothesis that colonization of the mouse gut by Oxalobacter formigenes could enhance enteric oxalate secretion and effectively reduce the hyperoxaluria associated with this genetic disease. Wild-type (WT) mice and mice deficient in liver alanine-glyoxylate aminotransferase (Agxt) exhibiting hyperoxalemia and hyperoxaluria were used in these studies. We compared the unidirectional and net fluxes of oxalate across isolated, short-circuited large intestine of artificially colonized and noncolonized mice. In addition, plasma and urinary oxalate was determined. Our results demonstrate that the cecum and distal colon contribute significantly to enteric oxalate excretion in Oxalobacter-colonized Agxt and WT mice. In colonized Agxt mice, urinary oxalate excretion was reduced 50% (to within the normal range observed for WT mice). Moreover, plasma oxalate concentrations in Agxt mice were also normalized (reduced 50%). Colonization of WT mice was also associated with marked (up to 95%) reductions in urinary oxalate excretion. We conclude that segment-specific effects of Oxalobacter on intestinal oxalate transport in the PH1 mouse model are associated with a normalization of plasma oxalate and urinary oxalate excretion in otherwise hyperoxalemic and hyperoxaluric animals.
Assuntos
Intestino Grosso/metabolismo , Oxalatos/metabolismo , Oxalobacter formigenes/metabolismo , Animais , Antiporters/metabolismo , Transporte Biológico , Ceco/metabolismo , Ceco/microbiologia , Colo/metabolismo , Colo/microbiologia , Modelos Animais de Doenças , Feminino , Genótipo , Hiperoxalúria/genética , Hiperoxalúria/metabolismo , Hiperoxalúria/microbiologia , Hiperoxalúria Primária , Intestino Grosso/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxalatos/sangue , Oxalatos/urina , Oxalobacter formigenes/crescimento & desenvolvimento , Fenótipo , Transportadores de Sulfato , Fatores de Tempo , Transaminases/deficiência , Transaminases/genética , Transaminases/metabolismoRESUMO
Epithelial oxalate transport is fundamental to the role occupied by the gastrointestinal (GI) tract in oxalate homeostasis. The absorption of dietary oxalate, together with its secretion into the intestine, and degradation by the gut microbiota, can all influence the excretion of this nonfunctional terminal metabolite in the urine. Knowledge of the transport mechanisms is relevant to understanding the pathophysiology of hyperoxaluria, a risk factor in kidney stone formation, for which the intestine also offers a potential means of treatment. The following discussion presents an expansive review of intestinal oxalate transport. We begin with an overview of the fate of oxalate, focusing on the sources, rates, and locations of absorption and secretion along the GI tract. We then consider the mechanisms and pathways of transport across the epithelial barrier, discussing the transcellular, and paracellular components. There is an emphasis on the membrane-bound anion transporters, in particular, those belonging to the large multifunctional Slc26 gene family, many of which are expressed throughout the GI tract, and we summarize what is currently known about their participation in oxalate transport. In the final section, we examine the physiological stimuli proposed to be involved in regulating some of these pathways, encompassing intestinal adaptations in response to chronic kidney disease, metabolic acid-base disorders, obesity, and following gastric bypass surgery. There is also an update on research into the probiotic, Oxalobacter formigenes, and the basis of its unique interaction with the gut epithelium. © 2021 American Physiological Society. Compr Physiol 11:1-41, 2021.
Assuntos
Intestinos , Proteínas de Membrana Transportadoras , Oxalatos , Trato Gastrointestinal/metabolismo , Humanos , Intestinos/metabolismo , Oxalatos/metabolismo , Oxalobacter formigenes/metabolismoRESUMO
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 14 C-oxalate and 22 Na+ 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 14 C-oxalate and 22 Na+ 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.
Assuntos
Mucosa Intestinal/metabolismo , Oxalatos/metabolismo , Trocador 3 de Sódio-Hidrogênio/metabolismo , Sódio/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Colforsina/farmacologia , AMP Cíclico/metabolismo , Inibidores Enzimáticos/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Transporte de Íons , Camundongos , Camundongos Endogâmicos C57BL , Oxalatos/urina , Trocador 3 de Sódio-Hidrogênio/genéticaRESUMO
Paper spray ionization mass spectrometry (PSI-MS) is a direct MS analysis technique with several reported bacterial metabolomics applications. As with most MS-based bacterial studies, all currently reported PSI-MS bacterial analyses have focused on the chemical signatures of the cellular unit. One dimension of the bacterial metabolome that is often lost in such analyses is the exometabolome (extracellular metabolome), including secreted metabolites, lipids, and peptides. A key component of the bacterial exometabolome that is gaining increased attention in the microbiology and biomedical communities is extracellular vesicles (EVs). These excreted structures, produced by cells in all domains of life, contain a variety of biomolecules responsible for a wide array of cellular functions, thus representing a core component of the bacterial secreted metabolome. Although previously examined using other MS approaches, no reports currently exist for a PSI-MS analysis of bacterial EVs, nor EVs from any other organism (exosomes, ectosomes, etc.). PSI-MS holds unique analytical strengths over other commonly used MS platforms and could thus provide an advantageous approach to EV metabolomics. To address this, we report a novel application representing, to our knowledge, the first PSI-MS analysis of EVs from any organism (using the human gut resident Oxalobacter formigenes as the experimental model, a bacterium whose EVs were never previously investigated). In this report, we show how we isolated and purified EVs from bacterial culture supernatant by EV-specific affinity chromatography, confirmed and characterized these vesicles by nanoparticle tracking analysis, analyzed the EV isolate by PSI-MS, and identified a panel of EV-derived metabolites, lipids, and peptides. This work serves as a pioneering study in the field of MS-based EV analysis and provides a new, rapid, sensitive, and economical approach to EV metabolomics.
RESUMO
OBJECTIVES: An intestinal permeability defect precedes type 1 diabetes mellitus and may be a permissive factor in its pathogenesis. Butyrate strengthens the intestinal tight junctions. We hypothesized that enteral administration of sodium butyrate (NaB) in preweaned rats would result in differences in the development of diabetes associated with decreased inflammation and pancreatic ß-cell destruction. MATERIALS AND METHODS: Using biobreeding diabetes-prone rat pups, oral NaB or saline was administered twice per day via micropipette from postnatal days 10 to 23. Rat pups were randomly assigned to 1 of 4 groups for the first experiment (control group, n = 7) and 3 different doses of butyrate groups (n = 8 for each group) and 2 groups for the second and third experiments (control n = 23; NaB at 400 mg · kg(-1) · day(-1), n = 20). Animals were studied into adulthood (up to day 140) for development of diabetes. RESULTS: The results showed that the survival rates were 28% versus 20% (butyrate vs control). No significant differences in survival were seen; however, there was a trend of delaying of onset of diabetes in the butyrate group. There were no differences of pancreatic histology score of islet inflammation between the 2 groups. Cytokine-induced neutrophil chemoattractant-1 was lower in the butyrate group at a dose of 400 mg · kg(-1) · day(-1) in the distal small intestine (P = 0.008) and in the liver (P = 0.01). There were no significant differences in the tracer flux measurements across the distal ileum and colon between the 2 animal groups. CONCLUSIONS: Oral NaB given during the preweaning period did not significantly decrease the subsequent development of death from diabetes in biobreeding diabetes-prone rats.
Assuntos
Butiratos/administração & dosagem , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/metabolismo , Enteropatias/complicações , Enteropatias/metabolismo , Mucosa Intestinal/metabolismo , Administração Oral , Animais , Quimiocina CXCL1/efeitos dos fármacos , Citocinas/efeitos dos fármacos , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Inflamação/complicações , Inflamação/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Intestinos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Permeabilidade/efeitos dos fármacos , Ratos , Ratos Endogâmicos BB , Cloreto de Sódio/administração & dosagem , Taxa de SobrevidaRESUMO
Oxalobacter sp. promotion of enteric oxalate excretion, correlating with reductions in urinary oxalate excretion, was previously reported in rats and mice, but the mechanistic basis for this affect has not been described. The main objective of the present study was to determine whether the apical oxalate transport proteins, PAT1 (slc26a6) and DRA (slc26a3), are involved in mediating the Oxalobacter-induced net secretory flux across colonized mouse cecum and distal colon. We measured unidirectional and net fluxes of oxalate across tissues removed from colonized PAT1 and DRA knockout (KO) mice and also across two double knockout (dKO) mouse models with primary hyperoxaluria, type 1 (i.e., deficient in alanine-glyoxylate aminotransferase; AGT KO), including PAT1/AGT dKO and DRA/AGT dKO mice compared to non-colonized mice. In addition, urinary oxalate excretion was measured before and after the colonization procedure. The results demonstrate that Oxalobacter can induce enteric oxalate excretion in the absence of either apical oxalate transporter and urinary oxalate excretion was reduced in all colonized genotypes fed a 1.5% oxalate-supplemented diet. We conclude that there are other, as yet unidentified, oxalate transporters involved in mediating the directional changes in oxalate transport across the Oxalobacter-colonized mouse large intestine.
Assuntos
Antiporters/metabolismo , Mucosa Intestinal/metabolismo , Oxalatos/metabolismo , Oxalobacter formigenes/metabolismo , Transportadores de Sulfato/metabolismo , Animais , Antiporters/genética , Ceco/metabolismo , Ceco/microbiologia , Colo/metabolismo , Colo/microbiologia , Fezes/microbiologia , Microbioma Gastrointestinal , Mucosa Intestinal/microbiologia , Masculino , Camundongos , Camundongos Knockout , Oxalobacter formigenes/isolamento & purificação , Eliminação Renal , Transportadores de Sulfato/genética , SimbioseRESUMO
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.