The role of Oxalobacter formigenes colonization in calcium oxalate stone disease

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1 clinical investigation & 2013 International Society of Nephrology see commentary on page 998 The role of Oxalobacter formigenes colonization in calcium oxalate stone disease Roswitha Siener 1, Ursula Bangen 1, Harmeet Sidhu 2, Ruth Hönow 3, Gerd von Unruh 4 and Albrecht Hesse 1 1 University Stone Centre, Department of Urology, University of Bonn, Bonn, Germany; 2 Oxthera, Alachua, Florida, USA; 3 Federal Institute for Drugs and Medical Devices, Bonn, Germany and 4 Department of Internal Medicine I, University of Bonn, Bonn, Germany About 75% of urinary stones contain oxalate. As Oxalobacter formigenes is a Gram-negative anaerobic bacterium that degrades oxalate in the intestinal tract, we assessed the role of O. formigenes in oxalate metabolism by evaluating its intestinal absorption, plasma concentration, and urinary excretion. Of 37 calcium oxalate stone formers, 26 tested negative for O. formigenes and were compared with the 11 patients who tested positive. Patients provided 24-h urine samples on both a self-selected and a standardized diet. Urinary oxalate excretion did not differ significantly on the self-selected diet, but was significantly lower in O. formigenes-positive than in O. formigenes-negative patients under controlled, standardized conditions. Intestinal oxalate absorption, measured using [ 13 C 2 ]oxalate, was similar in the patients with or without O. formigenes. Plasmaoxalate concentrations were significantly higher in noncolonized (5.79 lmol/l) than in colonized stone formers (1.70 lmol/l). Colonization with O. formigenes was significantly inversely associated with the number of stone episodes. Our findings suggest that O. formigenes lowers the intestinal concentration of oxalate available for absorption at constant rates, resulting in decreased urinary oxalate excretion. Thus, dietary factors have an important role in urinary oxalate excretion. The data indicate that O. formigenes colonization may reduce the risk of stone recurrence. Kidney International (2013) 83, ; doi: /ki ; published online 27 March 2013 KEYWORDS: calcium; colon; diet; oxalate; urinary calculi; urolithiasis Correspondence: Roswitha Siener, University Stone Centre, Department of Urology, University of Bonn, Sigmund-Freud-Strasse 25, D Bonn, Germany. Roswitha.Siener@ukb.uni-bonn.de Received 14 September 2012; revised 12 January 2013; accepted 17 January 2013; published online 27 March 2013 About 75% of urinary stones are predominantly composed of calcium oxalate. 1 Urinary oxalate is considered a crucial risk factor for calcium oxalate stone formation. As the molar oxalate-to-calcium ratio is normally at 1:10, even slight changes in urinary oxalate concentration exert much larger effects on crystallization and stone formation than comparable changes in the calcium concentration. 2 Urinary oxalate is predominantly derived from endogenous production of oxalate from ingested or metabolically generated precursors and from the diet. It has been suggested that dietary contribution to urinary oxalate excretion is up to 50%. 3 Some foods, particularly vegetables and cereals such as spinach, mangold, rhubarb, sorrel, and wheat bran, contain high amounts of oxalic acid. 4 7 A high dietary intake of oxalate can significantly increase urinary oxalate excretion already in healthy individuals without disturbances in oxalate metabolism. 8 Intestinal hyperabsorption of oxalate can considerably contribute to urinary oxalate, even in the absence of gastrointestinal diseases. An increased absorption of oxalate has been demonstrated in 46% of patients with calcium oxalate stone disease. 9 O. formigenes is a Gram-negative anaerobic bacterium that degrades oxalate in the intestinal tract. 10 Treatment with antibiotics may markedly affect colonization with O. formigenes A deficiency of oxalate degradation by O. formigenes might be expected to promote the risk of calcium oxalate stone formation by increasing urinary excretion of oxalate However, a case control study found no difference in median urinary oxalate excretion in patients who tested positive or negative for O. formigenes, despite a strong inverse association between O. formigenes colonization and risk of recurrent calcium oxalate stone formation. 20 The understanding of the role of O. formigenes in oxalate metabolism is still limited. It has been hypothesized that the absence of this bacterium could be associated with higher intestinal oxalate absorption. 21 However, there is still a significant lack of knowledge on the mechanism of the action of O. formigenes colonization in patients with idiopathic calcium oxalate stone disease. Therefore, the primary goal of our study was to assess the role of O. formigenes in oxalate metabolism, i.e., intestinal absorption, plasma concentration, and urinary excretion of oxalate in calcium oxalate stone patients, and to determine the risk of recurrent stone 1144 Kidney International (2013) 83,

2 R Siener et al.: Oxalobacter formigenes and oxalate metabolism clinical investigation formation. To exclude exogenous factors, patients provided 24-h urine specimens not only on a self-selected diet but also under controlled, standardized conditions. Table 1 Patients characteristics Sex a Men (n) 7 19 Women (n) 4 7 Age (years) b 49.3± ± Height (cm) b 167±9 172± Weight (kg) b 69±13 81± BMI (kg/m 2 ) b 24.8± ± GFR (ml/min per 1.73 m 2 ) b 78.0± ± Duration of disease (years) b 12.9± ± Antibiotic treatment (n) a,c 1/8 (13%) 17/21 (81%) Abbreviations: CSF, colonized stone formers; GFR, glomerular filtration rate; NSF, noncolonized stone formers. a Number of patients; Fisher s exact test. b Mean±s.d.; Mann Whitney U-test. c Data not available for all patients. RESULTS Of the 37 idiopathic calcium oxalate stone patients, only 11 (30%) tested positive for O. formigenes, whereas 26 (70%) were negative (Table 1). No significant differences were observed between colonized and noncolonized stone formers regarding distribution of gender, age, anthropometric parameters, and duration of disease. Glomerular filtration rate, calculated using the Modification of Diet in Renal Disease study equation, was similar in both groups. Whereas 13% of O. formigenes-positive patients had taken antibiotics, 81% of O. formigenes-negative patients had received antibiotic treatment before the start of the study. There were no statistically significant differences in baseline urinary parameters between colonized and noncolonized stone formers on self-selected diets, except potassium (Table 2). Urinary oxalate excretion was higher in O. formigenes-negative patients than in those who were O. formigenes-positive, although the difference was not statistically significant (0.399 vs mmol/24 h; P ¼ 0.054), probably because of the relatively small sample size. The comparatively large magnitude of differences in urinary phosphate and citrate and the significant difference in potassium excretion between colonized and noncolonized stone formers on self-selected diets provide evidence that dietary factors may strongly affect urine composition. Intestinal oxalate absorption did not differ with the presence or absence of O. formigenes colonization (11.5 vs. 11.3%; P ¼ 0.727) (Table 3). Hyperabsorption of oxalate, defined as absorption exceeding 10%, was observed in 64% (n ¼ 7/11) of O. formigenes-positive and in 54% (n ¼ 14/26) of O. formigenes-negative patients (P ¼ 0.723). However, plasma oxalate concentrations were significantly higher in patients without O. formigenes (5.79 mmol/l) than in colonized stone formers (1.70 mmol/l) (P ¼ 0.003). Urinary oxalate excretion was significantly lower in O. formigenespositive patients (0.318 and mmol/24 h) than in those who were O. formigenes-negative (0.454 and mmol/ 24 h, respectively) on both days under controlled, standardized diet (P ¼ and P ¼ 0.043, respectively). Serum parameters of the study population were within normal ranges (Table 4). No differences were found between colonized and noncolonized patients. Urinary parameters of the study population of 2 days on the standardized diet are reported in Table 5. Except for urinary oxalate excretion, all other urinary variables did not significantly differ between both groups. Analysis of data revealed a significant inverse association between the number of stone episodes and the colonization Table 2 Urinary parameters in CSF and NSF on self-selected diets (mean±s.d.) Volume (l/24 h) 1.930± ± ph 6.15± ± Density (g/cm 3 ) 1.010± ± Sodium (mmol/24 h) 160±70 171± Potassium (mmol/24 h) 47±18 66± Calcium (mmol/24 h) 6.56± ± Magnesium (mmol/24 h) 4.56± ± Ammonium (mmol/24 h) 28.9± ± Chlorid (mmol/24 h) 141±55 169± Phosphate (mmol/24 h) 27.3± ± Sulfate (mmol/24 h) 19.7± ± Creatinine (mmol/24 h) 11.44± ± Urate (mmol/24 h) 3.01± ± Citrate (mmol/24 h) 2.288± ± Oxalate (mmol/24 h) 0.307± ± Table 3 Plasma oxalate concentration, [ 13 C 2 ]oxalate absorption, and urinary oxalate excretion under controlled, standardized conditions in CSF and NSF (mean±s.d.) Plasma oxalate (mmol/l) 1.70±1.40 a 5.79± [ 13 C 2 ]oxalate absorption (%) 11.5± ± U-oxalate, day 1 (mmol/24 h) 0.318± ± U-oxalate, day 2 (mmol/24 h) 0.367± ± a n ¼ 10 patients. Table 4 Serum parameters in CSF and NSF (mean±s.d.) Sodium (mmol/l) 141±2 141± Potassium (mmol/l) 4.5± ± Calcium (mmol/l) 2.35± ± Magnesium (mmol/l) 0.90± ± Phosphate (mmol/l) 1.06± ± Chloride (mmol/l) 103±3 102± Urate (mmol/l) 301±70 323± Creatinine (mmol/l) 90±15 86± Kidney International (2013) 83,

3 clinical investigation R Siener et al.: Oxalobacter formigenes and oxalate metabolism Table 5 Urinary parameters in CSF and NSF under controlled, standardized conditions (mean±s.d.) CSF CSF NSF NSF P P (n ¼ 11) (n ¼ 26) CSF versus NSF CSF versus NSF Day 1 Day 2 Day 1 Day 2 Day 1 Day 2 Volume (l/24 h) 2.415± ± ± ± Density (g/cm 3 ) 1.010± ± ± ± Sodium (mmol/24 h) 128±45 131±52 149±57 154± Potassium (mmol/24 h) 61±27 66±34 61±17 62± Calcium (mmol/24 h) 6.08± ± ± ± Magnesium (mmol/24 h) 5.20± ± ± ± Phosphate (mmol/24 h) 26.4± ± ± ± Sulfate (mmol/24 h) 17.9± ± ± ± Creatinine (mmol/24 h) 12.41± ± ± ± Citrate (mmol/24 h) 3.223± ± ± ± Oxalate (mmol/24 h) 0.318± ± ± ± Patients (%) O. formigenes-negative O. formigenes-positive 1 Stone 2 4 Stones 5 Stones Figure 1 Association between the number of stone episodes and O. formigenes colonization rate (n ¼ 37). with O. formigenes (P ¼ 0.029; 1 vs. 2 4 stone episodes: odds ratio 4.00, P ¼ and 1 vs.z5 stone episodes: odds ratio 10.67, P ¼ 0.027) (Figure 1). DISCUSSION Colonization with O. formigenes, a substrate-specific oxalatedegrading bacterium that colonizes the intestinal tract, has been reported to reduce the risk of recurrent calcium oxalate stone formation by 70%. 20 Our study confirmed a significant inverse association between colonization with O. formigenes and the rate of stone recurrence. The prevalence of colonization with O. formigenes decreased from 67, 33, and 16% in patients with one, two to four, and more than four stone episodes, respectively. The results are in agreement with previous studies, which have noted decreased colonization rates in recurrent stone formers as compared with first-time stone formers. 15,16,19 Consistent with findings from experimental and clinical studies, the prevalence of colonization with O. formigenes was related to the use of antibiotics ,20,22 The absence of O. formigenes colonization in women could be attributed to frequent use of antibiotics owing to recurrent urinary tract infections, which have been found to be associated with increased oxalate excretion. 23 Although a lack of colonization with O. formigenes increases the risk of calcium oxalate stone recurrence, the role of this bacterium in the metabolism of oxalate, i.e., intestinal absorption, plasma concentration, and urinary excretion of oxalate, is unclear. A key unanswered question is whether the absence of O. formigenes increases the risk of calcium oxalate stone formation by increasing urinary oxalate excretion. Hyperoxaluria is considered a major risk factor for calcium oxalate stone formation. Oxalate degradation by O. formigenes might be expected to reduce the extent of urinary oxalate excretion. Whereas several studies have linked the absence of O. formigenes to higher urinary oxalate excretion, 11,17,18,24 others revealed no significant difference in urinary oxalate excretion between patients who tested positive or negative for O. formigenes. 20,25 However, a major limitation is that none of these studies in stone formers were conducted under controlled conditions. In our study, urinary oxalate excretion was significantly lower in O. formigenes-positive patients than in those who were O. formigenes-negative under controlled, standardized conditions. Although urinary oxalate excretion did not statistically significantly differ between colonized and noncolonized stone formers on self-selected versus controlled diet, the differences are all of similar magnitude. As a few more subjects might have yielded a significant result for urinary oxalate excretion on the free diet, a study with larger patient numbers is needed to confirm the present findings. Results of a study in healthy subjects, who were naturally colonized or noncolonized with O. formigenes, suggested that differences in urinary oxalate excretion on self-selected diets may have been masked by differences in dietary calcium or oxalate intake. 26 It has been hypothesized that the absence of O. formigenes could alter the degree of oxaluria by increasing intestinal absorption of oxalate. 15,27 In our study, no difference in mean oxalate absorption, measured by [ 13 C 2 ]oxalate absorption test, was observed between O. formigenes colonized and noncolonized patients. Intestinal oxalate absorption was 11.5±4.6% in O. formigenes-positive patients and 11.3± 5.9% in O. formigenes-negative patients, which is consistent with mean intestinal absorption of 10.2±5.2% in 120 recurrent idiopathic calcium oxalate stone formers. 9 Similarly, Knight et al. (2011) 25 reported in 38 calcium 1146 Kidney International (2013) 83,

4 R Siener et al.: Oxalobacter formigenes and oxalate metabolism clinical investigation oxalate stone formers that absorption was not significantly different in colonized (5.2±2.2%; n ¼ 6) and noncolonized subjects (9.3±6.2%; n ¼ 32) (P ¼ 0.13). However, limitations of their study, which may have contributed to variability, include small number of colonized patients and that subjects were not placed on controlled diets. Overall, these findings suggest that intestinal oxalate absorption is not modulated by O. formigenes. To our knowledge, this is the first study investigating the effect of colonization with O. formigenes on plasma oxalate concentration in idiopathic calcium oxalate stone patients. Interpretation of studies to date on the role of O. formigenes in oxalate metabolism has been limited by the lack of a reliable plasma oxalate assay. We used a selective and sensitive method for the determination of plasma oxalate concentration. 28 In our study, mean plasma oxalate concentration was more than threefold higher in patients without O. formigenes than in colonized stone formers. Oxalate is a toxic end product of metabolism that must be excreted or sequestered. Under physiological conditions, renal oxalate excretion occurs primarily by glomerular filtration and tubular secretion. 29 Whereas glomerular filtration of oxalate directly depends on plasma oxalate concentration, tubular oxalate handling is mediated by several SLC26 transporting proteins. Bergsland et al. (2011) 30 identified tubular secretion of oxalate, next to glomerular filtration, as a key mediator in calcium stone formers in maintaining plasma oxalate concentrations in a tight range, as a strong correlation was observed between highest urinary oxalate excretions and renal tubule secretion, whereas plasma oxalate was similar between patients and normal controls. As plasma oxalate concentration differed significantly between noncolonized and colonized patients, while glomerular filtration rate was similar in both groups, the results of our study suggest that differences in urinary oxalate excretion was not apparently associated with either changes in glomerular filtration or tubular secretion of oxalate. A hypothetical explanation for the higher plasma oxalate concentration in noncolonized stone formers is that the absence of O. formigenes is attributable to altered intestinal oxalate transport. Rat models have suggested that Oxalobacter, in addition to degrading dietary sources of oxalate, interacts physiologically with colonic mucosa by inducing active secretion of endogenously produced oxalate. 31 Hatch et al. (2006) 31 demonstrated that this bacterial enterocyte interaction results in a significant reduction in urinary oxalate excretion owing to this enteric oxalate shunt. The latter study was also the first to demonstrate that endogenously derived oxalate can sustain Oxalobacter colonization. In patients with primary hyperoxaluria, oral administration of O. formigenes has been shown to significantly induce enteric oxalate secretion, possibly by contributing to a transepithelial gradient favoring intestinal secretion of endogenous oxalate. 32,33 Results of these studies showing a reduction in plasma oxalate concentration in the majority of patients with primary hyperoxaluria treated orally with O. formigenes 32,33 could not be confirmed in a multicenter trial in 42 patients with primary hyperoxaluria. 34 Besides the number of stone episodes, plasma concentration, and urinary excretion of oxalate, no differences between O. formigenes colonized and noncolonized patients were observed. A limitation of this study is the relatively small sample size. However, because patients were kept under controlled, standardized conditions, it is unlikely that metabolic or dietary factors other than the intestinal colonization with O. formigenes could explain the difference in plasma concentration and urinary excretion of oxalate between both groups. Further studies examining the role of O. formigenes colonization status on enteric oxalate secretion in idiopathic calcium oxalate stone formers are necessary to fully explain the potential role of O. formigenes in recurrent urinary stone disease. In conclusion, the present results indicate that colonization with O. formigenes is associated with a reduced risk of calcium oxalate stone formation, probably resulting from a decreased excretion of urinary oxalate. Findings of our study provide evidence that colonization with O. formigenes may reduce urinary oxalate excretion, attributed to increased intestinal oxalate degradation, leaving less oxalate available for absorption at a constant intestinal absorption rate. A study with a larger sample size is needed to confirm the present findings. The observation that active enteric secretion of endogenously produced oxalate may be induced or enhanced by O. formigenes, thereby decreasing plasma oxalate concentration, may have important consequences for future hyperoxalemia treatment strategies. MATERIALS AND METHODS Patients The study comprised 37 patients, including 26 men and 11 women aged years (mean±s.d.: 48.9±15.5 years), attending our stone clinic with idiopathic calcium oxalate urolithiasis (at least one documented event). Stone analysis was performed by infrared spectroscopy, demonstrating more than 60% calcium oxalate. Recurrences were considered to be symptomatic. A symptomatic recurrence was defined as typical renal colic, or the expulsion or removal of a previously undiscovered stone. If a symptomatic recurrence was documented on the basis of renal colic, the recurrence had to be confirmed radiographically or by ultrasound examination. Patients with apparent predisposing conditions, e.g., primary hyperparathyreoidism, inflammatory bowel diseases, bowel resection, or other specific disorders, were not eligible. The subjects were instructed to avoid taking medications that might influence calcium and oxalate metabolism or acid base status. A history of the use of antibiotics within the previous 6 months was obtained. The study was approved by the Ethics Committee of the Medical Faculty of the University of Bonn. Informed consent was obtained from each participant before trial onset. Study procedure The presence of O. formigenes in the feces was determined to classify patients into colonized and noncolonized. Venous blood samples were obtained in the morning after an overnight fasting period. Kidney International (2013) 83,

5 clinical investigation R Siener et al.: Oxalobacter formigenes and oxalate metabolism Patients collected one 24-h urine sample on self-selected diets to assess baseline urinary excretions. The [ 13 C 2 ]oxalate absorption test was conducted and two 24-h urine samples were collected under controlled, standardized conditions. Determination of O. formigenes in feces Determination of O. formigenes was carried out by culture as well as by polymerase chain reaction (PCR). 11 Each patient provided a fresh stool specimen. Approximately mg of feces was inoculated into anaerobically sealed vials containing 10 ml of O. formigenesspecific growth medium B supplemented to 30 mmol/l with potassium oxalate. The vials were incubated at 37 1C for 1 week, and then the loss of oxalate from the culture medium was assessed with a calcium chloride precipitation method in which 50 ml culture medium is mixed with 100 ml 0.1% CaCl 2 solution plus 3.0 ml distilled water, and the absorbance of this mixture at 600 nm was analyzed by spectrophotometry. DNA was isolated from individual fecal cultures using guanidine thiocyanate. 11 The isolated DNA was used as a template in PCR amplification with 5 0 and 3 0 primers specific for the oxc gene of O. formigenes. PCR products were separated by size with electrophoresis through a 1.2% agarose gel containing ethidium bromide and visualized with ultraviolet light. Each set of PCR was controlled with a reaction that contained the PCR components, except the template DNA. PCR-amplification products were confirmed by Southern blot analysis and by hybridization with internal probes specific for the Oxalobacter genus. Plasma oxalate concentration and serum profile Plasma oxalate concentration was measured using an highperformance liquid chromatography enzyme reactor method. 28 Blood samples were obtained in lithium heparin tubes and immediately centrifuged at 1600 r.p.m. (1000 g) for 10 min at 4 1C. Ultrafiltration was carried out in a Centrisart-I-tube (cutoff 20,000 D, Sartorius, Göttingen, Germany) at 3900 r.p.m. (2500 g) for 50 min at 4 1C. To remove proteins and macromolecules, 0.5 ml plasma was added to 0.5 ml distilled water in the outer tube. To prevent in vitro generation of oxalate, 12.5 ml of 1 N hydrochloric acid was added to the inner tube. The yielded ultrafiltrate was analyzed for oxalate concentration using high-performance liquid chromatography enzyme reactor. Analysis of serum parameters was performed by routine methods. [ 13 C 2 ] oxalate absorption test Gastrointestinal oxalate absorption of the patients was measured using the standardized [ 13 C 2 ]oxalate absorption test. 35 The test was conducted over a period of 2 consecutive days on a standardized diet. The patients ingested 50 mg sodium [ 13 C 2 ]oxalate, corresponding to 33.8 mg [ 13 C 2 ]oxalic acid in the fasting state on the morning of day 2, and collected fractional 24-h urine samples. Labeled and unlabeled oxalate was quantified by gas chromatography mass spectrometry. Absorption is expressed as a percentage of labeled oxalate dose. Twenty-four-hour urinary excretion profiles Urine volume, ph, density, and concentrations of creatinine (Jaffé reaction), sodium, potassium, and calcium (ion selective electrode), magnesium (xylidyl-blue reaction), chloride (coulomb metric titration), inorganic sulfate (nephelometry), inorganic phosphate (phosphate molybdate reaction), ammonium (ion-selective electrode), citrate (enzymatically, citrate lyase), uric acid (enzymatically, uricase), and oxalate (enzymatically, oxalate oxidase) were measured. Laboratory quality certification was available for each parameter. Statistics Comparisons between groups were done using the Mann Whitney U-test for nonparametric unpaired data. Categorical variables were compared with Fisher s exact test or Cochran Armitage trend test, where appropriate. Data are presented as means±s.d. All reported P-values are two-sided. Differences were considered significant at Po0.05. 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