Abstract
Introduction
Lower bone mineral density (BMD) and reduced Oxalobacter formigenes colonization are common findings in urolithiasis patients. But none of the studies conducted investigated the relationship between decreased bone mineral density and reduced Oxalobacter colonization. Here we evaluated the relation between BMD and O. formigenes colonization in urolithiasis patients.
Materials and Methods
50 stone formers (48.9 ± 11.9 years) and 50 control (47.2 ± 13.4 years) adult male subjects were included in the study. Alterations in O. formigenes colonization were determined as absolute O. formigenes count from fecal samples by real time polymerase chain reaction using species specific primers. BMD was evaluated from t- and z- scores calculated by using dual energy absorptiometry in the total femoral neck and lumbar spine (L2-L4).
Results
Low BMD was observed in 18 (36%) urinary stone forming patients and in 7 (14%) control subjects in the lumbar area (p < 0.05). The mean O. formigenes count in stone formers and control subjects were 19,257 (5,791 ± 1,117.93) and 143,850 (2,815,725 ± 3,946,044.7) (p < 0.05) respectively. We observed a correlation between decreased lumbar BMD and O. formigenes colonization and testosterone levels in stone formers. Our results indicated that diminished O. formigenes colonization in the gut of urinary stone forming subjects was associated with reduced BMD.
Key Words: Urolithiasis, Bone mineral density, Oxalobacter formigenes, Oxalate degradation
Introduction
Urolithiasis is one of the most common benign urological diseases. Epidemiological data indicate an increase in the incidence and prevalence of urolithiasis [1]. The lifetime prevalence of kidney stone disease is estimated to range between 1% and 15%. Adult males are affected 2 or 3 times more frequently than adult females, but the gender gap has been declining in recent years. Occurrence of urolithiasis is relatively rare before age 20, and peaks in the fourth and sixth decades of life. Accumulated evidence from studies showed that the prevalence and incidence of urinary stone disease are in direct correlation with the patient's body mass index (BMI) in both sexes [2].
Urinary calculi are composed of several elements with calcium being the most prevalent constituent. Calcium oxalate stones make up about 60% of mixed calcium oxalate stones while hydroxyapatite constitutes 20% of all urinary stones [3]. Studies show that urinary oxalate excretion is a significant factor in stone formation [4, 5]. Recent studies investigating oxalate urolithiasis mainly focused on the role of the colonic gram negative-anaerobic bacterium Oxalobacter formigenes. Several studies support the role of O. formigenes in regulation of serum oxalate levels while reporting an inverse correlation between O. formigenes colonization and stone formation and recurrence [6, 7]. This bacterium uses oxalate as its main carbon and energy source. As a result of this, colonization of the colon by O. formigenes decreases the intestinal oxalate concentrations and oxalate hypoabsorption resulting in reduced risk of calcium oxalate stone formation [8].
Several studies have shown that bone mineral density (BMD) is reduced in calcium oxalate urolithiasis [9, 10, 11]. Despite the fact that previous studies have shown that both decreased BMD and O. formigenes colonization are associated, the relation between O. formigenes colonization and BMD in urinary stone forming patients has not been investigated. Therefore we decided to explore this possible association with a real time polymerase chain reaction (PCR) based system using a species specific primer pair.
Material and Methods
Patients and Controls
Fifty male calcium oxalate urolithiasis patients and 50 non-stone forming control subjects were included in the study. Following the approval of the local ethics committee, necessary informed consent forms were obtained from all participants. All control subjects were non-stone forming adults and in good health, as evidenced by their medical history and their normal profile on a complete metabolic serum panel. Participant who used any antibiotics in the last 3 months were excluded from the study since the use of antibiotics could decrease or in some cases completely abolish O. formigenes colonization [12]. Since menopausal status also affects BMD, both female stone formers and controls were excluded from the study in addition to any individual with systemic or metabolic disease which may affect bone mineral density. To minimize the uncontrolled impact on testosterone levels, patients undergoing anti-androgen therapy or those who were taking testosterone preparations due to androgen deficiency and 5-α reductase inhibitors for benign prostate hyperplasia were also excluded from the study. Total testosterone levels were measured from morning samples to adjust for the circadian rhythm in testosterone release.
Urinary Calcium and Oxalate Levels
Urinary and serum calcium levels and serum testosterone levels were measured using an automated analyzer (Model 7170; Hitachi High-Technologies, Tokyo, Japan). Urinary oxalate excretion levels were determined by using a commercial enzyme kit (Sigma Diagnostics Inc., MO, USA) from urine specimens obtained via 24-hour collection. Baseline urinary calcium and oxalate levels were compared with previously reported normal values (calcium 70–180 mg/24 h, oxalate 0.08–0.49 mmol/24 h for men). O. formigenes counts were quantified from stool samples which were stored at −80°C until analysis. All subjects provided at least 3 stool samples.
Quantitative Analysis of O. formigenes
Bacterial DNA was extracted from each sample at least three times using 1g stool per sample with the i-genomic Stool DNA Extraction Kit (iNtRON Biotechnology) according to the manufacturer's specifications. The exact amount of O. formigenes in each sample was determined by single color real time PCR with previously described species-specific primer pairs. We used Power SYBR Green Master Mix (Life Technologies) for real time PCR and all cycling conditions were modified accordingly except annealing temperatures which were previously reported by Kumar et al. [13]. In addition to 45 cycles of amplification, we performed a disassociation curve analysis in order to confirm amplification specificity. A single melting point of 85°C was considered to indicate amplicon specificity. Primers were checked for specificity using Primer BLAST [14] and were shown to be specific for the Oxc gene of O. formigenes. Each reaction was run against a set of dilutions containing pure O. formigenes DNA (1 ng DNA = 4.82 × 105 cells) in order to obtain a standard curve. The lower limit of detection was determined as 5 × 103. When a sample yielded a quantity at the lower limit we performed conventional multiplex PCR both with the aforementioned primer pair and a universal bacterial primer pair. Universal primers were used to check if the absence of an amplicon was due to an unsuccessful PCR or to the actual absence/scarcity of O. formigenes. Multiplex reactions were run on agarose gels and the existence of a single band of expected size for universal primers indicated the success of the reaction and absence of bacteria. None of the multiplex reactions indicated an unsuccessful PCR.
Determination of Bone Mineral Density
BMD (g/cm2), bone area (cm2), and bone mineral content (g), were measured for the femur neck, and total lumbar vertebra (L2-L4) using dual-energy X-ray absorptiometry (DEXA, Hologic Inc, Waltham, QDR Elite W 4500) in both groups. Obtained data were corrected for age, height, and the BMI. These measurements were used to calculate T and Z-scores. The Z score was obtained by comparing a subject's bone density to those with the same gender and age, while the T score was obtained by comparing the bone density to 30 year old subjects with the same ethnicity and gender. The obtained values were used to express the decrease in BMD. All scores were evaluated according to the classification criteria set by the World Health Organization and expressed as the mean standard deviation [15].
Statistics
We used the Pearson Chi-square test for statistical analysis of lumbar and femoral BDM values and the Independent Samples T-test for the evaluation of other measurements except O. formigenes counts. Since the Shapiro-Wilk test showed that the number of O. formigenes counts was not normally distributed among patients and healthy controls (p < 0.05), the Mann-Whitney U test was used to assess significant differences in O. formigenes counts among subjects. Linear regression was used to determine the association between BDM in the lumbar region and age, BMI, testosterone levels, and O. formigenes counts. All statistical tests were two-tailed, and statistical significance was defined as p < 0.05. All analyses were conducted using SPSS version 15.0 (SPSS Inc., Chicago, Illinois, USA).
Results
Median O. formigenes counts in patients and healthy subjects were 19,257 (mean 5,791 ± 111.93) and 143,850 (mean 2,815,725 ± 3,946,044.7) respectively. Our results indicated a strong association between the number of O. formigenes counts and calcium oxalate kidney stone formation (Mann-Whitney U = 661, n1 = n2 = 50, p < 0.001). Urinary calcium levels were higher in patients but the difference was not statistically significant. Serum testosterone and urinary citrate concentrations were lower in patients when compared with control subjects while urinary oxalate levels were higher in patients. The differences in serum testosterone levels, and oxalate and citrate concentrations were found to be statistically significant. BMD measurements in the lumbar region yielded significantly lower values in patients (p < 0.05). BMD measurements in the femoral neck area also yielded lower values but the difference was not statistically significant (p > 0.05). We observed lower O. formigenes counts in stone formers in comparison to non-stone formers (table 1).
Table 1.
Comparison of clinical findings and other characteristics in patients and controls
| Characteristics | Patients | Controls | p1 |
|---|---|---|---|
| Age (year) | 48.9 ± 11.9 | 47.2 ± 13.4 | 0.066 |
| BMI (kg/m2) | 26.7 ± 4.5 | 26.1 ± 3.3 | 0.47 |
| Low total testosterone2 | 18 (36%) | 7 (14%) | 0.008 |
| Urine calcium (mg/24h) | 267.2 ± 103,8 | 229.4 ± 104.5 | 0.073 |
| Urine citrate (mg/24h) | 260.1 ± 121.2 | 533.4 ± 263.9 | < 0.001 |
| Urine oxalate (mg/24h) | 39.9 ± 16.4 | 29.5 ± 15.8 | 0.002 |
| Lumbar T score < −1 | 19 (38%) | 8 (16%) | 0.013 |
| Femoral T score < −1 | 10 (20%) | 6 (12%) | 0.275 |
| Median OF count | 19,257 | 143,850 | < 0.001 |
OF = Oxalobacter formigenes; BMI = body mass index.
p values were separately calculated for comparison of each parameter.
A total testosterone concentration of < 285ng/ml is considered low.
We also evaluated the association between BMD, age, BMI, testosterone levels, and O. formigenes counts using linear regression. In urinary stone forming patients, we observed a significant correlation between lower BMD and testosterone levels (p = 0.011, Beta = 0.34) and decreased O. formigenes counts (p = 0.02, Beta = 0.32). In addition, we also observed a negative correlation between BMD and age but this correlation was not statistically significant (table 2).
Table 2.
BMD in the lumbar region is significantly correlated with testosterone levels and O. formigenes colonization in urinary stone forming patients
| Lumbar BMD | r | p |
|---|---|---|
| Age (years) | 0.07 | 0.625 |
| BMI (kg/m2) | 0.17 | 0.243 |
| Testosterone (ng/ml) | 0.34 | 0.011 |
| O. formigenes count | 0.32 | 0.02 |
BMD = Bone mineral density; BMI = body mass index.
When the same comparison was performed for BMD at the femoral neck region both for healthy subjects and stone formers no significant association was detected. Upon observing a significant difference in testosterone levels we categorized patients into 2 groups as the low testosterone (< 285 ng/ml) and normal testosterone (≥ 285 ng/ml) groups and compared O. formigenes counts. This analysis showed a significant association between decreased O. formigenes colonization and low testosterone (table 3).
Table 3.
Comparison of O. formigenes counts and total testosterone levels
| Total test < 285ng/ml | Total test ≥ 285 ng/ml | p | |
|---|---|---|---|
| Urolithiasis (+) | 0.025 | ||
| median OF | 5,510 | 28,610 | |
| mean OF ± SD | 110,818.9 ± 349,991 | 215,627.4 ± 460,553.9 | 0.624 |
| Urolithiasis (−) | |||
| median OF | 163,920 | 137,400 | |
| mean OF ± SD | 961,760 ± 2,122,535 | 1,037,891 ± 2,352,958 |
OF = Oxalobacter formigenes; SD = standard deviation.
Discussion
Urinary oxalate excretion has a crucial role in the formation of calcium oxalate stones. Even normal concentrations of urinary oxalate have been shown to affect calcium oxalate stone formation in terms of recurrence and severity [7]. O. formigenes colonization has been suggested to have an impact on calcium oxalate urolithiasis since this bacterium uses oxalate as its main energy source. Batislam et al. [16] reported that O. formigenes colonization is significantly lower in stone formers and subjects with significantly diminished colonization have higher rates for the coexistence of hyperoxaluria and hypercalciuria. Other studies corroborated these results, reporting reduced urinary oxalate concentrations in calcium oxalate stone forming patients colonized with O. formigenes in comparison to non-colonized stone formers [17]. Our results concerning O. formigenes colonization are in accordance with previous findings. Urolithiasis not only affects the urinary system, but also other systems including the skeletal system. Stone formers have been reported to have a lower BMD than non-stone formers [9, 10, 11]. To the best of our knowledge our study is the first one to investigate the association between bone mineral density and O. formigenes colonization. Due to the lack of an established threshold value for O. formigenes counts in the gut we used absolute quantification to compare O. formigenes counts in both groups [8, 19]. We detected significantly higher O. formigenes counts in control subjects in comparison to calcium oxalate stone formers. In addition, we observed decreased O. formigenes colonization in patients with a lower BMD in the lumbar region than in those with a normal BMD. However we could not confirm these findings in the femoral neck region. In non-stone former males there was no significant difference in O. formigenes colonization regardless of the osteoporotic status which indicated that O. formigenes has little to no effect on BMD in non-stone forming men. The marked decrease in BDM in the patient group may be a consequence of the impaired calcium absorption due to the formation of non-absorbable calcium-oxalate complexes [20]. Such complexes are more likely to occur when reduced O. formigenes concentrations result in higher oxalate concentrations in the gut. In addition to urinary calcium oxalate excretion, BMD is also affected by testosterone levels in men [21, 22]. Therefore we measured serum testosterone concentrations in patients and control subjects and determined significantly lower testosterone concentrations in lithogenic men. This finding implies that reduced BMD may be attributed to the decreased total testosterone levels in lithogenic men when other parameters do not show a significant difference including O. formigenes counts. Interestingly, previous studies investigating the role of endogenous steroids in urolithiasis have produced contradictory results. Naghii et al. [23] reported significantly higher testosterone levels in stone forming men while 2 other studies conducted by Zhao et al. [24] and Watson et al. [25] showed no correlation between testosterone levels and urolithiasis. In contrast with these studies we observed an inverse correlation between calcium-oxalate stone formation and testosterone levels. When we investigated the relation between the O. formigenes concentration and low testosterone levels, we observed a marked decrease in the O. formigenes colonization in patients with low testosterone levels. While it is possible for plasma oxalate concentrations to have an impact on the testosterone levels via an unknown mechanism, this discrepancy is probably a result of the limitations of this study. Limitations of this study include small sample size and the lack of dietary control. The effect of diet on oxalate concentrations could have been compensated for by using a food questionnaire. In conclusion, based on our findings we suggest that O. formigenes may play an essential role in calcium oxalate metabolism, affecting the bone mineral density in oxalate stone formers.
Acknowledgements
The authors declare no conflict of interest. This project was supported by Istanbul University Research Fund (Project No: 24949). Finally, we would like to express our gratitude to Dr. John Knight for providing the Oxalobacter formigenes strain and associated data.
References
- 1.Turney BW, Reynard JM, Noble JG, Keoghane SR. Trends in urological stone disease. BJU Int. 2012;109:1082–1087. doi: 10.1111/j.1464-410X.2011.10495.x. [DOI] [PubMed] [Google Scholar]
- 2.Taylor EN, Stampfer MJ, Curhan GC. Obesity, weight gain, and the risk of kidney stones. JAMA. 2005;293:455–462. doi: 10.1001/jama.293.4.455. [DOI] [PubMed] [Google Scholar]
- 3.Wilson DM. Clinical and laboratory approaches for evaluation of nephrolithiasis. J Urol. 1989;141:770–774. doi: 10.1016/s0022-5347(17)41006-8. [DOI] [PubMed] [Google Scholar]
- 4.Batz M, Kohlbecker G, Significance of serum and urinary oxalate in calcium stone formers . Oxalate in Human Biochemistry and Clinical Pathology. In: Rose GA, Robertson WG, Watts RW, editors. London: The Wellcome Foundation Ltd; 1979. pp. pp181–185. [Google Scholar]
- 5.Yriberri J, Posen S. A semi-automatic enzymic method for estimating urinary oxalate. Clin Chem. 1980;26:881–884. [PubMed] [Google Scholar]
- 6.Jiang J, Knight J, Easter LH, Neiberg R, Holmes RP, Assimos DG. Impact of dietary calcium and oxalate, and Oxalobacter formigenes colonization on urinary oxalate excretion. J Urol. 2011;186:135–139. doi: 10.1016/j.juro.2011.03.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Siener R, Bangen U, Sidhu H, Honow R, von Unruh G, Hesse A. The role of Oxalobacter formigenes colonization in calcium oxalate stone disease. Kidney Int. 2013;83:1144–1149. doi: 10.1038/ki.2013.104. [DOI] [PubMed] [Google Scholar]
- 8.Kaufman DW, Kelly JP, Curhan GC, Anderson TE, Dretler SP, Preminger GM, Cave DR. Oxalobacter formigenes may reduce the risk of calcium oxalate kidney stones. J Am Soc Nephrol. 2008;19:1197–1203. doi: 10.1681/ASN.2007101058. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Tugcu V, Ozbek E, Aras B, Ozbay B, Islim F, Tasci AI. Bone mineral density measurement in patients with recurrent normocalciuric calcium stone disease. Urol Res. 2007;35:29–34. doi: 10.1007/s00240-006-0074-0. [DOI] [PubMed] [Google Scholar]
- 10.Arrabal-Polo MA, Arrabal-Martin M, Arias-Santiago S. Bone and metabolic markers in women with recurrent calcium stones. Korean J Urol. 2013;54:177–182. doi: 10.4111/kju.2013.54.3.177. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Arrabal-Polo MA, Arrabal-Martin M, de Haro-Munoz T, Lopez-Leon VM, Merino-Salas S, Ochoa-Hortal MA, Garrido-Gomez J, Lahoz-Garcia C, Zuluaga-Gomez A. Mineral density and bone remodelling markers in patients with calcium lithiasis. BJU Int. 2011;108:1903–1908. doi: 10.1111/j.1464-410X.2011.10167.x. [DOI] [PubMed] [Google Scholar]
- 12.Sidhu H, Hoppe B, Hesse A, Tenbrock K, Brömme S, Rietschel E, Peck AB. Absence of Oxalobacter formigenes in cystic fibrosis patients: a risk factor for hyperoxaluria. Lancet. 1998;352:1026–1029. doi: 10.1016/S0140-6736(98)03038-4. [DOI] [PubMed] [Google Scholar]
- 13.Kumar R, Ghoshal UC, Singh G, Mittal RD. Infrequency of colonization with Oxalobacter formigenes in inflammatory bowel disease: possible role in renal stone formation. J Gastroenterol Hepatol. 2004;19:1403–1409. doi: 10.1111/j.1440-1746.2004.03510.x. [DOI] [PubMed] [Google Scholar]
- 14.Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden TL. Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics. 2012;13:134. doi: 10.1186/1471-2105-13-134. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Orimo H, Hayashi Y, Fukunaga M, Sone T, Fujiwara S, Shiraki M, Kushida K, Miyamoto S, Soen S, Nishimura J, Oh-Hashi Y, Hosoi T, Gorai I, Tanaka H, Igai T, Kishimoto H. Diagnostic criteria for primary osteoporosis: year 2000 revision. J Bone Miner Metab. 2001;19:331–337. doi: 10.1007/s007740170001. [DOI] [PubMed] [Google Scholar]
- 16.Batislam E, Yilmaz E, Yuvanc E, Kisa O, Kisa U. Quantitative analysis of colonization with real-time PCR to identify the role of Oxalobacter formigenes in calcium oxalate urolithiasis. Urol Res. 2012;40:455–460. doi: 10.1007/s00240-011-0449-8. [DOI] [PubMed] [Google Scholar]
- 17.Troxel SA, Sidhu H, Kaul P, Low RK. Intestinal Oxalobacter formigenes colonization in calcium oxalate stone formers and its relation to urinary oxalate. J Endourol. 2003;17:173–176. doi: 10.1089/089277903321618743. [DOI] [PubMed] [Google Scholar]
- 18.Prokopovich S, Knight J, Assimos DG, Holmes RP. Variability of Oxalobacter formigenes and oxalate in stool samples. J Urol. 2007;178:2186–2190. doi: 10.1016/j.juro.2007.06.045. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Kwak C, Kim HK, Kim EC, Choi MS, Kim HH. Urinary oxalate levels and the enteric bacterium Oxalobacter formigenes in patients with calcium oxalate urolithiasis. Eur Urol. 2003;44:475–481. doi: 10.1016/s0302-2838(03)00318-x. [DOI] [PubMed] [Google Scholar]
- 20.Borghi L, Nouvenne A, Meschi T. Probiotics and dietary manipulations in calcium oxalate nephrolithiasis: two sides of the same coin? Kidney Int. 2010;78:1063–1065. doi: 10.1038/ki.2010.345. [DOI] [PubMed] [Google Scholar]
- 21.Chin KY, Ima-Nirwana S. Sex steroids and bone health status in men. Int J Endocrinol. 2012;2012:208719. doi: 10.1155/2012/208719. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Gielen E, Vanderschueren D, Callewaert F, Boonen S. Osteoporosis in men. Best Pract Res Clin Endocrinol Metab. 2011;25:321–335. doi: 10.1016/j.beem.2010.08.012. [DOI] [PubMed] [Google Scholar]
- 23.Naghii MR, Babaei M, Hedayati M. Androgens involvement in the pathogenesis of renal stones formation. PloS One. 2014;9:e93790. doi: 10.1371/journal.pone.0093790. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Zhao Z, Mai Z, Ou L, Duan X, Zeng G. Serum estradiol and testosterone levels in kidney stones disease with and without calcium oxalate components in naturally postmenopausal women. PLoS One. 2013;8:e75513. doi: 10.1371/journal.pone.0075513. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Watson JM, Shrewsberry AB, Taghechian S, Goodman M, Pattaras JG, Ritenour CW, Ogan K. Serum testosterone may be associated with calcium oxalate urolithogenesis. J Endourol. 2010;24:1183–1187. doi: 10.1089/end.2010.0113. [DOI] [PubMed] [Google Scholar]