Impact of intestinal flora on calcium oxalate stones / 中南大学学报(医学版)

Kidney stone is one of the common diseases of the urinary system. About 80% of kidney stones are mainly composed of calcium oxalate. As a huge bacterial network, the interaction of gut microbes is complex. Intestinal microbes may play a role in the pathogenesis and prevention of kidney stones. The intestinal flora of patients with calcium oxalate stones possess unique distribution of gut microbes.

Role of Oxalobacter formigenes in preventing calcium oxalate kidney stones / 中华肾脏病杂志

Objective To screen Oxalobacter formigenes (OxF) from fresh feces of healthy adults,and study its effect on the the prevention of calcium oxalate kidney stones.Methods OxF was screened and cultured from fresh feces of healthy adults.The rat model of calcium oxalate stone was established by esophageal gavage of 0.8％ of ethylene glycol.Rats were divided into a control group and four groups of rats with ethylene glycol-induced calcium oxalate kidney stones according to random number table.Three groups were treated with 106 CFU,107 CFU,108 CFU viable OxF every day,respectively,for 4 weeks.The blood and 24-hour urine samples were collected to detect the serum creatinine,urea nitrogen,serum and urine calcium,phosphorus,magnesium and urine oxalate every week.At the end of the 4th week,the rats were sacrificed and the kidney tissues were stained with HE and Yasue.The deposition and content of calcium oxalate crystals were observed under a light microscope.Results The bacteria strain isolated from fresh feces of healthy adults was 100％ as same as the known ATCC35274 bacteria strain,which means the strain screened is OxF.Among the 5 groups,there were no significant differences in body weight,Scr,BUN,serum calcium,blood magnesium,blood phosphorus,urinary magnesium and urinary phosphorus.The 24-hour urinary calcium excretion in the model group was significantly lower than that of the control group (P ＜ 0.05).After intervention with OxF solution,the 24-hour urinary calcium excretion in the 108 CFU OxF group was significantly higher than that in the model group (P ＜ 0.05),while there was no significant difference between the other intervention groups and the model.The oxalic acid excretion of 106 CFU OxF group and 107 CFU OxF group was lower than that of the model,but the difference did not reach statistical significance (P＞ 0.05).The 24 h oxalic acid excretion in the 108 CFU OxF group was significantly lower than that of the model at the end of first week (P ＜ 0.05),and continued to decrease for the next 3 weeks.After 4 weeks of intervention,no crystal formation was observed in the control group under the deflection microscope,but a large amount of calcium oxalate crystals were formed in the renal cortex and renal medulla.The crystals were piled up and connected to each other.Yasue staining coincided with the calcium oxalate crystal in the same part of the kidneys.Compared with the model,there was no significant change in the score of calcium oxalate crystal in the kidneys of 106 CFU OxF group and 107 CFU OxF group,while the score of calcium oxalate crystal in the kidneys of 108 CFU OxF group was significantly lower (P ＜ 0.05).Conclusions OxF are successively screened from healthy adults.Daily administration of 108 CFU OxF can safely and effectively reduce the urinary oxalic acid excretion,prevent the formation of calcium oxalate crystals and inhibit the formation of stones in kidneys of rats.

Construction of oxalate-degrading adult bone marrow mesenchymal stem cell lines / 中国组织工程研究

BACKGROUND: High oxalic acid urine is a risk factor for stone formation. Constructing cel lines with high oxalate metabolic ability using genetic engineering and stem cel technology wil become the effective method to prevent and treat calcium oxalate stone. OBJECTIVE: To construct the adult bone marrow mesenchymal stem cel lines that can decompose the oxalic acid, through co-transfecting the oxalic acid degradation genes Frc and Oxc of oxalobacter formigenes into the normal adult bone marrow mesenchymal stem cells. METHODS: Frc and Oxc were amplified by PCR, and the eukaryotic expression vectors of pLEGFP-N1-myc-Frc and pBaBE-puro-flag-Oxc were constructed, then co-transfected into the normal adult bone marrow mesenchymal stem cells. The non-transfected bone marrow mesenchymal stem cells and the cells transfected with empty vectors were as control. Western blot was performed to detect the expression of the objective genes;the concentration of oxalate in the culture medium after transgenic was determined by ion chromatography. RESULTS AND CONCLUSION: Restriction enzyme digestion and sequencing results showed that the Frc and Oxc genes were successful y amplified, and the vectors of pLEGFP-N1-myc-Frc and pBaBE-puro-flag-Oxc were constructed. After tranfected into the bone marrow mesenchymal stem cells, the Western blot results showed that transfected bone marrow mesenchymal stem cells could stably express the target protein myc-formyl coenzyme A transferase enzyme and the flag-oxalyl coenzyme A decarboxylase; ion chromatography test results showed with the prolonging of the culture time, the concentration of oxalic acid in the human bone marrow mesenchymal stem cel culture medium transfected with target gene was decreased gradual y. While there was no target protein expression in the non-transfected human bone marrow mesenchymal stem cells as wel as the cells transfected with empty vectors. The cells had the ability of oxalate-degradation. The experiment successful y constructs the adult bone marrow mesenchymal stem cel lines that can decompose the oxalic acid, and the cel lines have the ability of oxalate-degradation and can stably express the oxalate decomposition proteins Frc and Oxc.

Development of Recombinant Escherichia coli expressing oxc and frc Gene of Oxalobacter formigenes / 대한비뇨기과학회지

PURPOSE: Oxalobacter formigenes (O. formigenes) is an obligate anaerobe, which may be important in the prevention of stone formation. O. formigenes degrades oxalates using oxalyl-CoA decarboxylase and formyl- CoA transferase encoded by the oxc and frc genes, respectively. Attempts were made to develop recombinant Escherichia coli (E. coli) expressing both the oxc and frc genes of O. formigenenes. MATERIALS AND METHODS: After the extraction of total RNA from O. formigenes, a reverse transcriptase-polymerase chain reaction (RT-PCR) was carried out using primers synthesized according to the oxc and frc genes reported in GenBank. The cloned cDNA encoding oxalyl-CoA decarboxylase and formyl-CoA transferase was introduced into the pET-22b (+) plasmid vector. The constructs were verified by restriction analysis and DNA sequencing. The plasmid vector containing the cDNA fragment was transformed into competent E. coli BL21 (DE3). The recombinant E. coli was then analyzed using SD-SPAGE for the protein expressions of oxc and frc gene products, and visualized by staining with Coomassie Blue. RESULTS: Restriction enzyme and sequence analyses showed the gene cloned into the pET-22b (+) plasmid vector was identical to the reported oxc and frc genes. After the transformation into the competent E. coli, the SDS-PAGE analysis showed the recombinant E. coli expressed the proteins migrating at 66 and 50KD, which was identical to the reported weight of oxalyl-CoA decarboxylase and formyl-CoA transferase. CONCLISIONS: A recombinant E. coli, expressing oxc and frc genes, was successfully produced. Further studies may be necessary to investigate their enzymatic activities on the degradation of oxalate in the development of a new therapeutic strategy for the prevention of stone formation.

Isolation and Purification of the Oxalate-Degrading Bacterium Oxalobacter formigenes in Human Feces / 대한비뇨기과학회지

PURPOSE: The absence of the oxalate-degrading bacteria, Oxalobacter formigenes, in the gastrointestinal tract correlates with the formation of calcium-oxalate urolithiasis. The aim of this study was to isolate Oxalobacter from human feces. MATERIALS AND METHODS: The OxB strain, isolated from sheep rumen, was incubated in selective growth media(medium B) in an anaerobic chamber, and its microbiological properties evaluated. Feces from volunteers, who were presumed to have O. formigenes from a polymerase chain reaction-based detection system, was incubated in medium B. The colonies isolated, primarily 7 days after incubation, were successively subcultured, and colony-PCR performed to isolate colonies from the successive subcultures. RESULTS: The colonies of OxB were Gram-negative, non-motile, non-spore forming, rod-shaped cells. The cells were often in pairs or chains. OxB DNA gave rise to an amplicon of the correct molecular size(416 bp) of O. formigenes. The morphology of the colonies from human feces, of which DNA was confirmed to have the same size amplicon of O. formigenes by PCR, was identical to the OxB, both grossly and by Gram stain. Although the morphology of the colonies isolated from the successive subcultures was no different from that of OxB, the PCR positivity of the isolated colonies decreased on successive subculturing, with no PCR-positive colonies from the fifth subculture. CONCLUSIONS: Our results suggest that the microbiological isolation and purification of Oxalobacter formigenes from human feces is a difficult procedure. Special culture conditions will be required to culture Oxalobacter species to reveal the link between O. formigenes and calcium oxalate urolithiasis in humans.

Correlation between Urinary Oxalate Levels and Enteric Oxalobacter formigenes in Patients with Calcium Oxalate Urolithiasis / 대한비뇨기과학회지

PURPOSE: We performed a prospective study to evaluate the intestinal colonization by Oxalobacter formigenes, and its relationship to the levels of urinary oxalate in patients with calcium oxalate stone disease. MATERIALS AND METHODS: One hundred and three patients with calcium oxalate urolithiasis, with a mean age of 47 years, ranging in age from 21 to 73, followed-up between August 2000 and September 2001, were enrolled in this study. Fresh stool and 24-hour urine samples were performed. The genus specific oligonucleotide sequences, corresponding to homologous regions residing in the oxc gene, which encodes for oxalyl-coenzyme A decarboxylase, were designed. In order to quantify the presence of O. formigenes in clinical specimens, a quantitative-PCR-based assay system, utilizing a competitive DNA template as an internal standard, was developed. Measurements of the urine volume, pH, creatinine clearance, oxalate, calcium, magnesium, phosphate, citrate and uric acid were performed. RESULTS: The intestinal Oxalobacterium was detected by PCR in 45.6% of the patients with calcium oxalate stones. In the patients with stones, who tested negative for the Oxalobacterium, the average urinary oxalate level was 0.36mmol/day, compared to 0.29mmol/day for those patients testing positive (p<0.05). The mean colony forming unit per gram of stool in all the patients was 1.1x107 (0 to 4.1x108). The 24 h urine oxalate level was significantly decreased with the increasing level of O. formigenes colony forming units (r= 0.517, p<0.001). CONCLUSIONS: Our results support the concept that O. formigenes is important in maintaining oxalate homeostasis, and its absence from the gut increases the risk of calcium oxalate urolithiasis, via increases in the level of urinary oxalate.

Molecular Identification of Oxalobacter formigenes with Polymerase Chain Reaction: Fresh vs. Frozen Stool / 대한비뇨기과학회지

PURPOSE: Oxalobacter formigenes is an intestinal flora degrading oxalate in the gut. However, microbiological detection of this organism is quite difficult. We tried to develop a simple, rapid and cost-effective PCR method for detecting Oxalobacter formigenes from fecal specimens and to determine whether O. formigenes could be detected from frozen fecal specimens as well as fresh stool. MATERIALS AND METHODS: Whole bacterial DNA was isolated directly from fresh and frozen stool samples obtained from 30 healthy volunteers known to be free from urolithiasis and from fresh stool samples obtained from 38 patients with urolithiasis. Genus specific oligonucleotide sequences corresponding to homologous regions residing in the oxc gene that encodes for oxalyl-coenzyme A decarboxylase were designed. A PCR-based assay was done in both fresh and frozen stool samples and the nucleotide sequences were analyzed to determine the details of oxc. RESULTS: PCR product of 416-bp molecular size encoding oxc gene was detected in 23 (77%) of 30 healthy volunteers and in 14 (37%) of 38 patients with urolithiasis. In healthy volunteers, the results of PCR for the fresh and the frozen stool proved identical in each subject. The nucleotide sequence analysis revealed that the sequence of the amplified product was compatible with that of oxc gene. CONCLUSIONS: O. formigenes could be identified easily and efficiently by this PCR-based detection system. Furthermore, as the PCR-based assay results in the frozen fecal samples were identical as that of fresh stool, immediate processing of the fecal samples may not be necessary to detect O. formigenes in the clinical setting.