| Background and objectiveKidney stone disease(KSD)is ranked number 3 in urinary system disease.The incidence of KSD will be further improved because of the aging population,the warming of global climate,changes in lifestyle and diet,the epidemic of obesity and more accurate diagnosis methods.Currently we can remove the stones mainly through all kinds of surgery,as we are lack of effective medical treatment.In the past,KSD is often considered as a kind of acute disease,but a growing body of evidence suggests that it also belongs to chronic systemic disease,which could eventually lead to end-stage renal disease(ESRD),myocardial infarction and so on.Lately scientists found that,compare with healthy adults,kidney stone patients have a higher risk in developing cardiovascular disease.Calcium oxalate stone is the most common type of KSD,accounting for 70% to 80% of all stones.Elevated urine oxalate concentration is an important factor in calcium oxalate stone formation.Excessive saturated oxalate combine the calcium in the urine to form insoluble calcium oxalate crystal.The aggregation of plenty of crystals leads to calcium oxalate stones.Oxalate in human body are both exogenous and endogenous,the exogenous oxalate comes from all kinds of foods we intake.The endogenous oxalate are toxic byproducts of metabolism of glycine and glyoxylate in liver.Oxalate in human body discharge in three ways: 5% ~ 10% of oxalate are secreted to the lumen,which combine the calcium in lumen to form insoluble calcium oxalate and discharge with faeces or degraded by intestinal microbes;degradation;90% ~ 95% of oxalate are discharged in urine through kidney.Therefore,increasing the degradation and discharge of oxalate in lumen may be one of the effective methods of prevention and treatment of calcium oxalate stone.In 1985,a scientist found a kind of obligate anaerobe which can degrade oxalate in human gut and named it Oxalobacter formigenes(OxF).There is a wide range in the level of colonization with Ox F in our intestine,ranging from zero to 108 cfu/gm.wet weight of intestine contents.If our intestine is colonized with 108 cfu/gm,0.5 to 1 gm.oxalate can be degraded everyday.In fact,the vast majority of calcium oxalate stone patients lack this kind of the bacteria in their gut,so the supplementation of the probiotics may lead to a significant reduction in the occurrence and recurrence of calcium oxalate stones.For now on,most research upon OxF are in foreign countries and few in domestic.The purpose of this study is to screen Ox F from intestines of healthy people,study its effect in the prevention of calcium oxalate stone and provide reliable basis for related drug development in our country.Methods1.Screen Ox F from fresh feces of healthy people.After the isolation,culture,purification,identification of the bacteria,preserve the strains and store them in-80°C.Culture the screened strain to different concentration(106 cfu/m L,107 cfu/m L,108 cfu/mL).2.Male Sprague-Dawley rats weighing approximately 180 to 200 gm are used in this study.EG is supplemented to 0.8% volume per volume to induce animal model of calcium oxalate stone.3.The animal model of calcium oxalate stone received an esophageal gavage of 1*106,107,108 viable OxF bacteria everyday for a 4-week period.Each dose consisted of 1 ml.of bacterial suspension.Each rat was followed for general health and changes in urinary oxalate.Weigh up the rats and collect their blood and 24-hour urine samples on weeks 1,2,3 and 4.Detect the serum creatine and blood urea nitrogen,and calcium,phosphorus,magnesium in blood and urine.4 weeks later,drug the rats with chloral hydrate and remove the kidney.After HE staining,observe crystals by polarizing microscope.After Yasue staining,observe calcium oxalate deposition.Results1.After 16 s rRNA sequence determination,the similarity of the bacteria strain we isolated from fresh feces of healthy people and the known ATCC35274 was 100%,which means the strain we screen is Ox F.2.We can see a significant increase in 24-hour urinary oxalate excretion in calcium oxalate stone models as early as 1 week after starting the EG supplemented water,while no such changes were observed in control rats.Also we can see a 50% decrease in 24-hour urinary oxalate in rats receiving 108 cfu OxF as early as 1 week,while there is no significant change in rats who received 106 cfu and 107 cfu OxF.3?After 4 weeks of intervention,there is a significant decrease in calcium oxalate score in rats receiving viable OxF comparing with animals,while we can see no significant change in calcium oxalate score in group 106 cfu and 107 cfu.4?There is no significant difference between animal models and blank controls in body weight,Scr and BUN,which indicate that the kidney function of animal models are not affected.There is also no difference in these areas between three treatment groups and animals which means the treatment was well tolerated.Throughout the study the rats remained healthy with no signs of toxicity.ConclusionsWe have successively screened OxF from healthy adults,however,the top concentration of the bacteria we culturaled only reached 107cfu/ml.Still we need to further optimize the bacteria,improve the concentration of fermentation,shorten the time of fermentation.Probiotic treatment of calcium oxalate stone rats with 108 cfu O.formigenes may significantly and safely reduce the level of oxalate in the urine and further prevent calcium oxalate stones. |
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