A critical analysis of the role of gut Oxalobacter formigenes in oxalate stone disease

Hyperoxaluria is a major risk factor for the formation of calcium oxalate stones, but dietary restriction of oxalate intake might not be a reliable approach to prevent recurrence of stones. Hence, other approaches to reduce urinary oxalate to manage stone disease have been explored. The gut‐dwelling obligate anaerobe Oxalobacter formigenes (OF) has attracted attention for its oxalate‐degrading property. In this review we critically evaluate published studies and identify major gaps in knowledge. Recurrent stone‐formers are significantly less likely to be colonized with OF than controls, but this appears to be due to antibiotic use. Studies in animals and human subjects show that colonization of the gut with OF can decrease urinary oxalate levels. However, it remains to be determined whether colonization with OF can affect stone disease. Reliable methods are needed to detect and quantify colonization status and to achieve durable colonization. New information about oxalate transport mechanisms raises hope for pharmacological manipulation to decrease urinary oxalate levels. In addition, probiotic use of lactic acid bacteria that metabolize oxalate might provide a valid alternative to OF.     

一水草酸钙与二水草酸钙结石形成机理的研究

目的　探讨一水草酸钙（ＣＯＭ） 与二水草酸钙（ＣＯＤ） 结石形成的机制。　方法　应用红外光谱仪对２５８ 块尿结石成分进行检测,同时检测３０ 例患者２４ｈ 尿液生化指标,对测定结果利用ＳＰＳＳ软件进行ｔ 检验。　结果　（１） 尿钙：ＣＯＭ 组（４．８３ ±１ ．９８）ｍ ｍｏｌ／２４ｈ,ＣＯＤ 组（９．８８ ±４ ．２８）ｍｍｏｌ／２４ｈ,Ｐ＜ ０ ．０１ ;（２） 尿磷：ＣＯＭ 组（１９ ．４０ ±９．６９）ｍ ｍｏｌ／２４ｈ,ＣＯＤ 组（２９．２０ ±１２．００）ｍ ｍｏｌ／２４ｈ,Ｐ＜ ０．０５,两组尿钙、尿磷差异有显著性。　结论　二水草酸钙结石患者尿钙、尿磷高于一水草酸钙结石患者,表明二水草酸钙的形成与高钙尿及磷酸盐异质成核有关,而一水草酸钙的形成可能与尿中抑制物缺乏有关。     

复发性草酸钙结石与尿内酸性粘多糖的关系

为了探讨酸性粘多糖（ＧＡＧｓ）对草酸钙结石形成的抑制作用机理，收集了１２例正常人及１５例复发性草酸钙结石病人２４小时尿，经超滤后，用ＰｒｏｎａｓｅＥ蛋白酶降解尿中糖蛋白，纯化ＧＡＧｓ，并分别测定了蛋白水解前后尿样中ＧＡＧｓ含量及种晶体系下ＧＡＧｓ对草酸钙晶体粒度分布。结果表明：（１）水解前后，正常人尿中ＧＡＧｓ含量均比结石病人高；（２）草酸钙晶体生长抑制指数（Ｉｇ）、聚集抑制指数（Ｉａ）与ＧＡＧｓ的含量成正比。     

Oxalobacter formigenes may reduce the risk of calcium oxalate kidney stones

Most kidney stones are composed primarily of calcium oxalate. Oxalobacter formigenes is a Gram-negative, anaerobic bacterium that metabolizes oxalate in the intestinal tract and is present in a large proportion of the normal adult population. It was hypothesized that the absence of O. formigenes could lead to increased colonic absorption of oxalate, and the subsequent increase in urinary oxalate could favor the development of stones. To test this hypothesis, a case-control study involving 247 adult patients with recurrent calcium oxalate stones and 259 age-, gender-, and region-matched control subjects was performed. The prevalence of O. formigenes, determined by stool culture, was 17% among case patients and 38% among control subjects; on the basis of multivariate analysis controlling demographic factors, dietary oxalate, and antibiotic use, the odds ratio for colonization was 0.3 (95% confidence interval 0.2 to 0.5). The inverse association was consistently present within strata of age, gender, race/ethnicity, region, and antibiotic use. Among the subset of participants who completed a 24-h urine collection, the risk for kidney stones was directly proportional to urinary oxalate, but when urinary factors were included in the multivariable model, the odds ratio for O. formigenes remained 0.3 (95% confidence interval 0.1 to 0.7). Surprisingly, median urinary oxalate excretion did not differ with the presence or absence of O. formigenes colonization. In conclusion, these results suggest that colonization with O. formigenes is associated with a 70% reduction in the risk for being a recurrent calcium oxalate stone former.     

中国人肠道产甲酸草酸杆菌oxc基因的克隆及其在真核细胞中的表达

目的 观察中国人肠道产甲酸草酸杆菌(Ox.F)草酰辅酶A脱羧酶基因(oxc)的分离、克隆及其在293细胞中的表达.方法 提取中国人肠道Ox.F的基因组DNA,PCR扩增oxc基因片段并克隆入真核表达载体pEGFP-C1,通过限制性内切酶酶切电泳和测序鉴定基因片段.将重组质粒脂质体转染至293细胞,利用RT-PCR和Western blot分别从mRNA和蛋白水平检测oxc基因在真核细胞中的表达.结果 中国人肠道产甲酸草酸杆菌oxc基因全长1707 bp,与Gene Bank中的序列比较,碱基序列的同源性为93.61%,氨基酸残基序列的同源性为97.18%.重组质粒转染293细胞后24～48 h,可观察到明亮的绿色荧光,从mRNA和蛋白水平上可以检测到oxc基因在真核细胞中的表达.结论 中国人肠道产甲酸草酸杆菌中可以分离出oxc基因;中国人肠道产甲酸草酸杆菌oxc基因存在一定的变异;oxc基因可在真核细胞293细胞中表达.     

表达产甲酸草酸杆菌草酸分解基因的人肝细胞系的构建

目的：构建表达产甲酸草酸杆菌（Ox．F）草酸分解基因的人肝细胞系,探索高草酸尿的治疗方法。方法：分离培养人肠道Ox．F并克隆其功能基因OXC和fre,构建同时表达OXC和fre的真核双表达载体质粒pIRES—oxc-frc,将pIRES-oxe—fre转染人正常肝细胞系L—02。用RT—PCR和Western blot技术了解转基因细胞的目的基因表达状况;用离子色谱法测定转基因后细胞培养液中草酸浓度,了解其草酸分解功能。结果：转基因后人肝细胞系L-02在mRNA和蛋白质水平均成功表达OXC和fre基因,其草酸分解功能较转染前明显增强（P〈0．01）。结论：Ox．F分解草酸的两个重要功能基因OXC和frc能在体外转入人正常肝细胞（L-02）中,并使后者草酸分解能力明显增强。转草酸分解基因的人肝细胞系的构建,有望用于高草酸尿,尤其是PH的病因治疗,值得进一步研究．     

肾结石大鼠肾组织bikunin mRNA的表达

目的 :研究bikunin在实验性肾草酸钙结石大鼠肾组织的表达及意义。方法 :采用乙二醇和氯化铵诱导大鼠肾草酸钙结石模型形成 ,检测各组大鼠肾功能、肾组织Ca2 + 含量和草酸钙晶体沉积、尿生化指标 ,并用逆转录聚合酶链反应 (RT PCR)检测bikuninmRNA在肾组织的表达情况。结果 :模型组大鼠的血清Cr、BUN、肾Ca2 + 含量、2 4h尿Ca2 + 、草酸 (Ox)分泌量和肾组织bikuninmRNA的表达均明显高于正常组 (P <0 .0 5 )。结论 :高草酸尿和草酸钙结晶的沉积能促使大鼠肾脏通过合成更多的bikunin来抑制大鼠肾组织草酸钙晶体的形成。     

Rapid reversal of hyperoxaluria in a rat model after probiotic administration of Oxalobacter formigenes

PURPOSE: The gut inhabiting bacterium Oxalobacter formigenes may be a negative risk factor in recurrent calcium oxalate kidney stone disease that apparently maintains oxalic acid homeostasis in its host via the degradation of dietary oxalate. The possibility of using this bacterium as probiotic treatment to reduce urinary oxalate was investigated in a rat model. MATERIALS AND METHODS: Male Sprague-Dawley rats were placed on a diet supplemented with ammonium oxalate to induce a state of severe hyperoxaluria. Subgroups of these rats received an esophageal gavage of 1 x 10(3), 10(5), 10(7) or 10(9) O. formigenes per feeding for a 2-week period. Each rat was followed for general health and changes in urinary oxalate. RESULTS: Rats with chronic hyperoxaluria resulting from high dietary oxalate that were treated with O. formigenes showed decreased urinary oxalate within 2 days of initiating probiotic supplementation. The amount of the decrease in a 2-week period proved directly proportional to the dose of bacteria. Urinary oxalate in rats receiving higher amounts of O. formigenes returned to almost normal. Throughout the study the rats remained healthy with no signs of toxicity, antibody development or a histopathological condition. CONCLUSIONS: Probiotic treatment of hyperoxaluric rats with O. formigenes may significantly and rapidly reduce the level of oxalate in the urine. This probiotic treatment appears to be safe and well tolerated. The approach may be feasible for treating calcium oxalate kidney stone disease.     

正常人和草酸钙结石病人尿草酸钙晶体基质

以改良Ｍｏｒｓｅ和Ｒｅｓｎｉｃｋ法提取１０例上尿路草酸钙结石病人和１１例正常人的尿草酸钙晶体基璺，用双向聚丙烯酰胺凝胶电泳对晶体基质及结晶前后大分子物的蛋白质组成进行了比较分析。     

Morphology of crystals in calcium oxalate monohydrate kidney stones

Both scanning electron microscopy and atomic force microscopy (AFM) have shown that calcium oxalate monohydrate kidney stones are made up from arrangements of sub micron crystals. The purpose of this investigation was to determine the morphology of these crystals which was obscured by the presence of organic matrix in our earlier study. Sections of stones were treated to remove the protein component of the matrix and then imaged using AFM. Images obtained after proteolysis show that the crystals are in the form of plates stacked on (100) surfaces. These results were confirmed by scanning electron microscopy observations from selected regions of calcium oxalate kidney stone surfaces. The observed crystal sizes are consistent with both the known matrix mass fraction and crystallite growth in the passage through the collecting duct.     

草酸钙结石患者肾乳头Randall斑的超微结构特点

目的 探讨草酸钙结石患者肾乳头Randall斑与草酸钙结石形成的关系. 方法经结石化学成分分析确诊为草酸钙肾结石患者12例.于经皮肾镜取石术中直视卜获取肾乳头Randall斑活检标本,分别行HE染色和锇酸固定,光镜和透射电镜下观察其组织病理和超微结构特点.结果 12例患者共检查肾乳头72处,肾乳头表面有Randall斑形成63处(87.5%),7例部分肾乳头表面有小结石附着.12例Randall斑活检标本光镜下肾乳头组织内见成团钙盐样沉积.2例电镜下肾乳头结缔组织中散在分布大小不均簇状草酸盐团聚体,典型结晶体呈针状,晶体轮廓周边电子密度高,晶体中央呈电子透亮区. 结论草酸钙结石患者肾乳头Randall斑主要是草酸盐结晶沉积,在Randall斑基础上草酸盐结晶进一步沉积可能促使草酸钙结石的形成.     

苄丙酮香豆素对实验性大鼠肾草酸钙结石形成的影响

目的：探讨Vit．K拮抗剂苄丙酮香豆素(商品名华法令)对大鼠肾草酸钙结石形成的影响。方法：采用乙二醇饮水和氯化铵灌胃作成石剂，30只Wistar大鼠随机分为对照组(A组)、成石组(B组)、华法令组(C组)。饲养4周后，检测大鼠肾组织钙含量和草酸钙晶体形成、24h尿钙、尿草酸含量及血生化指标。结果：成石组和华法令组肾组织中钙、镁含量，24h尿草酸及尿钙、镁排泄量差异无显著性意义；镜下观察发现：华法令组大鼠肾脏草酸钙结晶形成多于成石组，但组间比较差异无显著性意义。结论：苄丙酮香豆素对大鼠肾草酸钙结石的形成无显著影响。     

钙拮抗剂抑制大鼠肾草酸钙结石生成的实验研究

目的观察不同剂量硝苯地平灌喂对大鼠肾草酸钙结石生成的影响。方法选用60只雄性SD大鼠,体重200~250g,随机分为6组,分别为空白对照组、单纯硝苯地平组、单纯诱石组、诱石 3、6、10mg·kg-1·d-1硝苯地平干预组,每组各10只。应用乙二醇诱导大鼠产生肾草酸钙结石,4周后观察大鼠肾小管结晶沉积情况、肾组织自由基水平、细胞凋亡情况和大鼠血和尿多项生化指标的变化。结果与单纯诱石组相比,各硝苯地平干预组的肾小管草酸钙结晶评分分别降低37.0%、55.6%、66.7%(P均<0.05),肾小管上皮细胞凋亡数分别减少30.2%、44.6%、48.7%(P均<0.05),两者有显著相关性(r=0.8251);肾组织中MDA含量也分别减少14.5%、20.4%、21.8%,而SOD活性增加3.5%、8.7%、12.6%(P均<0.05);量效关系呈正相关。结论硝苯地平通过减少高尿草酸所致的肾小管上皮细胞凋亡,能有效抑制饮用诱石剂大鼠的肾小管结石生成。     

近端肾小管上皮细胞损伤后对草酸钙结晶的促进作用

目的 研究过氧化氢(H2O2)对人肾小管上皮细胞(HKC)的氧化损伤作用,探讨结晶时间对损伤HKC调控草酸钙(CaOxa)晶体生长的影响.方法 通过检测细胞成活率和细胞中丙二醛释放量的变化评价HKC的损伤程度;利用扫描电镜研究HKC损伤对CaOxa结晶的影响.结果 0.3 mmol/L H2O2作用HKC 1 h后,细胞活性降为79.0%,作用2 h后,细胞活性仅37.8%(P＜0.05).正常HKC形态饱满,细胞连接成片,鞭毛、突触等均完好.0.3 mmol/L H2O2作用1 h后,HKC发生明显皱缩,细胞表面粗糙,周围出现细胞碎片;作用2 h后,细胞皱缩更明显,部分细胞脱落.对照组细胞只诱导少量二水草酸钙形成,损伤细胞不仅诱导一水草酸钙(COM)形成,而且增加CaOxa晶体的数量和聚集程度;用CaOxa过饱和溶液长时间孵育对照组细胞后亦可以产生损伤.结论 H2O2能使HKC产生氧化性损伤,促进COM晶体成核和聚集;晶体在尿路中长时间滞留是肾结石形成的危险因素.

Abstract：

Objective To investigate the injury caused by hydrogen peroxide (H2O2) on human renal tubular epithelial cell (HKC) and its effect on calcium oxalate (CaOxa) crystal crystallization time before and after the injury. Methods The injury degree of HKC by H2O2 was measured by detecting the cell survival rate and the concentration change of malonaldehyde (MDA). CaOxa crystallization was investigated by scanning electron microscopy (SEM). Results Control cells induced only a small amount of calcium oxalate dihydrate (COD) crystals, while the injured cells not only induced calcium oxalate monohydrate (COM) crystals, but also increased the number and aggregation of CaOxa crystals. After incubating with CaOxa supersaturated solution, the control group HKC cells could be injured as well. Conclusions H2O2 can cause oxidative damage on HKC. The injured HKC promotes the nucleation and aggregation of COM crystals. In the body environment, the long-term presence of crystals in urinary tract is a risk factor for stone formation.     

晶体表面结合蛋白对草酸钙晶体生长的抑制作用

为观察晶体表面结合蛋白（ＣＳＢＰ）对草酸钙晶体生长的影响，用层析法分离提取正常人和草酸钙结石病人ＣＳＢＰ，应用种晶体技术检测ＣＳＢＰ、尿凝血酶原激活肽Ｆ１片段（ＵＰＴＦ１）和白蛋白在体外对草酸钙晶体生长的作用。结果发现正常人ＣＳＢＰ和ＵＰＴＦ１能显著抑制草酸钙晶体的生长，结石病人ＣＳＢＰ的抑制活性低于正常人。提示病人ＣＳＢＰ中ＵＰＴＦ１减少致抑制活性降低与结石形成有关     

鱼油抑制实验鼠草酸钙结晶形成

目的 了解鱼油在尿石形成中的作用。方法 ６０只大鼠随机分４组,饮用１％乙二醇（ＥＧ）水,同时喂饲不同剂量的鱼油。４周后检测各组大鼠肾功能、草酸钙结晶、２４小时尿钙和尿草酸。结果 加服鱼油组鼠肾积水、组织水肿减轻,肾组织内草酸钙结晶数及含钙量明显减少,２４小时尿钙排出减少;尿尿素氮、肌酐排出明显增加,而血中尿素氮、肌酐浓度显著低于成石组。结论 鱼油能抑制实验性高草酸尿症大鼠体内草酸钙结晶形成,减少尿     

凝血酶原分子中γ-羧基谷氨酸对草酸钙结晶的抑制作用研究

目的：探讨凝血酶原(PT)分子中γ-羧基谷氨酸(Gla)在抑制草酸钙结晶形成中的作用。方法：采用加入不同浓度的吗啉和甲醛(1：10000)或(1：100)，体外化学修饰PT，将其分子内8个或2个γ-羧基谷氨酸残基修饰转变成γ-亚甲基谷氨酸残基(γ-MGlu)，草酸钙种晶抑制试验测定化学修饰后PT对草酸钙结晶抑制作用的变化。结果：PT蛋白转变为2γ-MGlu的PT后对草酸钙结晶的抑制活性从正常对照的14．2降低至128(10％)；PT蛋白转变为8γ-MGlu后其抑制活性降低至5．0(65％)。结论：凝血酶原分子内γ-羧基谷氨酸(Gla)在抑制草酸钙结晶形成中具有重要作用。