Role of Oxalobacter formigenes in preventing calcium oxalate kidney stones

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.     

Investigations on macromolecular precipitation inhibitors of calcium oxalate

Summary Certain important aspects of the urine oxalate tolerance test (OTT) have been revised. The stirring system has been changed and the test has been adapted to the kinetics of calcium oxalate precipitation. True equilibrium conditions are now ensured during the measurements. Furthermore, the endogenous oxalate concentration is determined and taken into consideration. As a result of these changes, the significance of the test results has greatly improved. The effects of the addition of small amounts of zinc on the precipitation of calcium oxalate have been used in a new variation of the OTT. This new test makes it possible to discriminate much faster and more simply between recurrent stone-formers and other subjects. Tamm-Horsfall protein (THP) has been tested for its effect on the precipitation of calcium oxalate by means of OTT. THP inhibits the precipitation of calcium oxalate, but THP of stone-formers has a diminished inhibitory activity. The inhibitory activity of this protein strongly depends on the method by which it is isolated.     

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。     

Glycosaminoglycans,uric acid and calcium oxalate urolithiasis

Summary The interaction between calcium and glycosaminoglycans (GAGs) was studied using a calcium ion-selective electrode. The Ca-binding capacity of GAGs involved 16% of total calcium in the presence of chondroitin sulphate and 28% in the presence of pentosan polysulphate. The action of GAGs on the nucleation of uric acid and sodium urate was examined and inhibitory effects were observed. The action of uric acid as a heterogeneous nucleant of calcium oxalate was studied, and considerable promotion of the heterogeneous nucleation of calcium oxalate by uric acid was found, which could be inhibited by the action of GAGs. From these summarised in vitro results, we conclude that uric can constitute an important risk factor for calcium oxalate urolithiasis through heterogeneous nucleation and the GAGs can play an important role as preventive agents.     

May enzyme activity in urine play a role in kidney stone formation?

Summary It has been found that calcium oxalate stone formers have low UGOT and UGPT activity compared to healthy individuals. The urine of 23 stone formers and 19 controls has been tested for combined UGOT and UGPT activity. The effect of L-aspartic acid, alanine and L-glutamic acid on calcium oxalate precipitation has been tested. Only L-glutamic acid exerted a significant retardation effect at physiological concentrations. As GPT and GOT convert alanine and aspartic acid respectively into glutamic acid, a possible mechanism of retardation of kidney stone formation involving enzyme steps via glutamic acid creation in situ is suggested.     

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。     

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。     

草酸钙晶体表面结合蛋白质的研究

目的 了解草酸钙晶体表面结合蛋白质在结石形成的过程中的作用。方法 用草酸钙过饱和结晶法制备正常人和草权钙结石患者尿草酸钙晶体表面结合物质（ＣＳＢＳ）,经ＤＥＡＥ－ＳｅｐｈａｒｏｓｅＣＬ－６Ｂ柱层析分离蛋白质和葡胺聚糖,用ＳＤＳ－聚丙烯酰胺凝胶电泳（ＳＤＳ－ＰＡＧＥ）测定蛋白质组成和分子量,用氨基酸自动分析仪测定蛋白质的氨基酸,结果;正常仍ＣＳＢＳ中主要含分子量为３１０００的尿凝血酶原激活肽片段１（     

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。     

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.     

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。     

Inhibition of calcium oxalate precipitation by bile salts

BACKGROUND: Both urinary and biliary stones can contain calcium. Bile salts (BA), which are known to bind Ca2+, are commonly used to dissolve the latter but not the former. METHODS: The effect of physiologic BA on calcium oxalate (CaOx) precipitation was evaluated by a recently developed method. RESULTS: The Ca2+ binding properties of BA were confirmed by small but significant decreases in pH observed following addition of CaCl2 to bile acids solutions. More importantly, BA inhibited CaOx precipitation with effective concentrations of approximately 10-3 mol/L. The clinical relevance of the latter observation is presently unknown but it is of note that in the same in vitro assay, the activity of BA appeared comparable to that of citric acid, the most common drug for urolithiasis. Although BA do not reach mmol/L levels in urine, they are known to change the physicochemical properties of this fluid, possibly slowing down the crystal growth process. However, the hypothetical therapeutic use of BA in former stone patients would present at least two major problems: (i) hepatotoxicity and (ii) lithogenic activity, due to hyperoxaluria subsequent to increased intestinal absorption of oxalate. CONCLUSION: The ability of BA in effectively binding calcium ions and in inhibiting the precipitation of CaOx appears of interest from both a physiopathologic and a pharmacologic point of view, even if it does not currently seem exploitable for prophylactic or therapeutic purposes.     

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     

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。     

Production of calcium oxalate monohydrate,dihydrate or trihydrate

Summary Procedures to obtain calcium oxalate monohydrate, dihydrate and trihydrate are presented and discussed. The influence of several additives and conditions in the formation of calcium oxalate dihydrate crystals are compartatively evaluated. It seems that the presence of colloidal phosphate favours the formation of calcium oxalate dihydrate crystals through heterogeneous nucleation.     

Determination of GOT activity on nucleation and crystal growth of calcium oxalate

Summary Crystal Size Distribution (CSD) and the yield of Calcium Oxalate Crystals in solutions with an admixture of 5 normal and 3 stone forming urines, were determined. A positive correlation was found between the median size, the number of particles and the overall inhibitory potentials of the urines toward calcium oxalate precipitation in vitro as reflected by Discriminating Index (DI) measurements. Incubation of two samples of stone formers' (SF) urines with glutamic-oxalacetic-transaminase (GOT) caused a reduction of aspartic acid concentration, an increase in glutamic acid concentration and a parallel decrease in the DI values. After 90 min of SF urine incubation with GOT the DI in three samples was improved and both the median size and number of particles reduced, by 28% and 45% respectively. These results could indicate that GOT activity changes the inhibitory power of the SF urine by transforming aspartic acid into glutamic acid, having thus most probably a part in the inhibition of CaOx stone fromation.     

Identification of proteins extracted from calcium oxalate and calcium phosphate crystals induced in the urine of healthy and stone forming subjects

The purpose of our study was to identify the proteins and investigate the differences, if any, between protein components of the matrices of calcium oxalate (CaOx) and calcium phosphate (CaP) crystals induced in␣vitro in whole human urine of healthy individuals and kidney stone patients. In addition, preliminary studies were performed to understand the effect of centrifugation and filtration of urine on its protein contents. Crystallization in urine was induced by addition of an oxalate or phosphate load. Crystals were collected, washed, and analyzed by scanning electron microscopy, X-ray diffraction, and energy dispersive X-ray microanalysis. Matrix proteins were obtained by demineralization with ethylene diamine tetraacetic acid (EDTA), analyzed by polyacrylamide gel electrophoresis, and identified by western blotting technique. No significant differences were detected between protein components of the matrices of CaOx and CaP crystals and between the crystal matrices obtained from the urine of normal and stone forming subjects. Albumin (AB), inter-α-inhibitor (IαI) related proteins, α-1 microglobulin (α-1 m), osteopontin (OPN), prothrombin (PT)-related proteins and Tamm-Horsfall protein (THP) were identified in matrices of both CaOx and CaP crystals induced in urine from both the normal subjects and stone formers. AB, PT-related proteins and OPN were the main constituents. The other proteins were present in smaller but detectable amounts. However, CaP crystal matrix, contained a large amount of THP. In addition CaP crystals contained significantly more proteins than CaOx crystals. Centrifugation and/or filtration of the urine resulted in reduction of many high molecular weight proteins including THP, AB and OPN in the urine. Received: 24 July 1997 / Accepted: 2 January 1998