Orthosiphon grandiflorum has a protective effect in a calcium oxalate stone forming rat model

This study amied to investigate the effects of Orthosiphon grandiflorum on the renal tubular cell injury induced by oxalate and the inhibitory effects of O. grandiflorum on urinary deposit formation in an animal model and compared the results with those from a potassium citrate treatment. Rats were divided into three groups: an untreated stone-forming group, an O. grandiflorum-treated stone-forming group and a potassium citrate-treated stone forming group. Ethylene glycol (0.5%) was administered to the rats during the last week, and vitamin D3 (0.5 μm) was force fed to induce hyperoxaluria and kidney calcium oxalate crystal deposition. Twenty-four hour urine samples were collected before and after inducing crystal deposits. Rats were killed and both kidneys were harvested after 3 weeks. Bisected kidneys were examined under a polarized light microscope to determine the number of crystals. The renal tissue superoxide dismutase and catalase levels were measured by Western blot. Oxidative stress was examined by 8-OHdG immunohistofluorescence. O. grandiflorum and potassium citrate have the ability to alkalinize urine. Among all groups, the number of crystal deposits and the level of 8-OHdG staining decreased significantly in the O. grandiflorum-treated stone forming group, as compared to the other groups. Superoxide dismutase and catalase levels also increased significantly in the O. grandiflorum-treated stone-forming group, as compared with the untreated stone-forming group. The results indicate that O. grandiflorum has a significant inhibitory effect on crystal deposition in the calcium oxalate-stone-forming rat model.     

睾酮和雌二醇对大鼠草酸钙结石模型的影响

目的　进一步明确性激素与肾结石的关系,探讨成石机制。　方法　健康成年雄性ＳＤ大鼠２４ 只,随机分成对照组、成石组、睾酮（Ｔ） 组和雌二醇（Ｅ２） 组,收集第１ 、３ 、７ 天的２４ 小时尿,测定尿晶体成分的浓度。　结果　实验第７ 天时,Ｔ 组和Ｅ２ 组尿钙和草酸明显低于成石组, 尿镁和柠檬酸明显高于成石组（ Ｐ＜ ０ ．０５）。偏光显微镜观察发现成石组鼠肾可见大量晶体沉积,Ｔ 组和Ｅ２ 组仅有散在晶体分布。　结论　性激素的直接作用可能抑制结石形成     

A porcine model of calcium oxalate kidney stone disease

PURPOSE: The pig has been extensively used in biomedical research because of the similarities in organ structure and function to humans. It is desirable to have an animal model of oxaluria and urolithiasis with physiological, anatomical and nutritional characteristics that more closely resemble those of man. In this study we determined if feeding pigs trans-4-hydroxy-l-proline (HP) increased urine oxalate levels and if it would serve as a model for human hyperoxaluria and stone disease. MATERIALS AND METHODS: Male Yorkshire-Durox cross-bred pigs were fed HP for up to 20 days. Urine was periodically collected and analyzed for oxalate levels and the presence of crystalluria. After 20 days of feeding the kidneys were removed and examined grossly and microscopically for indications of injury, crystal deposition and stone formation. RESULTS: Feeding pigs 10% HP (weight per weight HP/food) produced hyperoxaluria, which reached a maximum and leveled off by day 6. Urine oxalate remained near this level until the study ended at 20 days regardless of the further increase in HP to 20% of the weight of the food. When the kidneys were removed and grossly examined, calcium oxalate encrustations were observed on multiple papillary tips. Histopathological observation of the papillary tissue showed tissue injury and crystal deposition. CONCLUSIONS: Pigs fed HP have hyperoxaluria and calcium oxalate crystalluria, and calcium oxalate papillary deposits form that may be precursors of kidney stones. The use of the pig as a model of human hyperoxaluria and stone formation should prove ideal for studies of these human diseases.     

Plasma oxalate concentration in calcium oxalate stone formers

A sensitive, simplified method for plasma oxalate determination by gas chromatography is described. After deproteinizing the plasma with 3N HC1 and 20% sulfosalicylic acid, the oxalate was methylated, extracted and analysed by gas chromatography. This method has three advantages i.e., smaller sample size (plasma 5.0 ml), rapidity (takes less than 3 hours) and accuracy. The recovery rate of oxalate added to plasma was 91.42 +/- 11.31% (SD) and the coefficient of variation of replicate determinations was 4.18%. The minimum detectable concentration of oxalate was 0.3 micrograms/ml (oxalate peak was higher than 5 mm). The mean oxalate concentration under fasting conditions from 16 healthy subjects was 1.37 +/- 0.39 micrograms/ml (SD), while that from 31 calcium oxalate stone formers was 1.45 +/- 0.39 micrograms/ml (SD). There was no significant difference in plasma oxalate concentration between the two groups. The dietary influence of oxalate on plasma and urinary oxalate was investigated in 5 healthy subjects and 5 calcium oxalate stone formers. When 100 g spinach (total oxalate 545.5 mg, soluble oxalate 381.5 mg) was given, the increase of plasma oxalate concentration was more prominent in stone formers; in stone formers it increased to 142% of control value at 2 hours (p less than 0.05) after spinach loading, to 163% at 4 hour (p less than 0.01) and to 232% at 6 hours (p less than 0.01); while in healthy subjects increased to 119% at 2 hours (ns) after loading, to 144% at 4 hours (p less than 0.05) and only to 167% at 6 hours (p less than 0.01). Urinary oxalate excretion increased promptly between 1 and 2 hours after loading in both groups, reaching peak levels between 2 and 4 hours after loading in healthy subjects and between 4 and 6 hours or later in stone formers. The mean renal clearance of oxalate was 18.0 ml/min in 6 healthy subjects and 19.0 ml/min in 4 calcium oxalate stone formers. There was no significant difference in oxalate clearance between the two groups. The mean ratio of oxalate/creatinine clearance was 0.22 for stone formers, which was equal to that for healthy subjects.     

Effect of high and low calcium diets on stone forming risk during liberal oxalate intake

PURPOSE: Recent studies suggest that a high calcium diet protects against calcium oxalate stone formation. We compared the effect of high and low calcium diets on urinary saturation of calcium oxalate during liberal oxalate intake. MATERIALS AND METHODS: A total of 10 healthy subjects (5 male, 5 female) participated in a 2-phase, randomized, crossover study comparing high (1,000 mg daily) and low (400 mg daily) calcium intake on a liberal oxalate diet (200 mg daily). During each phase subjects adhered to an instructed diet for 3 days followed by a controlled, metabolic diet for 4 days. Blood and 24-hour urine specimens collected on the last 2 days of each phase were analyzed for serum biochemistry studies and stone risk factors, respectively. RESULTS: Urinary calcium was higher (mean +/- SD 171 +/- 64 vs 124 +/- 49 mg daily, p = 0.002) and oxalate was lower (25 +/- 4.8 vs 27 +/- 4 mg daily, p = 0.02) on the high vs low calcium diet. Overall, the urinary relative saturation ratio of calcium oxalate was higher on the high compared with the low calcium diet (3.3 vs 2.5, p <0.0001) even after adjusting for confounding variables. CONCLUSIONS: In normal subjects urinary saturation of calcium oxalate was higher on a high calcium diet than a low calcium diet during liberal oxalate intake because the decrease in urinary oxalate did not overcome the effect of increased calcium. A high calcium diet during liberal oxalate intake may pose an increased risk of calcium oxalate stone formation.     

Genes in idiopathic calcium oxalate stone disease

An examination of the urinary excretions of 101 normal subjects indicated that the major genetic influence on calcium excretion is a codominant pair of alleles giving rise to three phenotypes, low, intermediate and high (hypercalciuric) excretors. This inference was based on variance, Hardy-Weinberg and segregation analyses. Similar independent gene pairs also appear to influence oxalate and citrate excretion, A 3-locus Hardy-einberg table using estimates of gene frequencies derived from the study of normals suggests that only 3 or 4 leading genes are involved in oxalate stone disease. Strong candidate genes identified from molecular and physiological studies cannot be proposed at present, but it is assumed that they influence the transport of these ions in either the intestine, kidney or both organs. The identification of the genes involved should be facilitated by the reduction of dietary influences on urinary excretions through the use of formula diets.     

Relationship between supersaturation and calcium oxalate crystallization in normals and idiopathic calcium oxalate stone formers

BACKGROUND: In an earlier study on recurrent CaOx stone formers with no detectable abnormalities, we found that the urine of these subjects had a lower tolerance to oxalate load than controls and that the removal of urinary macromolecules with a molecular weight greater than 10,000 D improved their tolerance to oxalate. METHODS: The effects on CaOx crystallization of reduced urinary supersaturation of calcium oxalate (CaOx), induced by night water load, were studied in 12 normal males and in 15 male OxCa stone formers who were free from urinary metabolic abnormalities. The effect of the macromolecules, purified and retrieved from the natural and diluted urine, were analyzed in a metastable solution of CaOx. RESULTS: The water load caused an increase in urine volume (from 307 +/- 111 to 572 +/- 322 ml/8 hr, P = 0.014 in normal subjects, and from 266 +/- 92 to 518 +/- 208 ml/8 hr, P = 0.001 in the stone formers) and a concomitant reduction of the relative CaOx supersaturation (from 8.7 +/- 2.5 to 5.1 +/- 2.5 ml/8 hr, P = 0.001 in normal subjects, and from 10.4 +/- 3.5 to 5.0 +/- 2.7 ml/8 hr, P = 0.001 in the stone formers). The decrease in CaOx supersaturation was accompanied by an increase of the permissible increment in oxalate, both in normal subjects (from 43.8 +/- 10.1 to 67.2 +/- 30. 3 mg/liter, P = 0.018) and in the stone formers (from 25.7 +/- 9.4 to 43.7 +/- 17.1 mg/liter, P = 0.0001), without any significant variations of the upper limit of metastability for CaOx (from 21.6 +/- 5.3 to 20.5 +/- 4.2 mg/liter in normal subjects, and from 18.7 +/- 4.5 to 17.1 +/- 3.7 mg/liter in the stone formers). The inhibitory effect of urinary macromolecules with molecular weight greater than 10,000 Daltons did not undergo any change when the latter were recovered from concentrated or diluted urine, either in normal subjects or in the stone formers. CONCLUSIONS: Reduced CaOx supersaturation by means of water load has a protective effect with regards to CaOx crystallization in subjects who do not present any of the common urinary stone risk factors.     

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（     

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     

Studies of dietary influence on urinary oxalate in calcium oxalate stone formers

In order to examine the effect of diet on the urinary excretion of oxalate, a spinach loading and milk loading experiment was performed in normal subjects and patients with single calcium oxalate stones and recurrent calcium oxalate stones after a rat experiment. When spinach (100 g, total oxalate 642.57 mg, insoluble oxalate 282.21 mg, taken oxalate 444.57 mg) was given with a low calcium diet to the patients, the increase of urinary oxalate was more prominent in those with recurrent stones; the mean urinary oxalate increased from 39.84 to 84.18 mg/day (P less than 0.01) in the group with recurrent stones, from 36.95 to 55.12 mg/day (P less than 0.05) in the group with single stones and from 33.99 to 42.78 mg/day in the control group. These increases in oxalate excretion could be ameliorated by the concurrent oral administration of milk (calcium 343 mg). Moreover, diurnal variation in oxalate excretion was observed. It was more evident under spinach load in the group with recurrent stones than in the control group. Urinary oxalate increased promptly, reaching peak levels between 4 and 6 hours after loading in the group with recurrent stones and single stones, and between 2 and 4 hours in the control group. The influence of the spinach load disappeared within 24 hours.     

Oxalate transport and calcium oxalate renal stone disease

Hyperoxaluria is considered to play a crucial role in calcium oxalate (CaOx) renal stone disease. The amount of oxalate excreted into the urine depends on intestinal absorption, endogenous production, renal clearance and renal tubular transport. Since a primary disorder has not been found so far in most CaOx stone formers and since oxalate is freely filtered at the glomerulus, most studies are presently focussed on alterations in epithelial oxalate transport pathways. Oxalate can be transported across an epithelium by the paracellular (passive) and transcellular (active) pathway. Oxalate transport across cellular membranes is mediated by anion-exchange transport proteins. A defect in the structure of these transport proteins could explain augmented transcellular oxalate transport. Little is known about the physiological regulation of oxalate transport. In this review cellular transport systems for oxalate will be summarized with special attention for the progress that has been made to study oxalate transport in a model of cultured renal tubule cells. Better understanding of the physiological processes that are involved in oxalate transport could yield information on the basis of which it might be possible to design new approaches for an effective treatment of CaOx stone disease.     

Uric acid-binding proteins in calcium oxalate stone formers and their effect on calcium oxalate crystallization.

OBJECTIVES: To study the effect of urinary uric acid-binding proteins of controls and stone formers on calcium oxalate crystal nucleation and aggregation. MATERIALS AND METHODS: Urine samples were collected over 24 h from 20 stone formers and from 20 age-matched normal controls. Uric acid crystallization was induced by adding equal volumes of 2.5 mmol/L uric acid. The bound proteins were separated on a cellulose column, and by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The effect of the separated fractions on calcium oxalate crystal nucleation and aggregation was assessed. RESULTS: The protein bound to unit mass of uric acid crystals was higher in hyperoxaluric urine than in control urine. On cellulose-column separation, the uric acid-crystal binding proteins produced three major protein peaks, i.e. fraction I (buffer), fraction II (0.05 mol/L sodium chloride in Tris-HCl buffer) and fraction III (0.3 mol/L sodium chloride in buffer), with a minor peak obtained on elution with increasing concentrations of sodium chloride in Tris-HCl buffer (pH 7.0). Fraction I derived from either stone formers or controls promoted calcium oxalate crystallization. Fraction II from the control samples was a strong inhibitor, whereas hyperoxaluric fraction II was less inhibitory. CONCLUSION: Uric acid-binding proteins isolated either from the urine of stone formers or controls modulated calcium oxalate crystal growth. Proteins isolated from stone formers were less inhibitory of crystal nucleation and aggregation. These proteins may act as a bridge, leading to the epitaxial deposition of calcium oxalate over a urate core.     

Studies on crystalluria in calcium oxalate stone formers

Summary The excretion of calcium oxalate and calcium phosphate crystals was studied in fractionated 24 h urine from 7 men with recurrent calcium oxalate stone disease, both before and during daily administration of 5 mg bendroflumethiazide. Urinary calcium, oxalate, magnesium, citrate, phosphate, pH, and inhibition of calcium oxalate crystal growth rate were analyzed in all samples. Exclusively calcium oxalate crystals were found in 30 per cent of the samples, all with a pH below 6.25, whereas calcium phosphate was the crystal type encountered in urine with a pH above 6.50. Bendroflumethiazide decreased the volume of calcium phosphate but not of calcium oxalate crystals. During the period of observation there was no correlation between calcium oxalate supersaturation and calcium oxalate crystal volume, but a relationship was demonstrated between calcium phosphate supersaturation and calcium phosphate crystal volume.     

Urinary phytate in calcium oxalate stone formers and healthy people--dietary effects on phytate excretion

The phytate urinary levels in a group of active calcium oxalate stone formers were studied and compared with those found in healthy people. Urinary phytate was significantly lower for stone formers. If deficit of the capacity to inhibit crystallization of calcium salts is considered an important factor related to calcium stone formation, the excretion of low phytate amounts could be an important risk factor in the development of this type of renal calculi. The influence of dietary phytate on urinary excretion was also studied. Clearly maintenance of a phytate-free diet significantly decreased the urinary excretion of phytate (about 50% after 36 h). This demonstrated the importance of dietary phytate in maintaining adequate urinary levels to permit effective crystallization inhibition of calcium salts and consequently preventing renal stone development.     

Comparison of urinary tract infection in calcium oxalate and calcium phosphate stone formers

To compare the frequency of urine infection in calcium oxalate and calcium phosphate stone formers, we reviewed charts from patients whose last renal stone submitted for analysis was predominantly composed of calcium phosphate in 118 and of calcium oxalate in 223. Positive cultures were commoner, but not significantly, in the phosphate than the oxalate stone formers, both in men (17 vs. 7.6%) and women (22 vs. 15%). Bacteria frequently producing urease were found in only 4% of the phosphate group. Urine leucocytes were slightly more frequent in the oxalate group for men and significantly so for women. The results do not support the concept that calcium phosphate stones are mainly due to infection with urease-producing or other bacteria.     

Conversion of calcium oxalate to calcium phosphate with recurrent stone episodes

PURPOSE: We have extended our previous observation that the percent occurrence of calcium oxalate stones decreased while that of calcium phosphate stones increased with each new stone event. MATERIALS AND METHODS: The National VA Crystal Identification Center has analyzed veteran patient urinary tract stones from VA hospitals throughout the United States since 1983. We reviewed the composition of 33,198 stones with emphasis on the changes in composition. More than 11,786 stones came from 5,088 recurrent stone formers. Stones were analyzed using high resolution x-ray powder diffraction and Fourier transform infrared spectroscopic techniques. When the stones were investigated as a function of time, it was determined that there was greater variability when samples were more than 30 days apart. RESULTS: The percent occurrence of whewellite, weddelite, apatite, brushite and uric acid in stones increased between 1.0% and 5.9% since our previous study. The percent occurrence of struvite decreased by 2.6%. The percent of calcium oxalate stones decreased while that of calcium phosphate stones increased with each new event. However, the total percent occurrence of all calcium containing stones did not significantly change with recurrent stone events. CONCLUSIONS: Our study suggests a strong trend for the conversion of stone disease from calcium oxalate to calcium phosphate containing stones, which could influence the progression and severity of disease.     

Calcium oxalate monohydrate binding protein: a diagnostic biomarker for calcium oxalate kidney stone formers

Urinary oxalate is a biomarker for calcium oxalate kidney stone disease; however, its assay is insensitive and nonspecific. Calcium oxalate monohydrate (COM) binding protein (45 kDa) is a promoter of calcium oxalate kidney disease, which is markedly upregulated by oxalate induced oxidative stress. The current study was carried out to evaluate whether COM binding protein can serve as a diagnostic marker for calcium oxalate kidney stone formers. COM binding protein was isolated, purified and antibody was raised against it in rabbits. Urine samples (24 h) were collected from patients suffering from various kidney diseases such as acute nephritis, chronic nephritis, nephrotic syndrome, calcium oxalate (CaOx) stone formers, uric acid stone formers, struvite stone formers and calcium phosphate stone formers. This COM binding protein was quantified by an in house ELISA method and the excretion was found to lie between 2 and 3 mg in control samples, while in CaOx stone formers it was detected between 11 and 19 mg. Urinary risk factors were assayed. We conclude that COM binding protein can serve as a diagnostic marker for CaOx stone formers.