We investigated the effects of a traditional Chinese herbal formula, Wulingsan (WLS), on renal stone prevention using an ethylene glycol-induced nephrocalcinosis rat model. Forty-one male Sprague-Dawley (SD) rats were divided into four groups. Group 1 (n=8) was the normal control; group 2 (n=11) served as the placebo group, and received a gastric gavage of starch and 0.75% ethylene glycol (EG) as a stone inducer; group 3 received EG and a low dose of WLS (375 mg/kg); and group 4 received EG and a high dose of WLS (1,125 mg/kg). Baseline and final 24 h urine samples were collected individually; biochemical data of urine and serum were also obtained at the beginning and at the end of the experiment. After 4 weeks, animals were killed and kidneys were harvested. The kidney specimens were examined by polarized light microscopy and the crystal deposits were evaluated by a semi-quantitative scoring method using computer software (ImageScoring). The results revealed that the rats of placebo group gained the least significant body weight; in contrast, the rats of WLS-fed groups could effectively reverse it. The placebo group exhibited lower levels of free calcium (p=0.059) and significantly lower serum phosphorus (p=0.015) in urine than WLS-fed rats. Histological findings of kidneys revealed tubular destruction, damage and inflammatory reactions in the EG-water rats. The crystal deposit scores dropped significantly in the WLS groups, from 1.40 to 0.46 in the low-dose group and from 1.40 to 0.45 in the high-dose group. Overall, WLS effectively inhibited the deposition of calcium oxalate (CaOx) crystal and lowered the incidence of stones in rats (p=0.035). In conclusion, WLS significantly reduced the severity of calcium oxalate crystal deposits in rat kidneys, indicating that Wulingsan may be an effective antilithic herbal formula.

Melamine-contaminated milk formula caused infant nephrolithiasis in some areas of China. Its combination with cyanuric acid causes crystallization in renal tubules. Following this renal damage and even renal failure that require long-term hemodialysis has been reported. Therefore, correct and timely diagnosis of these complex diseases is critical. Melamine containing stone is a combination of equal molar ratios of common stone compositions that has been reported from previous animal studies. We have previously identified the compositions of urinary tract stones with infrared (IR) spectroscopy. We hypothesized that the absorbance of wavelength of IR can identify melamine/cyanuric acid in the presence of mixing human stone compositions. In this study, we made an artificial stone composition and examine under IR absorbance by mixing equal molar ratios of melamine/cyanuric acid with different types of human urinary stones, and established a reference of IR analysis for the identification of melamine/cyanuric acid-containing human urinary tract stones. Knowledge of the precise stone composition allowed institution of appropriate prophylactic dietary and medical therapy and this may help in the prevention of urinary stone recurrence. The results are promising that melamine and cyanuric acid can be identified clearly in a low percentile (approximately 1%) of stone mixture pellet. Therefore, IR seems to be an ideal tool for the identification of melamine/cyanuric acid-containing stones.

Ethylene glycol (EG), a frequently used lithogenic agent, causes calcium oxalate crystal deposits in the renal cortex and causes death if over-dosage. In this study, we compared the protein expression pattern of rat renal lithiasis induced by EG with that of normal renal tissues using two-dimensional electrophoresis (2-DE). Sixteen male Sprague-Dawley rats were divided into 2 groups. In control group (n = 8), rats were fed with normal drinking water; in EG group (n = 8), rats were fed with 0.75% EG in drinking water. After 4 weeks, rats fed with EG had calcium oxalate deposited in renal cortex. The renal cortex proteins were extracted and isolated by 2-DE. Comparative analysis of the respective spot patterns on 2-DE were identified by matrix-assisted laser desorption ionization-time of flight-mass spectrometry technique. Among

We extracted proteins with a molecular weight of 60 kDa from human renal calcium oxalate stones. One-dimensional electrophoresis (SDS-polyacrylamide gel electrophoresis) with Coomassie blue stain was used for molecules separation. After in-gel digestion by trypsin, protein samples were analyzed using Nanospray liquid chromatography coupled to a quadrupole time-of-flight mass spectrometer. Peptide sequence analysis was performed by MASCOT Web searching and National Center for Biotechnology Information database comparison. Our results revealed that the matrix possess only 2.5% of stones in weights. Three most abundant 60 kDa proteins named myeloperoxidase, lactoferrin, and albumin were identified. Because albumin has been previously studied by many researchers including our team, we are highly interested to study the remaining two proteins. Later, their in vitro activities of crystallization were studied by employing methods such as nucleation, crystal growth, and aggregation. The mass spectra pattern of myeloper-oxidase and lactotransferrin precursor (lactoferrin) revealed modifications and deamidations, and their calcium oxalate stones crystallization studies revealed no effects on the nucleation, crystal growth, and aggregation. The mass spectra pattern of albumin indicated no amino acid modifications. In conclusion, we found three commonly seen 60 kDa proteins (albumin, myeloperoxidase and lactoferrin) in calcium oxalate stones in this study. Albumin has been tested before and has effect on stone formation. However, purchased myeloperoxidase and lactoferrin have no effect in the crystallization process; their role play in the process of stone formation should be further elucidated.