| BackgroupStudies show that the reason of a large number of pets poisoning and renal failure occurred in March 2007 in the United States is due to the pet food, which mixed with 8.4% melamine and 5.3% cyanuric acid; In September 2008, nearly 300,000 Chinese infants and young children appear urinary calculi and urinary tract abnormalities by taking " False Milk Powder ", which contains a large number of varying concentrations melamine. At present,studying the pathological process and pathogenesis of induced kidney damage and urinary stone, searching the effective ways of preventment and treatment, is a hot direction for the domestic and foreign experts to explore actively.Melamine (MA), molecular formula C3N6H6, is a triazine nitrogen-containing heterocyclic compounds. It is appearance of pure white powder monoclinic crystal, slightly soluble in water, low toxicity, rat oral lethal dose greater than 3g/kg body weight. Melamine mainly used for synthesis of melamine-formaldehyde resin to produce composite panels, plastic, paint, commercial filters, glues or adhesives, as well as tableware and kitchen utensils. In the melamine manufacturing process, some melamine may be generated cyanuric acid, Ammelide and Ammeline. If the melamine is not fully metabolized into ammonia and carbon dioxide, may also form its homologue by bacterial metabolism.Cyanuric acid (CA) is white crystal, also known for isocyanuric acid, derived from urea polymer. Molecular formula:C3H3N3O3.It is also an important nitrogen-containing triazine heterocyclic organic compounds. It is slightly soluble in water and nearly non-toxic.The rat LD50 is 7.7g/kg. Cyanuric acid is used for organic synthesis of formaldehyde resin, coatings, adhesives, pesticides, herbicides, metal cyanide corrosion inhibitor, polymer modifiers and for the production of halotrizinol.The recent studies show the Trichloromelamine, which is widely used to disinfect food production equipment, vegetables and fruits in many countries including the United States,will be degraded into melamine. Cyromazine, a pesticide can also be left on the surface of fruits and vegetables and degrade into melamine. Dichloroisocyanurate, disinfectant of drinking water and swimming pool water, can produce cyanuric acid by metabolizm. Therefore, melamine and cyanuric acid have been in close contact with human life.Previous studies have shown that melamine and cyanuric acid are inert metabolism in mammals, that is, they will not be converted and accumulate in the body but discharge from the urine in prototype. The half-life of Melamine in vivo is about 2.7h, cyanuric acid is 2.5h, so the urinary system is easily become the damage target organ of melamine and cyanuric acid. Foreign animal experiments showed that long term and high-dose single intake of melamine or cyanuric acid cause bladder cancer, bladder stones, renal tubules epithelial hyperplasia, expansion and renal fibrosis. In 2008, Dobson and others had confirmed that rats die of acute renal failure after intake melamine and cyanuric acid at the same time, and the pathological anatomy prompted a large number of crystals block in the renal tubules, other organs unaffected. Detection found that the crystals in kidney tissue is melamine and cyanuric acid in 1:1 combination. Dobson and others'study also demonstrated that a separate intake of melamine or cyanuric acid did not cause damage in rat kidneys, and melamine or cyanuric acid will not cause toxic effect on kidney cells within the limits of the concentration. Currently there is no data about Ammelide and Ammeline inducing renal damage and stone.As a high nitrogen content of melamine, it has been illegally added to pet food, milk and other protein products to falsely increase protein content. However, low purity melamine is often mixed with cyanuric acid, or melamine can be converted into a small amount of cyanuric acid in the food processing. After "False Milk Powder" incident,a nationwide special inspection of infant milk powder was carried out by Chinese General Administration of Quality Supervision.A certain brand milk. powder was detected the melamine up to 2563mg/kg, but was not reported to contain cyanuric acid. In addition,the studies of theFirst Affiliated Hospital of Peking University found that the incidence of children fed with melamine powder suffer urinary tract stones was 7 times higher than normal infants and young children. The urinary tract stones from children fed with melamine powder were analyzed by the Beijing Children's Hospital,the result was melamine and uric acid. Therefore, the kidney stones occurred in infants have always been considered caused by melamine and uric acid in China.The pathogenesis of Acute Renal Failure (ARF) can be divided into prerenal, renal and Postrenal, while the melamine and cyanuric acid were proved to be no direct renal toxicity, its main reason for kidney damage may be postrenal obstruction ARE The pathogenesis and types of urinary stone is complicated, present studies have shown that melamine-related stones may be related to uric acid stone, which may be related to the impact of triazine compounds in vivo metabolism, as well as its molecular structure is able to induce uric acid to form stones easily. Therefore we need to analysis the key factors that melamine and cyanuric acid induced uric acid stones on uric acid excretion, urine pH value and urine output.The mechanisms how the "False Milk Powder" damaged urinary system are not yet entirely identify.Based on the highest dose of melamine in "False Milk Powder" containing (2563mg/kg),and according to the average weight of the smallest babies (6kg), the average daily intake of milk amount (150g) and the pharmacological conversion formula of rat and human (about 6.3:1), we calculate the maximum daily intake melamine of rat model should be about 403.7 mg/kg. At the same time,refer to the dose of foreign animal experiments, we designed the dose for this experiment. In this study, we gavaged rats with melamine and cyanuric acid individually and combination to to set up models of kidney damage, aiming to find out the mechanism of kidney damage and stones formation induced by melamine and cyanuric acid, and to provide help for the further seeking prevention and treatment methods.Objectives:1. To reseach the mechanisms of rat renal damage and urinary system stones formation induced by melamine and cyanuric acid individually,and to analyze the chemical characterization of the stone crystals.2. To reseach the mechanisms of rat renal damage and urinary system stones formation induced by melamine and cyanuric acid combination,and to analyze the chemical characterization of the stone crystals.3. Base on the rats experimental study,to find the possible mechanism of infant urinary calculus that induced by "False Milk Powder".Methods:1.60 SD rats were randomly divided into group A (control group), group B(melamine 400mg/kg/d), group C (cyanuric acid 400mg/kg/d), group D (melamine 400mg/kg/d+cyanuric acid 400mg/kg/d), group E(melamine.30 mg/kg/d+cyanuric acid 30 mg/kg/d),12 rats in each group, administered by gavage.2. The 24-hour urine was collected to measure 24-hour urine volume, pH value and 24-hour uric acid excretion volume for the first 3days, and the urine sediment smears were observed by a microscope. On the fourth day and the thirty-first day, before gavaging drugs,the blood samples of 6 rats in each group were collected to test the serum creatinine, blood urea nitrogen and uric acid.The kidneys were observed by surgical microscope,and then produced to fast-frozen sections and paraffin sections(HE staining) to observe kidney histopathology and stone crystal formation. Frozen tissue samples were sectioned to produce a thickness of 5μm to be determined by FTIR microspectroscopy.3. Data are presented as the mean±SD.The urine data were assessed using Repeated Measures and the blood data using Factorial Analysis. Differences between groups were assessed using Multiple Comparisons.All of the statistical analyses were performed using SPSS, and p<0.05 was considered significant.Results:Serum and urinary biochemistryCompared with the group A, the 24-hour urine volume and 24h uric acid excretion volume in groupB and group C increased significantly, the differences has statistically significant (P<0.05); while group D and E urine output decreased, PH value step down,the amount of 24h uric acid excretion reduced, the differences were statistically significant (P<0.05); group D changed more obviously than group E, the difference had statistically significant (P<0.05).Each group compared with the group A at the corresponding time, serum creatinine, serum uric acid and urea nitrogen in group B and group C were both increased, but only blood urea nitrogen difference had statistically significant on the 4th day (P<0.05), blood urea nitrogen and serum uric acid differences had statistically significant on the 31st day (P<0.05).All the measured items of group D significantly increased on the 4th day, the differences had statistically significant (P <0.05); serum creatinine and urea nitrogen of group E increased significantly on the 4th day, the difference has statistically significant (P<0.05), all the items significantly increased on the 31st day, the differences have statistically significant (P <0.05). Changes of group D was more obviously than group E, difference had statistically significant (P<0.05), at the mean while all the items increased more obviously on the 31st day than 4th day, the differences had statistically significant (P <0.05).Urinary sediment smearsObserved to the urinary sediment smears:group A had no significant crystal; group B showed suspected melamine crystals; group C had no significant crystal, but a few small sand-like particles; group D and E had the light yellow circle-specific crystal.Renal pathological changesMacroscopic Renal Changes:Group A:The renal envelope was smooth and dark,and dissect observation had no significant pathological changes. Group B and C: Renal cortex and medullary structure were clear, kidney calices and renal pelvis were no abnormalities, no stone crystal deposition. Group D:kidneys were significantly swelling, appearance greyish-yellow, many scattered yellow spots on envelope, renal cortical become thinning, renal calices and pelvis structural was damaged. Many light yellow stone crystals deposit in the kidney tissue. Group E: Renal cortex and medullary structure were still clear, a lot of yellow stone crystals deposition were visible on the 4th day. On the 31st day, kidney became reddish-brown, renal cortex and medullary structure become blurred, a large number of yellow particles were deposition in the renal tissue. No bladder stones was observed in each group.Pathological section:Group A were no significant pathological changes. Kidney paraffin sections and frozen sections of group B and C were no crystal and obviously pathological changes on the 4th day.There were no crystals on the 31st day, only a small amount of mild expansion of renal tubular, epithelial cell swelling and a small amount of tube shape formation. Kidney paraffin sections and frozen sections of groupD and E were deposition many yellow, radial concentric circular crystals, mainly concentrated in the renal tubules, but also found in collecting ducts, interstitial and subcapsular, occasionally found in glomerulus capsule. Most of crystals appear in the renal tubules in irregular fragments. In addition, renal tubular swelling, epithelial damage, and a variety of tube formation, a large number of inflammatory cells in the glomerular cysts, renal tubule and interstitium; Group E can also can see a lot of fiber cells filled with interstitial on the 31st day. In addition, the number and size of crystal stones, as well as pathological changes are larger than that in group E on the 4th day; The number of stone crystals, size as well as pathological changes on the 31st day was larger than those on the 4th day.Chemical Characterization of the Renal Stone CrystalsVarious forms of stone crystals in the rat kidney was detected by FTIR microspectroscopy to collect infrared spectra.The spectra were almost similar. By professional analysis,the chemical identity of crystals in rat kidney tissue was determined to be melamine cyanurate only,no uric acid and others.Conclusion:1. Melamine and cyanuric acid mixture into the body at the same time can form a water-insoluble melamine cyanurate crystal in the kidney, which will block the renal tubular, cause renal damage and even acute renal failure.In addition, as a stone core together with uric acid and other components, the melamine cyanurate crystal will induce urinary calculi.2. Both melamine and cyanuric acid are less kidney damage, and the mechanism is similar. Individually intake of melamine or cyanuric acid,there is no stone crystal formation in rat kidney,but the long-term intake will cause kidney function damage for a long-term osmotic diuresis leading to kidney tissue physical damage. 3. Melamine and cyanuric acid combined effect on the kidney damage is more larger than their individual effect, and more easily induced urinary uric acid stones. And there is a dose-effect in their combined effect.4. The mechanism of urinary calculi in infants caused by "False Milk Powder" is more complex, but melamine and cyanuric acid concomitance in milk powder may be the main predisposing factors.
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