An Experimental Study On SDrat Renal Damage Induced By Melamine Cyanurate

BackgroupIn the melamine was illegal added to the pet feed, the milk as well as other protein product creates massive pets to be poisoned in the United States in March,2007as well as nearly300,000babies and infants occurs the urolithiasis in China in September,2008, the toxic effects of melamine on the human body and the mechanism for melamine stone formation and renal failure attracted wide attention at home and abroad. Numerous studies showed that the ingestion of either melamine or cyanuric acid alone does not have any effect on renal function of rats; however, there is a severe renal damage of rats because of the formation of an insoluble melamine cyanurate by physical blockage of the renal tubule after ingestion of the mixture of melamine and cyanuric acid. Melamine and cyanuric acid, a kind of white powder, which slightly dissolving in water and nearly non-toxic, is a triazine heterocyclic organic compounds, so it is an important organic industrial products, which is widely used in adhesives, coatings, textile production. 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. Melnick et al. 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. In2008, 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 in1:1combination. 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. In previous study, we gavaged rats with melamine and cyanuric acid combination to to set up models of kidney stone, and the stone crystals in the rat kidney was detected by FTIR microspectroscopy, which show that the insoluble crystal is melamine cyanurate(MCA). Melamine cyanurate is a white crystalline powder, odorless and tasteless, greasy feel insoluble in water, soluble in formaldehyde and ethanol, which complex formed from al:1mixture of melamine and cyanuric acid. As a fire retardant, melamine cyanurate has a stable chemical stability and insoluble in water. The formation of crystals in the urinary tract is a complex process, but upon mixing melamine and cyanuric acid, a cloudy solution of melamine cyanurate was rapidly formed, and the present researches consider that the main mechanism is the dehydration reaction between melamine and cyanuric acid. Kim et al. suggested that that the oral mixture of melamine-cyanuric acid mixture (M+CA1:1) into the body were absorbed, respectively, and then in the kidney, they re-formed the insoluble melamine cyanurate crystals, inducing acute renal toxicity through physical blockage of the renal tubule. Currently, there is no data about why melamine and cyanuric acid only recombine in the kidney not in circulatory system. Is the kidney damage ascribable to the blockage of the renal tubule by melamine cyanurate crystals or to the nephrotoxicity on kidney tissue of it?Melamine cyanurate is indefinitely stable, and is of low acute toxicity. A toxicology study performed in the1980s in the Soviet Union showed that melamine cyanurate is more toxic than either melamine or cyanuric acid alone. The ingested LD (50) of the melamine cyanurate, melamine, cyanuric acid to rats and mice was4.1g/kg,6.0g/kg,4.3g/kg, respectively. Although melamine and cyanuric acid in combination could create insoluble renal crystals in the kidney, no data could be found that have determined the potential nephrotoxicity of melamine cyanurate crystals. In this study, we investigated the nephrotoxicity of melamine cyanurate when fed to SD rats gain a novel insight into the mechanism underlying M+CA-caused renal damage.Objectives:1. To discover if MCA ingestion could lead to crystal formation in kidney and induce rats renal damage, and to analyze the chemical characterization of the stone crystals.2. To reseach the nephrotoxicity of melamine cyanurate.Methods:1.150healthy SD rats (SPF), male/female, weight200g±10g, were randomly assigned into5groups (30each) according to the doses of melamine cyanurate they received (0,10,60,200,400mg/kg/day), which were repectively given daily by gavage for60consecutive days. 2. The24-hour urine was collected to measure24-hour urine volume, pH value and24-hour uric acid excretion volume for the first3days, and the urine sediment smears were observed by a microscope. Before drug administration, the blood samples of6rats in each group were collected to test the serum creatinine, blood urea nitrogen, serum cysctain C and uric acid.The kidneys were observed by surgical microscope,and then produced to paraffin sections(HE staining) to observe kidney histopathology and stone crystal formation.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<0.05was considered significant.Results:Scrum and urinary biochemistryThe24-hour urinary volume of first week except4th day after the rats were treated with melamine cyanurate (10mg/kg,60mg/kg,200mg/kg) increased significantly in comparison with those in the controls(P<0.05), the60mg/kg group decreased more significantly than the10mg/kg group and200mg/kg group (P<0.05). However, the decrease of the24-hour urinary volume was significant on day7in400mg/kg group (P<0.05). Urinary pH within24h:except the400mg/kg group, all other groups there were no significant difference compared with the control group (P<0.05). The24-hour urinary excretion of uric acid significantly decreased gradually in first week as time went and dose increased(P<0.05)(Table3-1,2,3).Serum creatinine, blood urea nitrogen (BUN) and serum cystatin C increased gradually according to the sequences of the10mg/kg,60mg/kg,200mg/kg and400mg/kg, and they all had the tendency to rise in the last three groups. Serum creatinine, blood urea nitrogen in the60mg/kg and200mg/kg groups since the31th day had obvious difference (P<0.05), and that in the400mg/kg group since the4th day had extreme difference (P<0.05). The serum cystatin C level of60mg/kg and200mg/kg groups since the14th day as well as400mg/kg group since the4th day were significantly higher than that of control groups(P<0.05). The times of blood uric acid begun to decreasing of four groups were respectively,31day in the lOmg/kg group,14day in the60mg/kg group,14day in the200mg/kg group and4day in the400mg/kg group.(Table3-4,5,6,7,8)Urinary sediment smearsObserved to the urinary sediment smears:control group had no significant crystal; The gross appearance of kidneys of rats in the10mg/kg/day melamine cyanurate treated group,60mg/kg/day-treated group (for14days) and200mg/kg/day-treated group (for14days) was similar to that of control group (FIG.3-1.A,B,C,D). In the60mg/kg/day-treated group (30～60days),200mg/kg/ day-treated group (30～60days) and400mg/kg/day-treated group, an typical appearance of "large, yellow and edematous kidney" was seen by grossly.(FIG.3-1.F,G,H,D).Renal pathological changesMacroscopic Renal Changes:Control group:The renal envelope was smooth and dark, and dissect observation had no significant pathological changes. Of all the rats dosed with lOmg/kg melamine cyanurate for60days, only one was found to have a little of Brownish-yellow, concentric crystals (FIG.3-2. B, C). No crystals have been found in pathological renal sections of rats treated with60mg/kg or200mg/kg melamine cyanurate for14days, while by31day there were a lot of granular sediment scattered in the cortex and medullar regions, particularly localized on renal medullary tubules, and the number and size of sediment increased with the dose or the time prolonged. Oral administration of400mg/kg melamine cyanurate for4days results in numerous characteristic, yellow and radial crystals especially in the cortex. All renal tissue damage resulting from crystals had similar appearance in different degrees of severity. Too many crystals in the kidney tissue for a certain days could cause serious injury to the kidneys, resulting in lesions characterized by tubular dilatation, wall thinned and rupture, more alveolar space, inflammatory cells infiltration and fibrosis in thinned renal interstitium. In one word, in the most severe case it looked like the shape of alveolus organization.(图3-3,4F,K,N,O)。Conclusion:1. Melamine cyanurate into the body 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, the melamine cyanurate crystal will induce urinary calculi.2. This study demonstrated that limitation of the function of kidney spherule filtration existed long before the crystals deposited, suggesting that the renal impairment is a progressive deterioration, and also show that it could decrease serum uric acid.3. Melamine cyanurate can be used to create kidney stone model and reduce the serum uric acid, it also offer a new perspective for study the melamine-associated crystal formation.4. This study suggested that the sanitary supervision department should improve detection techniques that can not merely detect melamine and cyanuric acid, and can also detect melamine cyanurate to make the vast consumers to have the safer foods.