| Urinary calculi are a common clinical problem in dogs.According to Minnesota stones Analysis Center reported that the incidence of calcium oxalate stones in dogs had increased from 5% in 1981 to 41% in 2007. However, the incidence of struvite calculus, the highest incidence of stones in the past, had decreased from 78% in 1981 down to 40% in 2007. The Minnesota stones Analysis Center recently released 2009 to 2010 and 1999 to 2000 the incidence of urinary stones in dogs compared to calcium oxalate urinary stones has significantly increased. The incidence of calcium oxalate stones in dogs in China is also increasing in recent years. Meanwhile, the calcium oxalate stone is also the highest incidence of stone types in human. There are important theoretical and applied significance in the study of the formation mechanism and prevention of the calcium oxalate urinary stone.Domestic and international studies showed that the high concentrations of urinary oxalate have cytotoxic effect. The oxalate in urinary acting on the renal tubular epithelial cells can cause lipid peroxidation which releases oxygen free radicals and results in cell damage. The calcium oxalate crystals were more easily adhere to the surface of damaged renal tubular epithelial cell, which may eventually induce the formation of calcium oxalate stones. It has been proved by Grases, who simulated the conditions of kidneys in vitro, that the free radicals can injury epithelial cells, resulting in a favorable environment for the growth of crystal. However, some antioxidants can inhibit the formation of calcium oxalate crystals. Selenium is now considered an important antioxidant trace element, which can protect cells from lipid peroxidation damage. Including organic and inorganic selenium in two forms, organic and inorganic selenium compared with toxicity, palatability, absorption and utilization advantages. But especially organic selenium impact on the formation of calcium oxalate urinary stones? How the mechanism? Few studies domestic and foreign.Domestic and foreign researchers found that OPN is a larger molecular organics that can inhibit the formation of urinary stones. OPN may disrupt the crystal lattice by involving in cell migration, and inhibits the growth of calcium oxalate crystals., but there are also the opposite view. To date, no studies have reported OPN expression in renal tissue and calculus relations on dogs.In this study, the incidence of urinary stones on 283 dogs in Nanjing in January 2009-December 2011 clinical cases were investigated. The compositions of urinary stones collected samples were analyzed. Ethylene glycol induced calcium oxalate urinary stones canine model was established. The blood biochemical indicators, urine biochemical indicators, the antioxidant indicator of the blood and kidney, renal histopathological changes and the expression level of OPN and the OPN-mRNA of canine model urinary stones by selenium intervention were studied. The purpose is to explore the role of selenium in particular organic selenium on mechanism to prevent the formation of calcium oxalate stones in dogs. In this study, the mechanism of calcium oxalate urinary stones and prevention provides a new research ideas.The contents of each test as follows:Test 1. Investigations the incidence of urolithiasis dogs of Nanjing in 2009-2011The main components of 283 dogs stone specimens collected from 5 large animal hospitals in Nanjing in January 2009 -December 2011 were analyzed by chemical methods. The epidemiological of the dogs suffering from urolithiasis was investigated. The results showed that chemical compositions of the 283 cases of canine urinary calculi are 38.16% (108/283) struvite (magnesium ammonium phosphate),32.51%(92/283) calcium oxalate, 18.73%(53/283) uric acid and urate,3.18%(9/283) calcium phosphate,7.42%(21/283) composite stones. Dogs suffering from calcium oxalate stones found significantly higher proportions. The sexual orientation, species trends, the average age and diet habit of dogs suffering from urolithiasis have a certain relationship with the composition stones and the site occurrence stones.Test 2. Establishment of the animal models of calcium oxalate urinary stones in dogs induced by the ethylene glycolMethod:Calcium oxalate urinary stones canine model induced by using two different concentrations (0.5% and 1%) of ethylene glycol.15 male dogs (1 year old, about 10 kg) were randomly divided into control group,0.5% ethylene glycol module group,1% ethylene glycol module group. The three groups were fed basal diet.The total calcium, oxalate, urea nitrogen, and creatinine in urine, and the calcium, urea nitrogen, and creatinine in blood for each dog in 24 h on the 1 st,9th, and 18th weeks were tested. Urine collected is analysed by chemical qualitative analysis, Fourier transform infrared spectroscopy and scanning electron microscopy.The results showed that the specific gravity of urine of ethylene glycol group was significantly higher (P<0.05) after adding ethylene glycol in drinking water. The urine pH of 0.5% and 1% ethylene glycol groups were significantly reduced (P< 0.05). The serum calcium of dogs in 0.5% and 1% ethylene glycol group were significantly lower (P<0.05) than the control group, and serum calcium of dogs in 1% ethylene glycol group decreased more significantly (P<0.05) than in 0.5% ethylene glycol group. The serum creatinine and urea nitrogen of dogs in ethylene glycol group was significantly higher compared to the normal control group (P<0.05). Throughout the study period, urinary oxalate of dogs in 0.5% and 1% ethylene glycol group were higher (P<0.05) than the control group,1% ethylene glycol group were significantly higher than 0.5% ethylene glycol group (P<0.05). The urine calcium of dogs in ethylene glycol group was significantly higher than the control group (P<0.05). On the 9th weeks, urine creatinine and urea nitrogen of dogs in 1% ethylene glycol group was significantly lower than the control group and 0.5% ethylene glycol group (P<0.05). The urine creatinine and urea nitrogen were decreased significant (P<0.05) for dogs in 0.5% ethylene glycol group on the 18th week. Urine crystal is shown that calcium oxalate crystals by chemistry qualitative analysis, Fourier transform infrared spectroscopy and scanning electron microscopy.On the 18th week, no any renal tubular dilation and deposition of calcium oxalate crystals were observed in normal dogs. The crystallization of piles and connection, the significant swelling, degeneration, necrosis, and dilat of epithelial cells were found for dogs in 1% ethylene glycol group. Dogs in 0.5% ethylene glycol group have only a few crystalline distribution, and small calcium oxalate crystals scattered in the lumen, the expand the lumen is also lighter. These indicated that kidney of dogs in 1% ethylene glycol group have crystallization group, which can be considered successful model.Test 3. The effects of different selenium sources on the antioxidant capacity and calcium oxalate urinary stones formation in dogs induced by the ethylene glycolThe effects of different selenium sources on the formation of calcium oxalate urinary stones in dogs induced by ethylene glycol were observed, and the mechanism of selenium prevent calcium oxalate urinary stone from formation was also explored. Method:Model of dogs with calcium oxalate urinary stones were established by adding 1% ethylene glycol in drinking water.20 male dogs (1 year old, about 10 kg) were randomly divided into control group, calcium oxalate stones module group, sodium selenite prevention group and selenium-enriched yeast prevention group. The four groups were fed basal diet. Among them, sodium selenite and selenium-enriched yeast prevention group drink the solution with 1% ethylene glycol, and their basal diets are added sodium selenite and selenium-enriched yeast (the addition level of selenium was 0.3 mg/kg), respectively. After 18 weeks, the samples of urine for the four groups of dogs were collected to analyze the oxalate, calcium and sediment. The paraffin sections were made for left kidney, and tested the crystallization after HE stain. The levels of selenium, calcium, urea nitrogen, creatinine, malondialdehyde (MDA), glutathione peroxidase (GPX), superoxide dismutase (SOD), catalase (CAT) in blood were measured. The levels of MDA, GPX, SOD and CAT in right kidney tissue were also measured. The results showed that, compared with the control group, the serum calcium and antioxidant enzyme activity decreased and the urine calcium, urine oxalate, serum creatinine and urea nitrogen, level of MDA in blood and kidney tissue increased in the stone group. The calcium oxalate crystal deposition in renal tissues was significantly reduced in dogs drunk the solution with 1%ethylene glycol after supplement selenium. The level of whole blood selenium and serum calcium, and the antioxidant enzyme activity in kidney tissues in sodium selenite and selenium-enriched yeast prevention group were significantly higher than in the stone group, while the urine calcium, serum creatinine, blood urea nitrogen, MDA in blood and kidney tissues were significantly lower. The MDA levels in blood and kidney tissue in selenium-enriched yeast prevention group were significantly lower than in sodium selenite prevention group, while the antioxidant enzyme activities were higher. The levels of serum creatinine, blood urea nitrogen, and urinary calcium in selenium yeast prevention group were slightly lower than in sodium selenite prevention group, while the whole blood selenium and serum calcium were slightly higher than in sodium selenite prevention group. Conclusion:To some extent, supplement with a certain the amount of selenium may reduce the formation of calcium oxalate urinary stones in dogs induced by ethylene glycol, and can improve the body’s antioxidant capacity of dogs. The effect of organic source of selenium (selenium yeast) is better than inorganic selenium source (sodium selenite).Test 4. The effects of different selenium sources on the content and expression level of the OPN-mRNA and OPN in kidneys of calcium oxalate urinary stones in dogs induced by the ethylene glycolThe effects of different selenium sources on the expression level of OPN in kidneys of dogs with calcium oxalate urinary stones induced by ethylene glycol were studied to explore the relationship between OPN expression and kidney stones.20 local dogs were divided into four groups:the normal group, stones models group (a stone group), sodium selenite prevention group, and selenium-enriched yeast prevention group. After 18 weeks, analyze the OPN and the OPN mRNA expression levels in kidney tissue. The results showed that, on the 18th week, compared with the control group, the OPN expression levels in kidney tissue of dogs in stone group were significantly increased. Compared with the stone group, the OPN expression levels in kidney tissue of dogs in sodium selenite and selenium-enriched yeast prevention group were significantly reduced. The effects of selenium-enriched yeast were better than sodium selenite.These results showed that the OPN expressed in kidney and secreted into the urine was significantly decreased. The expression levels of OPN in kidney tissue of dogs in selenium-enriched yeast prevention group were lower than sodium selenite prevention group indicated that selenium can inhibit the formation of calcium oxalate crystals, thereby feedback reduce the OPN expression. The selenium-enriched yeast was better than sodium selenite. |
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