Establishment Of New Rat Models Of Hyperuricemia Under High Temperature-Humidity Environment And Its Mechanism Study

ObjectiveAccording to a large number of clinical evidences,the incidence rate of hyperuricemia was increasing under the high temperature-humidity environment.Moreover,there is no animal model of hyperuricemia established by environmental temperature and humidity treatment in the world at present.This study intends to establish hyperuricemia rat models by simulating high temperature-humidity environment,and detects their biochemical indicators such as serum uric acid（SUA）levels,as well as observe the damage of kidney tissue.Furthermore,this experiment also plans to evaluate the morphological changes of other major tissues,such as liver,heart and ankle joint,and detect the expression of uric acid transporters to further analyze the potential mechanism of increased SUA levels.MethodsIn this experiment,rat models of hyperuricemia induced by high temperature-humidity combined with chemical drugs were established and compared with the traditional chemical drug induced models.30 SPF-SD male rats,150～200g,6～7w,were divided into following five groups:control group（CON,n=6）,potassium oxonate group（PO,n=6）,high purine diet group（HPD,n=6）,high temperature-humidity group（HTH,n=6）,and compound factor group（CF,n=6）.The rats from CON group was only given 0.5%sodium carboxymethyl cellulose solution by gavage.PO group was gavaged with 250mg/kg potassium oxonate suspension,while HPD group was gavaged with 250mg/kg potassium oxonate suspension and fed with 20%yeast feed,thus establishing a rat model of hyperuricemia induced by traditional chemical drugs.On the basis of the above two groups of traditional chemical medicine modeling methods,HTH group and CF group were treated in high temperature-humidity environment,that is,irradiated in a special constant temperature incubator with a temperature of 37±0.5℃and a relative humidity of 80±1%for1 h/d（potassium oxonate+incubator irradiation in HTH group and potassium oxonate+yeast feed+incubator irradiation in CF group）.And the experiment lasted for 12continuous weeks.Every 2 weeks,all rats were fasted for at least 12h before collecting blood samples so as to test their SUA and creatine levels.three rats from each groups were anesthetized to collect their bilateral renal tissues,heart,liver and ankles after modeling for6 weeks and 12 weeks respectively.This study is divided into four parts.（1）The hyperuricemia rat models were established by simulating high temperature and high humidity environment,then analyzed the rising trend and duration of SUA values.（2）Observing the renal histopathological changes and ultramicrostructure changes through HE,masson,gomori staining and transmission electron microscopy.（3）Evaluating the histppathological changes of vital tissues,such as ankle and liver of the hyperuricemia models,by using the HE staining skill.（4）The expression levels of reabsorbing uric acid transporter URAT1 and excretory uric acid transporter ABCG2 in proximal tubules of kidney were detected by western blot and immunohistochemistry,so as to analyze the influence of high temperature-humidity environment on uric acid transporters and explore its mechanism of increasing blood uric acid level.Results（1）In this experiment,the survival rates of all groups was 100%after 12 weeks of continuous modeling.After 2 weeks,the SUA levels of all rat models had reached their peak,and that of CF group,HTH group,HPD group PO group are significantly higher than that of CON group（155.9±32.3μmol/L、133.9±17.8μmol/L、118.5±5.9μmol/L、107.6±12.4μmol/L VS 85.7±4.1μmol/L,P<0.05）,among them,the SUA value of HTH group was higher than PO group（133.9±17.8μmol/L VS 107.6±12.4μmol/L,P<0.05）,and CF group was higher than HPD group（155.9±32.3μmol/L VS 118.5±5.9μmol/L,P<0.05）.At the 12^thweek of the experiment,the SUA values of PO group and HPD group dropped seriously,while those of HTH group and CF group remained at a relatively high level.As the experimental time went on,the serum creatinine（CRE）levels of rats in each group increased slightly,which all reached their peak an the 8^thweek.At that time,the CRE levels of CF group,HTH group,HPD group and PO group were higher than CON group（50.7±4.0μmol/L、50.7±3.0μmol/L、50.5±7.8μmol/L、43.8±4.6μmol/L VS 30.7±3.2μmol/L,P<0.05）,then declined.（2）Under light microscope,the renal tissue morphology of PO group and HPD group showed tubular dilatation,while there were a large number of epithelial cells of proximal convoluted tubules swelled in HTH and CF groups,lead to the lumen gap narrowed,and some tubule spaces showed significant inflammatory infiltration.In addition,there were some renal tubular epithelial cells atrophy in CF group.Focal fibrosis of renal tubulointerstitium was observed in PO group and HPD group,while brush border fibrosis of renal tubular epithelial cells was relatively serious in HTH group and CF group.The staining area of urate crystal deposition was thicker in HTH and CF groups than that of PO and HPD groups.At the 6^thweek of the experiment,some glomerular podocytes in HTH group and CF group were swollen and fused.At the 12^thweek,mesangial cells in HTH group and CF group proliferated,mesangial matrix increased,glomerular basement membrane was significantly thickened,and some mitochondria in renal tubules were swollen or even vacuolated.（3）Under light microscope,the hepatic sinuses in PO group and HPD group were slightly dilated,while the liver cells in HTH group and CF group were swollen and the hepatic sinuses were narrowed.In CF group,the arrangement of myocardial myofilaments was slightly disordered,and the structure of ankle cartilage layer was not smooth,but other tissue structures were not obviously abnormal.（4）Western blot and immunohistochemical experiments showed that,the expression of ABCG2 protein in HPD,HTH group and CF group decreased compared with CON group（P=0.006、0.001、0.001<0.05）,that in HTH group was lower than PO group（P=0.001<0.05）,and CF group was lower than HPD group（P=0.001<0.05）.At the meanwhile,the expression of URAT1 protein in HPD,HTH group and CF group increased compared with CON group（P=0.043、0.001、0.001<0.05）,that in HTH group was higher than PO group（P=0.001<0.05）,and CF group was higher than HPD group（P=0.001<0.05）.Concusion（1）The results of this study preliminarily showed that the hyperuricemia rat models induced by high temperature-humidity environment pessessed more stable SUA levels.（2）The pathological morphology and ultrastructure of kidney in this model changed obviously,but it has not progressed to renal injury,while the changes of liver,heart and ankle joint were slight.（3）It had been further found that the increased expression of reabsorptive uric acid transporter URAT1 and the decreased expression of excretory uric acid transporter ABCG2 may be one of the important potential mechanism for the increase of SUA levels under high temperature-hmidity environment.（4）The increasing of temperature and humidity modeling methods in this study can be further applied to the research on the pathogenesis and drug treatment of hyperuricemia caused by high temperature humidity environment.