The Effect Of High Purine Diet And Rosiglitazone On The Blood Uric Acid Level Of OLETF Rat And The Potential Mechanism

Objective:To observe the effect of high purine diet and the intervention of rosiglitazone on the blood uric acid and the expressions of gene OAT1 and UAT in the renal epithelial cells of OLETF rats.Methods:30 male OLETF rats of 4 weeks age were raised up to 25 weeks age, and were divided randomly into control group(n=10)and high purine diet group(n=20).The high purine diet group was administered with adenine(50mg/kg/d) by gavage and was fed ground standard rat chow containing 10%yeast.30 male LETO rats of 4 weeks age were processed the same way with OLETF.TG,TC,UA,CRE,BUN and 24h urine volume and UUA were detected.Insulin were detected by radioimmunity.The incidence rate of hyperuricemia was compared between the OLETF and LETO rats.Then devide the OLETF high purine diet group into high purine diet group(n=10) and intervention group(n=10).Both were given high purine diet,besides the intervention group were given rosiglitazone(3 mg/kg/d) by gavage daily.Keep on monitoring the SUA until there were significant difference.Then extracte total RNA from the all rats'cortex of kidney.OAT1,UAT and GAPDH mRNA were detected by real time fluorescent quantification RT-PCR.The expression of OAT1,UAT were detected by immunohistochemistry.Results:①On the 11^th day of high purine diet, blood uric acid level of both OLTEF and LETO high purine diet group were significantly higher than their corresponding control group(P=0.000).Moreover,blood uric acid of OLTEF high purine diet group was much higher than that of LETO high purine diet group(P =0.008 );②The incidence rate ofhyeruricemia of OLTEF high purine diet group(84.21%) was significantly higher than that of LETO high purine diet group(36.84%)(P＜0.05);③On the 11^th day of high purine diet,OAT1 expression level was significantly decreased in the OLTEF high purine diet group compared with the OLTEF control group(t' =21.690,P =0.000).There was no difference between the high purine diet group and the control group in LETO rats(t'=-1.541,P =0.162).OAT1 expression level of the OLTEF high purine diet group was significantly decreased than that of LETO high purine diet group(P =0.000);④On the 11th day of high purine diet,UAT expression level of both OLTEF and LETO high purine diet group were significantly decreased than their corresponding control group(P =0.000),UAT expression level of the OLTEF high purine diet group was significantly decreased than that of LETO high purine diet group(P=0.035);⑤On the 21^th day of intervention,blood uric acid level of the intervention group was significantly decreased than that of the high purine diet group(P=0.002),and there was no difference between the intervention group and the control group;⑥Both OAT1 and UAT expression level of the intervention group were significantly higher than that of the high purine diet group(P＜0.05 ),but still were significantly decreased than that of the control group(P＜0.05 );⑦The expression of OAT1 and UAT were detected in all slices.Absorbance values of both OAT1 and UAT of the intervention group were significantly higher than that of the high purine diet group(P＜0.05),but still were significantly decreased than that of the control group(P＜0.05);Conclusion:(1) The incidence rate of hyeruricemia led by high purine diet in OLTEF rats is significantly higher than of LETO rats.It is probably because that under the same dosage of purine loads,OAT1 and UAT expression level of OLTEF rats is significantly decreased than that of LETO rats.(2) The high blood uric acid level of OLTEF rats led by high purine diet can be significantly reduced by rosiglitazone.It is probably because that rosiglitazone can indirectly up regulate the expression of OAT1 and UAT by improving insulin resistance. PART TWO Role of Lipid Metabolic Disorder in the Expression of hUAT and hOAT1 Gene in HK-2 CellsObjective:To observe the role of different concentration of lipoprotein and free fat acid in the expression of hUAT and hOAT1 mRNA in HK-2 cells.Methods:All experiments were performed with HK-2 cells cultured in vitro.According to the concentration of lipoprotein in culture medium,growth-arrested cells were devided into 3 groups.And according to the concentration of VLDL,HDL,cells treated with lipoprotein were devided into 1,2,group.①Control group:DMEM/F-12②VLDL group: V1:DMEM/F-12+50ug/ml VLDL.V2:DMEM/F-12+400ug/ml VLDL..③HDL group:H1: DMEM/F-12+50ug/ml HDL.H2:DMEM/F-12+40Oug/ml HDL.In each culture medium, 6 bottle of cells were cultured for 48h to get the mean.Then all were collected,counted and total RNA was extracted.1 ug mRNA was reverse transcripted to eDNA and hUAT, hOAT1 and GAPDH were amplified by real-time fluorescent quantitative PCR.Then relative expression quantity of hUAT and hOAT1 mRNA was analyzed.Repeat the whole experiment.The data was analyzed by t-test and one-factor ANOVA and P＜0.05 was considered significant.Results:①hUAT and hOAT1 mRNA could be detected in all the samples.②hUAT mRNA expression decreased with the increase of VLDL concentration after treated for 48h. The hUAT mRNA of control groups was significantly higher than that of V1,V2 group(P=0.000) and there was significant difference between V1,V2 groups(P＜0.05).③hOAT1 mRNA expression decreased with the increase of VLDL concentration after treated for 48h.The hOAT1 mRNA of control groups was significantly higher than that of V1,V2 group(P=0.000) and there was no significant difference between V1,V2 groups(P=0.927 ).④With the increase of HDL concentration,hUAT mRNA expression decreased after treated for 48h.hUAT mRNA of control group was significantly higher than that of H1,H2 groups (P=0.000).There was significant difference between H1,H2 groups(P＜0.05).⑤With the increase of HDL concentration,hOAT1 mRNA expression increased after treated for 48h. hOAT1 mRNA of control group was significantly lower than that of H1,H2 groups (P=0.000).There was significant difference between H1,H2 groups(P＞0.05). Conclusions:Increased concentration of VLDL,HDL down-regulate the expression of hUAT mRNA,which may be an important pathogenesis in hyperuricemia induced by lipid metabolic disorder.HDL can increase the expression of hOAT1 mRNA,Which indicate that HDL maybe can increase the excretion of uric acid.