A Study On Characteristics And Mechanisms Of Uric Acid Metabolism In Streptozotocin-induced Diabetic Rats

BackgroudAs one of the most common microvascular complications of diabetes mellitus (DM),diabetic nephropathy (DN) seriously affects life quality and survival in these patients.Previous studies found that hyperuricemia (HUA) closely relates to occurrence anddevelopment of DN in either type1(T1DM) or type2(T2DM) diabetes mellitus. While adecreased serum uric acid (SUA) level may effectively retard the progress of DN and chronickidney disease (CKD).Our preceding animal experiments investigated relationship between uric acid and renaldamage by lowering SUA level with febuxostat (Fx) in streptozotocin(STZ)-induced diabeticrats. The results showed that diabetic rats developed obviously HUA, while lowering SUAconspicuously ameliorates renal damage. Most interestingly, the underlying mechanism forHUA in DM rats might be mainly resulted from increased uric acid synthesis. Similarconclusion can also be drawn in our clinical research. Therefore, it’s very necessary to studycharacteristics and possible mechanisms of uric acid metabolism in DM for exploring the newtherapeutic target of DN.Uric acid is the end product of purine metabolism in human body. Three key enzymes,Phosphoribosylpyrophosphate synthetase1(PRPPS1), phosphoribosylpyrophosphateamidotransferase(PRPPAT) and xanthine oxidase (XO) are involved in the de novo anabolismand catabolism of purine. Certainly they are the key enzymes of uric acid anabolism at thesame time. It’s very important to detect mRNA expression，enzyme content and enzymeactivity of these enzymes in liver for studying uric acid anabolism.The process of tubular uric acid reabsorption and excretion depends on coordination ofseveral urate transporters. Among them, urate-anion transporter1(URAT1) and glucosetransporter9(GLUT9) play the most important role for reabsorption. URAT1is specificallyexpressed in the brush border side of epithelial cells of renal proximal convoluted tubules, andpromotes urate transporting into epithelial cells from renal tubular cavity. GLUT9has twosubtypes, respectively expresses in renal proximal tubule epithelial basement membrane andluminal membrane. It’s mainly responsible for transporting urate from renal tubular epithelialcells to interstitium across the cellular basement membrane. GLUT9coordinates with URAT1 in the whole process of renal urate reabsorption. For further understanding mechanisms ofHUA in STZ-induced diabetic rats, renal alterations of URAT1and GLUT9are also necessarymarkers to investigate.Summarily, our study focuses on key enzymes involved in liver uric acid anabolism andtwo major uric acid transporters in kidney in STZ-induced diabetic rats, for the sake ofmaking the exact mechanism of HUA clear.Part I Preceding research overview—Effect of Lowing Serum Uric Acid by Febuxostat on the Development ofRenal Damage in Streptozotocin-induced Diabetic RatsObjective To observe characteristics of uric acid metabolism in STZ-induced DM rats, andeffects of lowering SUA on the renal damage.Methods Twenty-four STZ-induced diabetic rats and twenty normal rats (N) were randomlydivided into febuxostat treatment group (DM+Fx, N+Fx) and control group (DM, NC).Fasting plasma glucose(FPG), blood urea nitrogen(BUN), serum creatinine(sCr), serum uricacid(SUA) and urinary albumin excretion(UAE), urinary uric acid(UUA), urinecreatinine(UCR), urine urea nitrogen(UUN) were measured at the baseline and the4th,8thweeks. All rats were sacrificed after intervention for8weeks. Livers and kidneys wereextracted and preserved in the-80℃refrigerator, part of the renal tissues were fixed byparaffin and embedded for room temperature preservation.Results Compared with NC group, STZ-induced diabetic rats had a significant increase inFBG, BUN, sCr, SUA;24h-UUA,24h-UCR,24h-UUN and24h-UAE were also significantlyhigher in diabetic rats(P<0.05). After treatment with febuxostat for8weeks, DM rats showeda conspicuous decline in SUA, BUN, sCr, and24h UAE, and more lower than those treatedwith placebo(P<0.05).Conclusions STZ-induced diabetic rats became hyperuricemia and renal damages.Febuxostat conspicuously lowered the SUA level, and obviously ameliorate the glomerulifiltration function in STZ-induced DM rats. The formation of HUA in STZ-induced DM ratsmay give priority to an increased uric acid synthesis. Part II The Study on Key Enzymes Involved in Liver Uric AcidSynthesis in Streptozotocin-induced Diabetic RatsObjective To detect levels of liver PRPPS1, PRPPAT, XO mRNA expression, enzymecontent and activity in STZ-induced diabetic rats.Methods Rat liver tissues weighed0.20~0.25g with nine times of normal saline werehomogenized by a high speed disperser, then centrifuged at2500r/min for10min.Supernatant was collected for measuring the quantity of PRPPS-1and XO with using ELISAmethod. XO activity was measured with the coomassie brilliant blue method. Rat livers weretaken out from the-80℃refrigerator. Total RNA of the liver tissue was extracted withTRIZOL reagent. Levels of PRPPS1, PRPPAT and XO mRNA expression were detected byreal-time polymerase chain reaction.Results Compared with normal control group, DM rats developed into higher levels ofPRPPS1content (23103.33±556.53vs10458.40±448.90, P=0.000); XO content (ng/ml)(125.59±3.04vs59.94±3.23, P=0.000); and XO activity(U/gpro)(24.42±2.95vs18.60±2.16,P<0.05); Liver XO content of DM+Fx group had no significant change after Fx treatment(P<0.05). The level of XO mRNA expression also increased significantly in DMgroup(P<0.05). However, PRPPAT mRNA and PRPPS1mRNA levels were comparedbetween DM and control groups(P>0.05). The mRNA expression levels of all three keyenzymes were not significantly affected after Fx treatment in either DM or controlrats(P>0.05).Conclusions In STZ-induced diabetic rats, liver content of several key enzymes and geneexpression of XO increased. XO activity also rose significantly.Part III The Study of Renal Expressions of URAT1and GLUT9inStreptozotocin-induced Diabetic RatsObjective To detect the levels of renal URAT1, GLUT9mRNA and their protein expressionin STZ-induced diabetic rats. Methods Paraffin sections of renal tissue were analyzed by immunohisto-chemical staining.Photographs were captured by high-definition color graphic analysis system of medicine.Average optical density values of URAT1and GLUT9in both DM rats and normal controlrats were detected. For gene measurement, rat renal tissues were taken out from the-80℃refrigerator. Total RNA of the renal tissue was extracted with TRIZOL reagent, levels ofURAT1and GLUT9mRNA expression were detected by real-time polymerase chain reaction.Results Average optical density values of URAT1, GLUT9had no significant difference inDM rats and normal control rats (URAT1:0.3140±0.0532vs0.3171±0.0643, P=0.744;GLUT9:0.2645±0.0498vs0.2612±0.0550, P=0.856). There were no differences aboutURAT1and GLUT9mRNA expression between DM rats and control rats (URAT1:0.3171±0.0643vs-0.76±0.93, P>0.05; GLUT9:2.78±0.84VS1.98±1.62, P>0.05).Febuxostat treatment exerted no any effects on either URAT1and GLUT9protein or mRNAexpression in both kinds of rats(P>0.05).Conclusions Renal protein and gene expressions of URAT1and GLUT9were bothcomparable between STZ-induced diabetic rats and normal rats. Febuxostat did not changeexpression levels of both urate transporters.Summary HUA in STZ-induced diabetic rats tend to be caused by increased uric acidsynthesis, instead of decreased excretion.