Effect Of Uric Acid On Adipose Tissue RAS And Its Association With Obesity Hypertension

Background:Hypertension is one of the most common findings in obese patients. Also, elevatedserum uric acid levels usually occur in obesity. In recent years, multiple evidences havedemonstrated that serum uric acid closely correlates with cardiovascular diseases andmetabolic diseases, such as hypertension, obesity, type2diabetes mellitus, and metabolicsyndrome. Among these diseases, the roles of uric acid in the pathogenesis of hypertensionattract more attention. Data from epidemiological studies, clinical trials and animalexperiments have indicated that hyperuricemia (HUA) contributes to the occurrence ofobesity-related hypertension, despite of the unclear underlying mechanisms.It is well documented that adipose tissue almost expresses all components of therenin-angiotensin system (RAS)[1]. It was found that angiotensinogen (AGT) andangiotensin Ⅱ(AngⅡ) derived from white adipose tissue contributed to circulation pool ofRAS[2], which in turn had an influence on blood pressure regulation. Mice with deficientAGT in adipose tissue had lower plasma AGT and decreased blood pressure[3]. In someobservational survey, plasma renin was found to be positively related to serum uric acidlevels in hypertensive patients[4]. However, almost no research pays attention to theassociation between plasma AGT concentration and serum uric acid levels in obese patientswith essential hypertension.Up to now, many factors were found to be associated with the regulation of adiposetissue RAS[5], including nutrition condition, insulin, glucocorticoid, free fatty acid,androgen, tumor necrosis factor alpha (TNFα), and cyclic adenosine monophosphate(cAMP). Uric acid was reported to exert effect on RAS expression in immortalized humanmesangial cells (ihMCs)[6], human vascular endothelial cells (HVECS)[7]and vascularsmooth cells (VSMCs)[8]. Obesity is characterized by overexpression of adipose tissue RAS[9]. This interesting phenomenon is commonly explained by increased fat mass in obesepatients. However, in view of the roles of both uric acid and adipose tissue RAS in obesity hypertension, it could be postulated that uric acid might also regulate the expression ofadipose tissue RAS.It is well known that obesity hypertension is one of oxidative stress-relatedcardiovascular diseases[10]. Experiments in vitro and in vivo have suggested that uric acidcould result in oxidative stress under some conditions. In particular, an increase inintracellular reactive oxygen species (ROS) was reported to be triggered by highconcentrations of uric acid in3T3-L1adipocytes[11]. Unfortunately, the molecular pathwayunderlying is not clear, through which uric acid upregulates NADPH oxidase (NOX)activity and then results in oxidative stress.Therefore, in the present study, we tried to solve the problems mentioned above bothin vitro experiments and in population study. First, we observed the effect of uric acid onadipose tissue RAS regulation using3T3-L1adipocytes as an experimental model. Next,we testified the hypothesis that adipose RAS could play a role in uric acid-inducedoxidative stress. Last but not the least, we investigated the association between serum uricacid levels and AGT concentration in untreated patients with obesity hypertension. Thesedata might provide new insights into the mechanism by which uric acid is involved incardiovascular diseases related with obesity, especially hypertension.Methods:1. The undifferentiated pre-adipocytes were cultured with uric acid (0、1、5、15mg/dl)for48hours. In addition, when the adipocytes were differentiating on the fourth day, uricacid (5、15mg/dl) were added into the medium for48hours. AGT mRNA was detected withreal time RT-PCR.2. The differentiated adipocytes were incubated with uric acid at differentconcentrations (0、1、5、15mg/dl) for48hours or with15mg/dl uric acid for differentperiods of time (0、24、48、96hours). In some plates, the adipocytes were cultured in thepresence of15mg/dl uric acid with or without probenecid (2mM) for48hours. The levelsof adipose RAS gene were detected with real time RT-PCR. The production of AngⅡprotein was determined by enzyme linked immunosorbent assay (ELISA).3. In the presence of15mg/dl uric acid, the differentiated adipocytes were culturedwith losartan (10-4M) or captopril (10-4M) for48hours. The levels of AGT mRNA andAngⅡ protein were measured by real time RT-PCR and ELISA, respectively. NOX activity was quantitatively detected with colorimetry method.4. The differentiated adipocytes were incubated under high concentrations of uricacid (5、15mg/dl) with or without RAS inhibitors (10-4M losartan or10-4M captopril) for48hours. Some plates were treated with10mM N-acetyl-L-cysteine (NAC) or200μMapocynin. The levels of intracellular ROS were detected using fluorescence probe2′,7′-dichlorofluorescin diacetate (DCFH-DA) or DHR (dihydrogen rhodamine123) or NBTassay. The fluorescence microscope was used for qualitative detection. Thefluorescence microplate was used for quantitative detection.5.162obese and162non-obese male patients with untreated essential hypertensionwere enrolled in the population study. Anthropometry indexes, including blood pressure,height, weight, waist circumference, and hip circumference, were measured by a speciallyassigned nurse. Biochemical indicators, including blood glucose, serum uric acid, creatinine,and blood lipid, were detected using a fully automatic biochemical analyser. Fasting insulin(FINS) levels were determined by radioimmunoassay method. Homeostasis modelassessment of insulin resistance (HOMA-IR) was used for assessment of insulin resistancestatus. Plasma AGT concentrations were assayed with ELISA method.6. Independent-samples t-test or chi-square test was used for comparison ofvariables between obesity hypertension group and non-obesity hypertension group.One-way ANOVA was used for comparing the differences among subgroups dividedaccording to uric acid tertiles. If a difference existed, further analysis was performed withBonferrroni’s post hoc test. The correlation coefficient between serum uric acid and othervariables was calculated with partial correlation analysis after adjustment for age, smokingratio and alcohol assumption ratio. Multiple variables regression analysis was used fordetermining the independent effect of uric acid on AGT levels in obese patients withhypertension. Statistical analysis was performed using SPSS17.0. Illustrations were drawnwith software GraphPad prism5.0.Results:1. Uric acid at different concentrations had no effect on AGT mRNA expression inundifferentiated3T3-L1adipocytes (P>0.05). However, uric acid, at high concentrations(5、15mg/dl), resulted in an increase in AGT mRNA in differentiating3T3-L1adipocytes(both P<0.05). 2. At physiological concentration (1mg/dl), uric acid played no role in the expressionof AGT mRNA in differentiated3T3-L1adipocytes (P>0.05). However, highconcentrations of uric acid (5、15mg/dl) significantly upregulated both RAS mRNAexpression and AngⅡ protein production (all P<0.05). AGT mRNA and AngⅡincreasedwhen differentiated adipocytes were cultured with15mg/dl uric acid for48and96hours(all P<0.05), but not for24hour. Moreover, the regulation effect of uric acid on adiposetissue RAS is in a dose-dependent and a time-dependent way. In addition, probenecid, akind of organic anion transporter (OAT) inhibitors, attenuated the effect of uric acid onadipose RAS regulation (P<0.05).3. When compared with15mg/dl uric acid, both10-4M losartan and10-4M captoprilblunted the increase in the over expression of AGT mRNA and AngⅡ protein induced byhigh concentration of uric acid (all P<0.05). Furthermore, RAS inhibitors couldsignificantly reduce NOX activity when mature adipocytes were cultured in15mg/dl uricacid (all P<0.05).4. The intracellular ROS was significantly increased by high levels of uric acid (5,15mg/dl) in mature3T3-L1adipocytes (all P<0.05). However, when cells at15mg/dl uricacid were treated with RAS inhibitors losartan or captopril simultaneously, an increased inintracellular ROS expression was preventable in differentiated3T3-L1adipocytes (allP<0.05). Moreover, both antioxidant NAC and NOX inhibitor apocynin could ameliorateROS excessive activation in adipocytes (all P<0.05).5. In addition to waist circumference, waist-to-hip ratio, weight and body mass index(BMI), there was a significant increase in alcohol consumption ratio, systolic bloodpressure (SBP), triglycerides, fasting blood glucose, serum uric acid, plasma AGT levels,fasting insulin, and HOMA-IR in obese patients compared to non-obese patients (all P﹤0.05). When patients were divided into three subgroups based on uric acid tertiles, it wasdemonstrated that AGT (P﹤0.001), FINS (P=0.002), and HOMA-IR (P=0.007) had amarked increase in the highest tertile (435.9~642.2μmol/L serum uric acid) compared tothe lowest tertile (282.7~373.6μmol/L serum uric acid). However, there was no obviouschange in levels of AGT, FINS, and HOMA-IR among non-obese patients when subdividedaccording to uric acid levels (P>0.05).6. After adjustment for age, smoking ratio and alcohol assumption ratio, partial correlation analysis showed that, in obesity group, serum uric acid positively associatedwith AGT, FINS, and HOMA-IR with correlation coefficient0.437(P﹤0.001),0.245(P=0.002), and0.237(P=0.003), respectively. However, AGT, FINS, and HOMA-IR werefound not to be related to serum uric acid levels in non-obese hypertensive patients(P>0.05). Furthermore, multiple variables analysis using stepwise regression modelindicated that obesity×uric acid (standardized coefficient0.257, P﹤0.001) independentlycontributed to plasma AGT levels in untreated hypertensive patients.Conclusions:1. Uric acid could upregulate adipose tissue RAS expression in differentiating anddifferentiated3T3-L1adipocytes, but not in undifferentiated preadipocytes. Uric acid mustenter into adiposytes via urate transporter to play its role.2. Uric acid could upregulate adipose tissue RAS, increase AngⅡproduction, andthen activate NOX, ultimately result in oxidative stress. These findings indicate that overactivation of adipose RAS is implicated in the pathogenesis of uric acid-induced oxidativestress in adipose tissue.3. Serum uric acid was found to be positively associated with plasma AGT levels inan obesity-dependent manner in essential untreated hypertensive patients. Also, serum uricacid obviously correlated with the elevated insulin levels and insulin resistance in obesehypertensive patients.