Effect Of PPARγ Agonists On AMPK And Antioxidant In Renal Tissue Of Obese Mice

Objective:Obesity is recognized increasingly as a major risk factor for kidney disease. We investigated previously that PPARγ agonist pioglitazone play a renal protective effect by increasing serum adiponectin levels in obese mice, and adiponectin has been reported to have anti-oxidative stress function, but whether the renal protective effect of PPARγ agonists is correlated with anti-oxidative stress is unclear.In this study, we investigated the effect of PPARγ agonists on AMPK and antioxidant in renal tissue of obese mice.Methods:24 male ob/ob mices and 8 male C57BL/6J mices which were 6 weeks old in this study, weighed the mice and calculated 24 hour microalbuminuria(24h m ALB) by collecting the urine of the mice after adaptive feeding 2 weeks, to make sure that weight and 24 h m ALB between ob / ob mice and C57BL/6J mice are significantly different.Randomly assigned ob / ob mice into 3 groups according to body mass: obesity group(M group, n=8), low dosage pioglitazone intervention group(T1 group, n=8), high dosage pioglitazone intervention group(T2 group, n=8), C57BL/6 mice were control group(C group) Mice in T1 group were administered with Pioglitazone(2.25mg·kg-1·d-1) by gavage daily,T2 group(6.75mg·kg-1·d-1).Mice in M group and C group were administered with the same volume of distilled water daily by the same way. Weighed the mice once a week and adjust the dose according to body weight for 12 weeks. Blood and urine samples were collected before and after the intervention to measure blood glucose, serum adiponectin(ADP) and calculate 24 h m ALB. After 12 weeks, the mice were sacrificed, and the right kidneys of mice were obtained and weighed. Observe the morphological changes of kidneys by HE staining, measure and compare glomerular diameters. Positioning AMP-activated protein kinase(AMPK) and Nicotinamide adenine dinucleotide phosphate Oxidase-4(NOX-4) by immunohistochemistry, evaluate and compare the level of expression of AMPK and NOX-4.And then compare the difference of the various indicators among the four groups,separately analysis the correlation of serum ADP,24h microalbuminuria, renal tissue expression levels of AMPK and NOX4 with other indicators.The statistical analysis of the data was done with the statistical package of SPSS 17.0. A value of P < 0.05 was considered statistically significant.Results:1.The general situation of mice 1.1 Comparison of the weight and 24 h m ALB between two kinds of mice before the experiment: The average body weight of ob / ob mice was significantly higher than that of C57 BL / 6 mice at 6 weeks old(P<0.01), what’s more, the average body weight of ob / ob mice was not only significantly higher than that of C57BL/6J mice(P<0.01), but more than 50% of the latter(> 20%), which means the ob / ob mice met the standards of obesity. Meanwhile, the 24 h m ALB of ob / ob mice was significantly higher than that of C57BL/6J mice,but the glucose of all the mice didn’t meet the standards of diabetes although the difference of glucose was significantly between two kinds of mice, then we can make sure that the apparent proteinuria of ob/ob mice was caused by obese but not diabetes. 1.2 The weight of mice before and after the experiment in each group: After grouping the average weight of mice in M and T1, T2 group were more than 20% of group C(P<0.01),which reached the standard of obesity, but there was no significant difference among the M and T1, T2 groups(P>0.05). At the end of the experiment, the weight of group M and T1, T2 mice is more obviously higher than that in group C(P<0.01),and group T2 was much higher than group T1(P<0.01). The weight of all groups after experiment were higher than that before(P<0.01). 2.Serum concentrations of ADP 2.1 Comparison of each group before and after the experiment: The serum adiponectin levels of mice of group M,T1 and T2 after the experiment were lower than that before(P<0.01) while that of group C didn’t change much(P>0.05). 2.2 Comparison among each group: The serum adiponectin levels of mice of group M, T1 and T2 were lower than that of group C(P<0.01),while that of group T1 and T2 were higher than group M(P<0.01), and group T2 was higher than group T1(P<0.01). 3.24 h m ALB3.1 Comparison of each group before and after the experiment:The 24 h m ALB of mice of group M,T1 and T2 after the experiment were higher than that before(P<0.01) while that of group C didn’t change much(P>0.05). 3.2 Comparison among each group: The 24 h m ALB of mice of group M,T1 and T2 were higher than that of group C(P<0.01)both before and after the experiment,while that of group T1 and T2 were lower than group M(P<0.01), and group T2 was lower than group T1(P<0.01). 4.Pathological changes of kidney 4.1 Kidney weight(right kidney): The kidney weight of mice of group M,T1 and T2 were higher than that of group C(P<0.01)both before and after the experiment,while that of group T1 and T2 were lower than group M(P<0.01), and group T2 was lower than group T1(P<0.01). 4.2 Pathological changes in renal tissue: The changes include Bowman’s capsule widened, glomerular basement membrane thickening, mesangial cells and mesangial matrix proliferation, glomerular volume increased, some accompanied by focal segmental glomerulosclerosis, tubular atrophy and mild interstitial fibrosis. These changes were most severely in group M, and group T1 and T2 were alleviate than group M. 4.3 Glomerular diameter: The glomerular diameter of mice of group M,T1 and T2 were bigger than that of group C(P<0.01) after the experiment, while that of group T1 and T2 were smaller than group M(P<0.01), and group T2 was smaller than group T1(P<0.01). 5.Expression of AMPK in renal tissue:AMPK could expressed in glomerular, renal tubules and renal interstitial; The expression of AMPK of mice of group M and T1, T2 were lower than that of group C(P<0.01),while that of group T1 and T2 were higher than group M(P<0.01), and group T2 was higher than group T1(P<0.05). 6.Expression of NOX-4 in renal tissue:NOX-4 could expressed in glomerular, renal tubules and renal interstitial; The expression of NOX-4 of mice of group M and T1, T2 were higher than that of group C(P<0.01),while that of group T1 and T2 were lower than group M(P<0.01), and group T2 was lower than group T1(P<0.05). 7.Linear correlation analysis 7.1 The 24h-m ALB was positively correlated to body weight, glomerular diameter, kidney weight(right kidney) and expression of NOX-4(r1=0.741;r2=0.936;r3=0.877;r4=0.878,P<0.001), was negatively correlated to serum concentrations of ADP and expression of AMPK(r5=-0.915;r6=-0.759,P<0.001). 7.2 The serum concentrations of ADP was negatively correlated to body weight, glomerular diameter, kidney weight(right kidney), 24h-m ALB and expression of NOX-4(r7=-0.772; r8=-0.821; r5=-0.915; r9=-0.958, P<0.001),was positively correlated to the expression of AMPK(r10=0.878,P<0.001). 7.3 The expression of AMPK was negatively correlated to body weight, glomerular diameter, kidney weight(right kidney), 24h-m ALB and expression of NOX-4(r11=-0.627;r12=-0.711;r13=-0.743;r6=-0.759;r14=-0.884,P<0.001), was positively correlated to serum concentrations of ADP(r10=0.878,P<0.001). 7.4 The expression of NOX-4 was positively correlated to body weight, glomerular diameter, kidney weight(right kidney) and 24h-m ALB(r15=0.671;r16=0.843;r17=0.794;r4=0.878,P<0.001),was negatively correlated to serum concentrations of ADP and expression of AMPK(r9=-0.958;r14=-0.884,P<0.001).Conclusions:1.The serum ADP levels of ob / ob mice was declined, AMPK expression in renal tissue was decreased, NOX-4 expression and oxidative stress were increased, and urinary microalbumin was increased. 2. PPARγ agonist pioglitazone could reduce urinary protein, play a renal protective role in obese mouse through elevated serum adiponectin levels and the expression of AMPK in renal tissue, then reduce the expression of NOX-4, alliviate the state of oxidative stress. 3.Compared to low-dose PPARγ agonists, high dose PPARγ agonist have more protective effect on the kidneys in obesity.