Nur77 Defends Renal Fibrosis By Inhibiting Activation Of P-Smad2/3 In Aging Mice

Objective:Chronic kidney disease（CKD）is an important risk factor that endangers the health of the aging people.Renal fibrosis is the common pathological process of various chronic kidney disease progressing to renal failure.Nerve growthfactor-induced gene B（Nur77）belongs to the NR4A subclass of nuclear hormone receptor,which plays an important role in the developing of virious aging-related diseases,but the mechanism of action in senile renal fibrosis is not clear.Therefore,this study explored the protective mechanism of Nur77 in senile renal diseases.Methods:In this study,5 month-old young and 20 month-old naturally aging WT and Nur77^-/-mouse models were fed freely,and metabolic cages were used to collect urine for 24 hours in order to detect urine volume（UV）,urinary albumin（ALB）,and biochemical indicators of blood serum such as blood urea nitrogen（BUN）,serum creatinine,Transforming growth factorβ（TGFβ）were tested with kits;H&E and Masson stainings were used to observe the morphological changes of renal tissues in mice.In vitro experiments at the same time,Nur77 over expression and knockdown stable cell lines were constructed;renal tubular epithelial cells were stimulated 10 ng/m L TGFβfor24 h,Western blot assay was used to analyze the protein expression in HK2 cells and renal tissue,including Smad2/3,phospho-Smad2/3（P-Smad2/3）,Collagen I,alpha-smooth muscle actin-positive（α-SMA）,RT-PCR detected the expression of Collagen I and ACTA2 m RNA level in HK2 cells and renal tissue.Results:The renal function of miced declined with age-related,and the renal function of Nur77^-/-aged mice decreased significantly compared with elderly WT mice at 20month-old（p<0.01）,which ALB,SCR and BUN were increased,and UV,endogenous creatinine clearance rate（CCr）were decreased.H&E and Masson results showed 20month-old elderly Nur77^-/-mice had renal degenerative diseases,glomerular volume increased,Bowman’s sac thickened,renal tubule atrophy,tubular cell swelling,renal tubule injury index increased,and renal fibrosis level increased compared with 5month-old Nur77^-/-mice.Compared with 20 month-old WT mice,20 month-old Nur77^-/-aged mice showed an increased level of P-Smad2/3 in renal tissues（p<0.01）,the change of total Smad2/3 protein was not obvious,the expressions of fibrosis associated protein Collagen I andα-SMA were increased（p<0.01）.After TGFβstimulation on HK2 cells,P-Smad2/3 increased（p<0.01）,the expressions of fibrosis associated protein Collagen I andα-SMA were increased（p<0.01）,the m RNA levels of Collagen I and ACTA2 were also significantly increased（p<0.01）.In conditions of Nur77 knockdown,P-Smad2/3was activated,and the protein and m RNA expressions of Collagen I andα-SMA were more obvious increased（p<0.01）,on the contrary,after the overexpression of Nur77,P-Smad2/3 was inhibited,and the expressions of Collagen I andα-SMA were down-regulated.Conclusion:Our project proved that the elderly mice renal morphology were abnormal and renal function declined;Nur77 knockout accelerated the progression of renal fibrosis in elderly mice.The mechanism of its action is that Nur77 protects senile renal function decline and renal fibrosis by inhibiting TGFβ/Smads signaling pathway.