Protective Role And Mechanism Of Adiponectin In Renal Injury Induced By Combining DOCA Salt And Angiotensin ?

Objective: Chronic kidney disease(CKD)is associated with a major public burden and its prevalence is rising throughout the world.Nowadays,the treatment of chronic kidney disease is still very limited,It is necessary to develop new treatment strategies to delay the progress of chronic kidney disease.Renal fibrosis is a final common pathway for the development from a variety of chronic kidney disease to renal failure.Progressive kidney disease is characterized by reduction in the number of podocytes and accumulation of extracellular matrix.Among many risk factors,Angiotensin II(Ang II),the main peptide of the renin angiotensin system,plays a crucial role in renal fibrosis.Ang II injuries podocytes,activates kidney mesangial cells and interstitial fibroblasts,recruits inflammatory cells,enhances matrix protein synthesis and increases proteinuria.Adiponectin(Ap N),an adipokine secreted by adipocytes,has been the recent research focus due to its insulin-sensitizing,anti-inflammatory and vasoprotective properties.Epidemiological studies have shown that Ap N is down-regulated in obesity and diabetes.High,rather than low,concentrations of Ap N are unexpectedly found in patients with CKD via an as yet unknown mechanism.Animal studies have supported a protective role of Ap N in the 5/6 nephrectomized model.Ap N knockout mice display exacerbation of albuminuria,glomerular hypertrophy,markers of oxidative stress,podocyte foot effacement and tubulointerstitial fibrosis in diabetes or a subtotal renal ablation model of progressive CKD,which are reversed by Ap N treatment.Over-expression of Ap N using adenovirus-mediated gene transfer alleviated the progression of proteinuria in streptozotocin-induced diabetic rats.Our recent data further shows that treatment with recombinant Ap N peptide reduced the increases in albuminuria and markers of glomerulosclerosis seen in db/db mice.This antifibrotic effect of Ap N is new and beyond its glucose-lowering and insulin-sensitizing actions and probably through its anti-inflammatory and angiotensin-antagonistic effects since human Ap N obliterates the stimulatory effects of Ang II and TGF-? on the expression of profibrotic markers in cultured glomerular mesangial cell.However,the inhibitory effect of Ap N on Ang IIinduced kidney injury in vivo and the underlying mechanism involved in action of Ap N has not been well explored.According to this findings,we hypothesized that Ap N may ameliorate deoxycorticosterone acetate(DOCA)-salt and Ang IIinduced kidney injury in vivo,and could be a promising new therapeutic candidate for the chronic kidney disease.To validate this hypothesis,we performed the following experiments by establishing an inducible human full-length Ap N transgenic mice and developed the animal model of DOCA-salt and Ang IIinduced kidney injury.We testified that whether Ap N could ameliorate DOCA-salt and Ang IIinduced renal injury to explore its underlying mechanism.Methods:1.The target gene is obtained by DNA recombination in vitro,which was excised,purified and introduced into C57BL/6J mouse single-cell embryos by micro-injection.Two positive female founders were identified by PCR.The founders with the transgenic expression in liver origin were breed with wild type C57BL/6J mice.The subsequent positive transgenic F1 mice were used to continually breed with wild type mice to establish the stable transgenic line.2.Three groups of uninephrectomized(UNX)male mice were assigned at 8-10 weeks of age and treated 1 week later as follows: wild type C57BL/6J mice as wild type(WT),C57BL/6J mice receiving DOCA,Ang II and 1%Na Cl in drinking water as wild type/DCOA+Ang II(WT/DOCA+Ang II),human full-length Ap N transgenic mice treated with 0.15% I3 C receiving DOCA,Ang II and 1%Na Cl in drinking water as Ap N transgenic/DOCA+Ang II(Ap N-Tg/DOCA+Ang II).All mice survived and were sacrificed at 3 weeks after Ang II infusion.Twenty-four-hour metabolic cage test every week,measure body weight,systolic blood pressure,water intake,urine output,urinary microalbumin/creatinine,urinary albumin excretion.3.Detect the expression of Ap N in liver,fat and serum in the Ap N transgenic mice induced by I3 C by western blot.The markers of renal fibrosis: renal expressions of PAI-1,FN,?-SMA,markers of renal inflammation: NF-?B-p65,p ERK1/2,oxidative stress indicators: NOX2,p47 phox and markers of podocyte injury B7-1 by western blot.4.The renal expression of TGF-?1,FN,COL I(COL IV),COL IV(COL I),megalin,cubilin,KIM-1,NAGL in different groups were detected by real-time reverse transcription-ploymerase chain reaction(real-time RT-PCR).5.Detect the renal deposition of Ap N,FN,podocin,nephrin,WT-1,Ki-67,F4/80 in different groups by Immunofluorescence method.6.The changes of glomerular mesangial proliferation in different groups were detected by periodic acid-shiff's reaction(PAS).7.The changes of tubulointerstitial fibrosis in different groups were detected by Masson's Trichrome.8.The changes of podocytes in different groups were observed by electron microscope.Results: 1.Construct liver specific expression vector p BSskplus-cyp1a1-human Ap N,generate the inducible human full-length Ap N transgenic mice.2.WT,WT/DOCA+Ang II,Ap N-Tg/DOCA+Ang II have no significant difference in body weight.Compared with WT group,the blood pressure,drinking water,urine output,urinary microalbumin/creatinine,urinary albumin excretion of the two groups of WT/DOCA+Ang II and Ap N-Tg/DOCA+Ang II group are significantly elevated.Compared with WT/DOCA+Ang II group,Ap N-Tg/DOCA+Ang II group have no significant difference in the blood pressure,drinking water,urine output.Compared with WT/DOCA+Ang II group,urinary microalbumin/creatinine,urinary albumin excretion of the Ap N-Tg/DOCA+Ang II group are significantly increased.3.Western blot analysis shows that 3.1 compared with WT+0.3%I3C group,expression of Ap N in liver in the two groups of Ap N-Tg+0.15% I3 C and Ap N-Tg+0.3% I3 C is significantly increased.The expression of Ap N in liver in the two groups of Ap N-Tg+0.15% I3 C group and Ap N-Tg+0.3% I3 C group has no significant difference.3.2 Compared with WT+0.3%I3C group,serum expression of Ap N in the two groups of Ap N-Tg+0.15% I3 C and Ap N-Tg+0.3% I3 C is significantly increased.Compared with Ap N-Tg+0.15% I3 C group,serum expression of Ap N in Ap N-Tg+0.3% I3 C group is significantly elevated.3.3 Three groups of WT+0.3%I3C,Ap N-Tg+0.15% I3 C,Ap N-Tg+0.3% I3 C show no significant difference in Ap N expression in adipose tissue.3.4 Compared with WT group,the serum expression of Ap N in WT/DOCA+Ang II group has no significant difference.Compared with WT and WT/DOCA+Ang II group,the serum expression of Ap N in Ap N-Tg/DOCA+Ang II group is significantly increased.3.5 Compared with WT group,the expression of PAI-1,FN,?-SMA,B7-1,p NF-?b-p65,NOX2 and p47 phox,p ERK1/2 in the renal cortex of WT/DOCA+Ang II group is significantly elevated.Compared with WT/DOCA+Ang II group,the expression of PAI-1,FN,?-SMA,B7-1,p NF-?B-p65,NOX2,p47 phox and p ERK1/2 in the renal cortex of Ap N-Tg/DOCA+Ang II group is significantly decreased.Compared with WT group,the expression of PAI-1,B7-1,p NF-?B-p65 is significantly decreased,the expression of FN,?-SMA,NOX2,p ERK1/2 is significantly elevated,the expression of p47 phox has no significant difference in the renal cortex of Ap N-Tg/DOCA+Ang II group.3.6 Compared with WT/DOCA+AII group,urine albumin in Ap N-Tg/DOCA+Ang II group is significantly decreased.4.Real-time RT-PCR result shows that 4.1 compared with WT group,renal cortex TGF-?1,FN,COL IV and COL I m RNA levels are markedly increased in WT/DOCA+Ang II and Ap N-Tg/DOCA+Ang II group.Compared with WT/DOCA+Ang II group,renal cortex TGF-?1,FN,COL IV,COL I,KIN-1,NGAL m RNA levels are significantly reduced in Ap N-Tg/DOCA+Ang II mice.4.2 Compared with WT group,renal cortex cubilin and megalin m RNA level is markedly decreased in WT/DOCA+Ang II and Ap N-Tg/DOCA+Ang II group.Compared with WT/DOCA+Ang II group,renal cortex cubilin and megalin m RNA levels are significantly increased in Ap N-Tg/DOCA+Ang II mice.5.Immunofluorescent staining result shows that 5.1 compared with WT/DOCA+Ang II group,Ap N staining can be detected in the Ap N-Tg/DOCA+Ang II group.5.2 Compared with WT group,the staining of FN in the renal cortex of WT/DOCA+Ang II group is significantly elevated.Compared with WT/DOCA+Ang II group,the staining of FN in the renal cortex of Ap N-Tg/DOCA+Ang II group is significantly reduced.Compared with WT group,the staining of FN in the renal cortex of Ap N-Tg/DOCA+Ang II group has no significant difference.5.3 Compared with WT group,the expression of nephrin,podocin and WT-1 in the renal cortex of WT/DOCA+Ang II group is significantly decreased.Compared withWT/DOCA+Ang II group,the expression of nephrin,podocin and WT-1 in the renal cortex of Ap N-Tg/DOCA+Ang II group is significantly increased.Compared with WT group,the expression of nephrin,podocin and WT-1 in the renal cortex of Ap N-Tg/DOCA+Ang II group has no significant difference.5.4 Compared with WT group,the expression of Ki-67 and F4/80 in the renal cortex of WT/DOCA+Ang II group and Ap N-Tg/DOCA+Ang II group is significantly increased.Compared with WT/DOCA+Ang II group,the expression of Ki-67 and F4/80 in the renal cortex of Ap N-Tg/DOCA+Ang II group is significantly decreased.6.PAS result shows that compared with WT group,accumulation of PAS-positive extracellular matrix in the glomeruli of WT/DOCA+Ang II and Ap N-Tg/DOCA+Ang II group is significantly increased.Compared with WT/DOCA+Ang II group,accumulation of PAS-positive extracellular matrix in the glomeruli of Ap N-Tg/DOCA+Ang II group is significantly decreased.7.Masson's Trichrome result shows that compared with WT group,tubulointerstitial fibrosis of WT/DOCA+Ang II and Ap N-Tg/DOCA+Ang II group is significantly increased.Compared with WT/DOCA+Ang II group,tubulointerstitial fibrosis of Ap N-Tg/DOCA+Ang II group is significantly decreased.8.Transmission electronic microscopy result shows that compared with WT group,podocyte foot process effacement is significant in WT/DOCA+Ang II.Compared with WT/DOCA+Ang II group,podocyte foot process effacement in Ap N-Tg/DOCA+Ang II group is apparently improved.Compared with WT group,podocyte foot process effacement in Ap N-Tg/DOCA+Ang II group has no significant difference.Conclusion: 1.Generate the inducible human full-length Ap N transgenic mice.Ap N was expressed in liver,secreted into the serum,and has no effect on the Ap N expression in fat tissue in the transgenic mice.2.Construct the animal model of DOCA-salt and Ang II induced renal injury,Ap N ameliorates proteinuria,urinary microalbumin/creatinine,urinary albumin excretion in the mice induced by DOCA-salt and Ang II infusion,but has no effect on the blood pressure,body weight,drinking water and urine output.3.Ap N ameliorates the markers of renal fibrosis,podocyte injury,oxidative stress and inflammation in the mice induce by the DOCA-salt and Ang II infusion.