| Background:Chronic heart failure（CHF）occurs as a result of a variety of cardiovascular diseases and the most common being is coronary heart disease,especially myocardial infarction.The prognosis of CHF is poor.Data show that the overall survival rate is even worse than malignant tumors.Life quality of patients decreased year by year and medical costs gradually increased,placing a really heavy medical burden to our society.Water retention is an important pathophysiological mechanism of heart failure.Therefore,diuretic is the cornerstone of standardized treatment of heart failure.The most commonly used diuretic in clinic is loop diuretics,it can specifically inhibit the distribution of medullary loop in the luminal membrane side of the Na+-K+-2CI--co-transporter,and suppress reabsorption of NaCl,resulting in a strong diuretic effect.However,hyponatremia is a risk factor for long-term prognosis of heart failure,and diuretic resistance is a common clinical problem.Therefore,finding of new diuretic targets and development of safe and effective diuretic drugs should be the main research directions of diuretic treatment on CHF patients.Aquaporin 2（AQP2）is distributed in the luminal membrane and intracellular vesicles of the principal cells of renal collecting duct and function as a key protein to regulate the water permeability.It mainly undertakes the reabsorption of water and maintains the concentration of urine.Therefore,upregulation of AQP2 in the luminal membrane will increase water reabsorption which is the most important pathophysiology in heart failure patients.Thus,new diuretic drugs developed for AQP2 regulatory pathway have many advantages and broad clinical applications.Obestatin is a kind of 23 amino acid polypeptide discovered by Dr.Zhang Jian of Stanford University in 2005,which is modified by amidation to become a bioactive form.It has a wide range of roles on the body’s appetite,body weight regulation,glycolipid metabolism and vascular endothelial function regulation.The central obestatin can inhibit the intake of water by acting directly on the thirst center,and reduce the water reabsorption of kidneys by inhibiting the synthesis and release of arginine vasopressin（AVP）.It was found that the obestatin level of peripheral circulation is increased in CHF.Thus it is worthy for further study whether the elevated obestatin in peripheral circulation has the regulatory effect on renal water reabsorption since peripheral circulating obestatin can not enter the nerve centre through the blood-brain barrier.Objective:Up to now,relationship between obestatin and AQP2 is rarely reported in the mechanism research of CHF.The aim of this study is to observe the long-term regulation of obestatin on the inner medullary AQP2 by in vivo and in vitro experiments,and predict the potential regulatory mechanisms by screening for differentially expressed genes through gene chip screening.Further molecular research is performed via shRNA technique in vitro.Methods:A four combined holder for vein puncture and new anti-backflow positioning evaluation device for orotracheal intubation were originally designed and utilized in the pre-experiment.The rat model of acute myocardial infarction was established by thoracotomy and ligating the left anterior descending（LAD）artery.After 6weeks,chronic heart failure models were selected by echocardiography with a standard of EF value less than 45%.Then heart failure rats were randomly divided into 7 groups:model control group,dDAVP group,V2 receptor antagonist group,high dose obestatin group,low dose obestatin group,NA-obestatin group and obestatin anti-serum group.Meanwhile,sham operation group were served as normal control.The rats were injected through tail vein with physiological saline,dDAVP,OPC31260,obestatin（200ug/kg）,obestatin（100ug/kg）,NA-obestatin and obestatin anti-serum respectively for 14 days.All rats were then sacrificed and kidneys were harvested and preserved in 4%paraformaldehyde or-80℃refrigerator.Urine volume and weight were measured periodically.In addition,echocardiography data,as well as blood and urine samples were collected before and after drugs intervention.The concentration of BNP and AVP in plasma were detected by ELISA.The expression of Aqp2 gene was detected by real-time quantitative PCR.And the expression of AQP2 protein in renal inner medulla was detected by immunohistochemistry and immunoblotting.Mouse IMCD3 cells were cultured in vitro,and were incubated for 12h in serum-free medium after cells density grown up to 70%fusion.Cells were then stimulated with obestatin(10-7mmol/L)for different time（0h,6h,12h,24h,36h and 48h）.On the other hand,cells were separately stimulated with PBS,obestatin(10-7mmol/L),obestatin(0.5×10-7mmol/L),NA-obestatin(10-7mmol/L),dDAVP(10-7mmol/L),OPC31260(10-7mmol/L),obestatin anti-serum（10μg/mL）for24h.The mRNA level and protein level of AQP2 were quantified by real-time quantitative PCR and western blotting independently.Microarrays（Gene chip）were performed to screen the differentially expressed genes of mIMCD3 cells treated with obestatin or PBS respectively.Meanwhile,potential regulatory mechanisms were predicted by bioinformatics analysis,seventy-three genes of interest were selected further for real-time qPCR verification.Three target genes,Pparg,V2r and Gpr39,which are closely related to water metabolism,were specifically knocked down via RNA interference,and the changes of V2R,PPARG and AQP2 expression regulated by obestatin were observed.Results:CHF models were successfully established in 51.16%（44/86）rats after myocardial infarction.No significant differences in LVIDd,LVIDs,LVVold,LVVols,EF%,FS%and other cardiac function indexes were observed among the modeling groups.Compared with before treatment,24h urine output increased significantly in the high dose obestatin group（75.18±8.51 vs.40.77±5.49 mL/kg,P<0.001）.Compared with the control modeling group,the plasma BNP concentration decreased significantly（1152.61±99.31,14.09±0.66 vs.1504.54±85.62 pg/mL,P<0.001）,whereas no significant statistical difference in the plasma AVP.Real-time qPCR and western blotting showed that the expression of AQP2 gene and protein in the renal inner medulla were also decreased significantly.After treating with obestatin for different durations in vitro,the expression level of AQP2 gene and protein in mIMCD3 cells showed similar trends.Compared with the control group（0h）,AQP2 expression decreased significantly at 6h,12h,24h,36h and 48h time point（P<0.05）.During 6h to 24h,the downward trend was obvious,but after 36h,it rebounded.After treating with different stimuli over 24h,the expression of AQP2 gene and protein significantly decreased（P<0.05）in high dose obestatin group,low dose obestatin group and OPC group compared with model control group.On the other hand,compared with high dose obestatin group,AQP2 expression of anti-serum group increased significantly both in gene and protein level.After the stimulation of obestatin,gene chip screening and the verification by real-time qPCR indicated that the expression of 6 genes in mIMCD3 cells were significantly changed,including Pparg,Aqp2,V2r,Fgf23,Myo1f,Slc8a1,together with FGF signaling pathway,which may be involved in the long-term regulation of renal AQP2.The results of shRNA studies revealed that the degree of AQP2protein downregulation was more remarkable after V2r and Pparg mRNA interference,but the inhibitory effects of AQP2,V2R,and PPARG induced by obestatin treatment were significantly attenuated after Grp39 knockdown.Conclusion:In vivo and in vitro experiments found that obestatin could downregulate the expression of AQP2 in renal inner medulla and the biochemical indexes of heart failure and diuresis were also improved in vivo.Bioinformatics analysis and quantitative PCR test of gene chip screening results found that obestatin could down-regulate the gene expression of AQP2 through V2R and PPARG pathway individually in IMCD3 cells.Moreover,in terms of molecular biological function,Myo1f gene and Slc8a1 gene may be involved in regulation of calmodulin binding and cytoskeletal protein binding.Further research of the genes knowdown suggested that GPR39 was the upstream target of obestatin through which obestatin could downregulate the expression of AQP2 protein via V2R and PPARG dual-pathway. |
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