Effects And Mechanisms Of Angiotensin Receptor-Neprilysin Dual Inhibition On Cardiorenal Syndrome Via Regulating TGF-?/Smad Signaling Pathway

Objective:Cardiorenal syndrome?CRS?is an important clinical condition leading to significant morbidity and mortality,placing a significant burden on healthcare systems.Its pathogenesis is complex,involving activation of the renin-angiotensin system?RAS?of both the heart and the kidneys.Natriuretic peptide system?NPS?has the functions of dilating blood vessels,natriuretic diuresis,inhibiting renin angiotensin system and sympathetic nervous system.Angiotensin receptor-neprilysin dual inhibition could suppress renin angiotensin system and activate natriuretic peptide system by blocking angiotensin??Ang??signal transduction and reducing natriuretic peptide decomposition to enhance the activity of natriuretic peptide.This study aims to illustrate the effects of angiotensin receptor-neprilysin dual inhibition on mouse model with cardiorenal syndrome in vivo,and verify the mechanisms of angiotensin receptor-neprilysin dual inhibition on renal tubular epithelial-mesenchymal transition?EMT?in vitro to provide a new therapeutic method and theoretical basis for cardiorenal syndrome.Methods:1.8 weeks old healthy male C57BL/6 mice were randomly divided into control group?CTL,n=8?and model group?MOD,n=24?.Abdominal aortic ligation was performed in model group and sham operation was performed in control group.At 4weeks after the operation,ELISA was used to determine the plasma N-terminal pro B-type natriuretic peptide?NT-proBNP?and urine neutrophil gelatinase-associated lipocalin?NGAL?of mice.Cardiac and renal ultrasonography were performed in mice.After confirming the successful establishment of the mouse model with cardiorenal syndrome,the mice in model group were randomly divided into cardiorenal syndrome group?CRS,n=8?,LCZ696 group?LCZ,n=8?and valsartin group?VAL,n=8?.Mice in LCZ696 group and valsartin group were given with LCZ696(60mg·kg^-1·d^-1)and valsartan(48mg·kg^-1·d^-1)respectively,and mice in control group and cardiorenal syndrome group were given the same amount of corn oil by gavage for 8 weeks.At 12 weeks after the operation,plasma NT-proBNP and urine NGAL of mice were determined by ELISA.Cardiac and renal ultrasonography and hemodynamic examination were performed in mice.Mice were sacrificed,left ventricular tissue and kidney tissue were taken for pathological examination,left ventricular myocardial cells,glomeruli and renal tubules were observed by HE staining,and left ventricular myocardial cell interstitial fibrosis and renal tubule interstitial fibrosis were observed by Masson staining.The protein and mRNA were extracted from kidney tissue of mice respectively.The protein expression levels of NGAL,kidney injury molecule-1?KIM-1?,Ang?,atrial natriuretic peptide?ANP?,transforming growth factor-??TGF-??,Smad2,Smad3,connective tissue growth factor?CTGF?and collagen??Col??in kidney tissue were assessed by immune protein imprinting analysis?Western blotting?.The mRNA expression levels of NGAL,KIM-1,Ang?and ANP in kidney tissue were assessed by reverse transcription-quantitative PCR?RT-qPCR?.2.Rat proximal renal tubular epithelial cells from the NRK-52E line were cultivated and treated with LCZ696 or valsartan,in the presence or absence of Ang II.NRK-52E cells were divided into control group,Ang?stimulation group,Ang?stimulus+LCZ696 intervention group,Ang?stimulus+valsartan intervention group,LCZ696 group,valsartan group?n=8 for each group?.After treating for 72 hours,the protein and mRNA were extracted from NRK-52E cells respectively.The protein expression levels of NGAL,KIM-1,TGF-?,Smad2,Smad3,CTGF and Col?in NRK-52E cells were assessed by Western blotting.The mRNA expression levels of NGAL,KIM-1 in NRK-52E cells were assessed by RT-qPCR.Results:1.At 4 weeks after the operation,plasma NT-proBNP and urine NGAL of mice in cardiorenal syndrome group,LCZ696 group,valsartan group were higher than control group?P<0.01?.Ultrasound examination showed interventricular septum thickness?IVST?,left ventricular posterior wall thickness?LVPWT?,left ventricular end-diastolic diameter?LVDd?,left ventricular isovolumic relaxation time?IVRT?of mice in cardiorenal syndrome group,LCZ696 group,valsartan group were higher than control group,renal blood flow velocity time integral?VTI?of them were less than control group?P<0.05?.At 12 weeks after the operation,plasma NT-proBNP and urine NGAL of mice in LCZ696 group decreased?P<0.01?.Ultrasound examination showed IVST,LVDd,IVRT and E peak deceleration time?EDT?of mice in LCZ696group decreased?P<0.05?,the kidney length?RL?of them were higher than those of cardiorenal syndrome group and their kidney blood flow VTI increased?P<0.05?.Histopathologic studies revealed left ventricular myocardial cell hypertrophy,myocardial interstitial fibrosis,higher myocardial cell cross sectional area?CSA?and myocardial interstitial fibrosis ratio?FR?,as well as glomerulosclerosis,renal tubulointerstitial fibrosis,higher renal tubulointerstitial fibrosis ratio in mice with cardiorenal syndrome.These abnormalities were significantly prevented by LCZ696and to a lesser extent by valsartan.NGAL,KIM-1,Ang?protein and mRNA expression levels in kidney tissue of LCZ696 group mice were lower than those of cardiorenal syndrome group?P<0.05?.ANP protein and mRNA expression levels in LCZ696 group were higher than those of cardiorenal syndrome group?P<0.05?.Compared with cardiorenal syndrome group,the protein expression levels of TGF-??P<0.05?,Smad2?P<0.01?,Smad3?P<0.05?,CTGF?P<0.01?and Col??P<0.01?in LCZ696 group were decreased significantly.The above effects were superior to those of valsartan group.2.Compared with Ang?stimulation group,LCZ696 downregulated NGAL,KIM-1 protein and mRNA expression levels in renal tubular epithelial cells?P<0.05?,and downregulated TGF-??P<0.01?,Smad2?P<0.05?,Smad3?P<0.05?,CTGF?P<0.01?,Col??P<0.05?protein expression levels.Valsartan downregulated Smad2,Smad3,CTGF protein expression levels?P<0.05?,and had no significant effects on NGAL,KIM-1,TGF-?,Col?protein expression?P>0.05?.Conclusions:Angiotensin receptor-neprilysin dual inhibition could significantly improve the cardiac function,alleviate hypertrophy of ventricular wall,enlargement of cardiac cavity,hypertrophy of cardiac myocytes and interstitial fibrosis,repair renal tubule injury,improve renal size,blood flow,glomerulosclerosis and renal tubulointerstitial fibrosis in mice with cardiorenal syndrome via enhancing the activity of natriuretic peptide system and inhibiting renin angiotensin system.Angiotensin receptor-neprilysin dual inhibition could alleviate renal tubular epithelial-mesenchymal transition,tubular injury and tubular interstitial fibrosis via downregulating the TGF-?/Smad signaling pathway.