Effect And Mechanism Of LCZ696 On Renal Interstitial Fibrosis In UUO Rats

Background:Chronic kidney diseases(CKD)is an irreversible and progressive disease characterized by persistent kidney injury and a significantly increased risk of cardiovascular events.However,therapeutic strategies to prevent or slow the progression of CKD remain limited.LCZ696 has been incorporated into clinical practice guidelines to improve the prognosis of patients with heart failure and has become a milestone for patients with heart failure.Given the complex and close relationship between CKD and heart failure,LCZ696 may be beneficial for the treatment of CKD.Renal interstitial fibrosis is the common pathway and the main pathological basis for the progression of multiple renal diseases to end-stage renal failure,but unilateral ureteral obstruction,UUO model is a mature experimental animal model for the study of renal tubulointerstitial fibrosis.Recent studies at home and abroad have shown that LCZ696 can improve diabetic cardiomyopathy and subtotal nephrectomy rats by inhibiting inflammation,oxidative stress,improving fibrosis and inhibiting apoptosis,and this effect is considered to be related to the regulation of MAPK signaling pathway.LCZ696 has not been reported on UUO rat model at home and abroad.Objective:This study aims to jointly explore the effect of angiotensin receptor neprilysin inhibitor(ARNI)LCZ696 on renal fibrosis of UUO rats through UUO rat renal fibrosis animal model and in vitro cytology experiment.In addition,ASK1/JNK/p38 MAPK signaling pathway was combined to further explore its mechanism,in order to provide a new strategy for the prevention and treatment of renal fibrosis.Methods:Part Ⅰ:The effect of LCZ696 on renal interstitial fibrosis in UUO ratsIn vivo experiment 1:1.Establishment of UUO model:30 SPF male Sprague-Dawley rats(8 weeks old,weight 240～250g)were randomly divided into 5 groups(n=6).(1)sham operation group(sham group):the left ureter was exposed and the skin was sutured again;(2)UUO group:left ureteral ligation for 7 days;(3)UUO+LCZ group:the left ureter was ligated and LCZ696(68mg/kg/day)was given by gavage for 7 days;(4)UUO+valsartan(VAL)group:the left ureter was ligated and VAL(31mg/kg/day)was given by gavage for 7 days;(5)UUO+GS group:the left ureter was ligated and GS-444217[a selective ATP-competitive apoptosis signaling regulated kinase 1(ASK1)inhibitor](30mg/kg/day)was injected intraperitoneally for 7 days.Administration in each treatment group began on the day of surgery and continued for 7 consecutive days,with the last administration on the day before kidney removal.2.Biochemical examination:blood samples were collected from the right internal jugular vein in all experimental groups during renal extraction,and serum creatinine,blood urea nitrogen and cystatin C were detected by automatic biochemical analyzer.3.Pathological examination:(1)Kidney tissue samples were collected,paraffin-embedded and sliced thick(4μm),and the pathological changes of kidney tissue in each experimental group were observed by HE staining.(2)Masson staining was used to observe the degree of tubulointerstitial fibrosis(TIF).(3)Ultrastructural changes of renal tissue were observed by transmission electron microscopy.4.Western blotting was used to detect the expression levels of fibrosis-related proteins TGF-β1 and CTGF in the renal tissues of rats in each experimental group.In vitro experiment 1:1.The in vitro model of renal interstitial fibrosis was established by HK-2 cells induced by TGF-β1.The expression of α-SMA and CTGF related proteins in renal interstitial fibrosis was detected by western blot and the morphology of HK-2 cells was observed by electron microscope to determine the optimal concentration of HK-2 cell fibrosis induced by TGF-β1.The concentration gradients of 5μM,10μM and 15μM were set.2.He determined concentration of TGF-β1 induced HK-2 cell fibrosis was determined as the modeling concentration,and LCZ696,VAL and GS-444217 were added according to experimental groups for 1h pre-treatment,and then co-incubated with TGF-β1 for 36h.The expression of α-SMA and CTGF proteins in HK-2 cells was detected by western blotting to evaluate the effect of LCZ696 on the renal interstitium fibrosis model of HK-2 cells induced by TGF-β1 in vitro.Part Ⅱ:Effects of LCZ696 on inflammation,oxidative stress,mitochondrial dysfunction and endoplasmic reticulum stress in UOO ratsIn vivo experiment 2:1.Biochemical examination:The 24-hour urine of rats in each group was collected the day before renal removal,and the level of 8-OHdG in urine of rats in each experimental group was detected by enzyme-linked immunosorbent assay(ELISA).2.Pathological examination:(1)ultrastructural changes of renal tissue were observed by transmission electron microscopy;(2)The specific marker antigen ectodermal dysplasial(ED-1)on the surface of monocytes/macrophages was detected by immunohistochemistry in the kidney tissues of rats in each experimental group.3.Western blotting was used to detect the expression levels of related proteins in the renal tissues of rats of all experimental groups,including pyrodeath related proteins,mitochondrial regulatory proteins,endoplasmic reticulum stress-related proteins and oxidative stress-related proteins.In vitro experiment 2:1.Human renal proximal tubule epithelial cells(HK-2)were cultured in DMEM medium containing 10%FBS(fetal bovine serum)and added 1%P-S(double antibody:penicillin 100μg/mL+streptomycin 100 μg/mL)in an incubator at 37℃ and 5%CO2.The experiment was divided into(1)control group(CON):the same volume of DMSO;(2)Module(H2O2 group):400μmol/L H2O2;(3)H2O2+LCZ group:400μmol/L H2O2+20μmol/L LCZ696;(4)H2O2+VAL group:400μmol/L H2O2+20μmol/L VAL;(5)H2O2+GS group:400μmol/LH2O2+16μmol/L GS-444217.2.The use of dichloro dihydrogen fluorescein double acetate(dichlorodihydrofluorescein diacetate,DCFH-DA)fluorescent probes and flow cytometry technology testing in vitro experiment by using the experimental group HK-2 intracellular reactive oxygen species(ROS)is produced.3.MitoSOX-red fluorescent probe was used to detect mitochondrial reactive oxygen species(mtROS)generation in HK-2 cells of each experimental group by flow cytometry.4.Cellular oxygen consumption(OCR)was measured by Seahorse XFp,an extracellular flux analyzer.Part Ⅲ:LCZ696 protects renal fibrosis in UUO rats by inhibiting apoptosis mediated by ASK1/JNK/p38MAPK signaling pathwayIn vivo experiment 3:1.Pathological examination:(1)TUNEL staining was used to observe the apoptosis of rat kidney tissue in each experimental group;(2)Immunohistochemical techniques were used to detect the expression of phospho-c-Jun N-terminal kinase(p-JNK)and p-p38 in renal tissues of rats in all experimental groups.2.Western blotting was used to detect the expression levels of related proteins in rat kidney tissues of each experimental group.Expression levels of apoptosis-related proteins(Bcl-2,Bax)and mitogen-activated protein kinase(MAPK)signaling pathway(p-ASK1,p-MEK3,p-MEK4,p-JNK,p-p38).In vitro experiment 3:1.CCK-8 reagent was used to measure the light absorption value at the wavelength of 450nm by enzyma-linked immunoassay,and the number of HK-2 cells in each experimental group was detected indirectly in vitro.2.Annexin V-FITC/PI staining was used,and flow cytometry was used to detect the apoptosis of HK-2 cells in each experimental group in vitro.3.The expression levels of apoptosis-related proteins(Bcl-2,Bax)and MAPK signaling pathway related proteins(p-ASK1,p-MEK3,p-MEK4,p-JNK,p-p38)in HK-2 cells of each experimental group were detected by western blotting.Results:PartⅠ1.There were no significant changes in renal function indexes such as serum creatinine,blood urea nitrogen and cystatin C in all experimental groups.2.Masson and HE staining results of UUO rat renal tissue showed tubular vacuolation,tubular atrophy,interstitial collagen deposition,and tubule interstitial expansion and fibrosis.The results of Masson staining and HE staining of UUO rat renal tissue treated with LCZ696 or VAL or GS-444217(selective ASK1 inhibitors)showed that renal tubule vacuolar atrophy,renal interstitial collagen deposition,tubule interstitial dilation,fibrosis and other morphological changes were improved,and these changes were confirmed by transmission electron microscopy.3.Western blot results showed that the expression levels of TGF-β1 and CTGF protein in kidney tissue of UUO rats were significantly increased,and treatment with LCZ696 or VAL or GS-444217(selective ASK1 inhibitors)could reduce the expression levels of TGF-β1 and CTGF protein;TGF-β1 induced HK-2 cells to establish an in vitro model of renal interstitial fibrosis.The expression of a-SMA and CTGF in the extracellular matrix and myofibroblasts induced by TGF-β1-induced HK-2 cells treated with LCZ696 or VAL or GS-444217(selective ASK1 inhibitor)decreased.LCZ696 can improve renal injury and renal interstitial fibrosis in UUO rats,and the improvement effect is stronger than VAL.Part Ⅱ1.Immunohistochemical results showed that ED-1 positive cell infiltration in renal tubule interstitial was significantly increased in UUO group,and ED-1 positive cell infiltration was significantly decreased after treatment with LCZ696 or VAL or GS-444217(selective ASK1 inhibitors).A large number of inflammatory cell infiltration was observed in the renal interstitial of rats in UUO group by transmission electron microscope,and the inflammatory cell infiltration was reduced after treatment with LCZ696 or VAL or GS-444217.2.Western blot assay showed that treatment with LCZ696 or VAL or GS-444217(selective ASK1 inhibitors)inhibited the expression of pro-inflammatory cytokines(pro-IL-1β,pro-IL-18,NLRP3,and TNF-α).The results showed that LCZ696 had a stronger inhibitory effect on renal tissue inflammation in UUO rats than VAL.3.ELISA was used to detect the level of 8-OHdG in the urine of rats in all experimental groups.The results showed that the level of 8-OHdG in the urine of rats in the UUO group was significantly increased,and the treatment with LCZ696 could reduce the level of 8-OHdG in the urine of UUO rats.4.Western blot test results showed that compared with Sham group,the expression of antioxidant enzymes MnSOD and TRX protein decreased significantly in UUO group,while the expression of iNOS and TXNIP increased.After treatment with LCZ696 or VAL or GS-444217(selective ASK1 inhibitors),the expression of antioxidant enzymes MnSOD and TRX protein increased,while the expression of oxidase iNOS and TXNIP decreased.5.Flow cytometry showed that HK-2 intracellular reactive oxygen species(ROS)and mitochondrial reactive oxygen species(mtROS)induced by H2O2 significantly increased HK-2 intracellular reactive oxygen species(ROS)and mitochondrial reactive oxygen species(mtROS).ROS and mtROS in HK-2 cells decreased after treatment with LCZ696 or VAL or GS-444217(selective ASK1 inhibitors),indicating that LCZ696 can improve oxidative stress.6.The results of in vivo observation of rat renal tissue of each experimental group by transmission electron microscope showed that the mitochondrial structural destruction in UUO group was manifested as mitochondrial volume reduction,mitochondrial number reduction,ridge breakage,focal vacuolation,endoplasmic reticulum destruction was also obvious,trachymic reticulum ribosome stripping and trachymic reticulum cisterna expansion.These changes were improved after treatment with LCZ696 or VAL or GS-444217(selective ASK1 inhibitors).7.Western blot results showed that treatment with LCZ696 or VAL or GS-444217(selective ASK1 inhibitors)could balance the expression imbalance of mitochondrial regulatory proteins(SDHA and Parkin)and ER stress-related proteins(CHOP and IRE-1α)in UUO rats.8.The detection of cell oxygen consumption rate(OCR)showed that LCZ696 or VAL or GS-444217(selective ASK1 inhibitors)could improve mitochondrial respiratory dysfunction,and LCZ696 could alleviate mitochondrial damage and endoplasmal reticulum stress in renal tissue of UUO rats.Part Ⅲ1.TUNEL staining was used to detect apoptotic cells in renal tissue of rats of all experimental groups in vivo.Apoptotic bodies of TUNEL-positive cells were mainly located in the renal tubular epithelial cells and interstitial fibrotic regions,and the apoptotic cells showed dense nuclear chromatin and concentrated cytoplasm.Treatment with LCZ696 or VAL or GS-444217(selective ASK1 inhibitors)reduced the number of TUNEL-positive cells in kidney tissue of UUO rats.2.Annexin V-FITC/PI staining was used to detect the apoptosis level of HK-2 cells in each experimental group in vitro experiment,and the results showed that the number of apoptotic cells in the H2O2-induced HK-2 cell apoptosis model increased significantly.The increase in apoptosis of HK-2 cells was reversed after treatment with LCZ696 or VAL or GS-444217(selective ASK1 inhibitors).In addition,the viability of HK-2 cells was detected by CCK8 kit,and the number of HK-2 cells induced by H2O2 increased significantly after treatment with LCZ696 or GS-444217.3.The expression of apoptosis-related protein Bcl-2/Bax was detected by western blotting,and the expression ratio of apoptosis-related protein Bcl-2/Bax decreased significantly in UUO group compared with Sham group.After treatment with LCZ696 or VAL or GS-444217(selective ASK1 inhibitors),the expression ratio of apoptosis-related protein Bcl-2/Bax in renal tissue of UUO rats increased.The results of western blot detection of apoptosis-related protein Bcl-2/Bax expression on H2O2-induced HK-2 cell apoptosis model were consistent with the results of in vivo experiments,indicating that LCZ696 has an inhibitory effect on cell apoptosis,and its mechanism of action is related to the inhibition of ASK1.4.Immunohistochemical detection of the major components of ASK1/JNK/p38MAPK signaling pathway in kidney tissue of UUO rats showed that compared with Sham group,the expression of p-p38 and p-JNK proteins in renal interstitial of UUO rats was significantly increased.After treatment with LCZ696 or VAL or GS-444217(selective ASK1 inhibitors),the expression levels of p-p38 and p-JNK protein in rat renal interstitial were decreased.5.Western blot results showed that the establishment of UUO model or H2O2-induced HK-2 cell apoptosis model could activate the expression of ASK1 and downstream MAPK-related proteins,such as MEK3/MEK4 and JNK/p38,while LCZ696 or GS-444217 inhibited their protein expression.Conclusion:1.LCZ696 attenuates renal fibrosis caused by UUO.2.LCZ696 inhibits inflammation,oxidative stress,mitochondrial damage,and endoplasmic reticulum stress caused by UUO.3.LCZ696 decreases apoptotic cell death caused by UUO.4.LCZ696 attenuates renal fibrosis caused by UUO probably through interfering with ASK1/JNK/p38 MAPK signaling pathway mediated apoptotic cell death.