Experimental Study Of MTOR Inhibitor Delaying The Progression Of Chronic Aristolochic Acid Nephropathy

Significance:Aristolochic acid nephropathy(AAN)is a rapidly progressive renal tubular interstitial disease caused by the ingestion of Chinese herbal medicines or plants containing aristolochic acid(AA)1.A multicenter retrospective cohort study in China showed that the incidence of acute kidney injury(AKI)was 11.6%among nearly 660,000 hospitalized patients.Approximately 40%of AKI cases result from the consumption of drugs,among which 16%are Chinese herbal medicines2.In Southeast Asia and other countries or regions where traditional medicine is popular,AA-containing medicines and plants are still widely used3.Because AAN involves a wide range of regions and populations,there is an urgent need to identify novel therapeutic drugs for AAN.Although it has been reported that steroid hormones can be used to treat AAN4,the effects and mechanisms involved remain unclear.A considerable proportion of patients with AAN progress to end-stage renal disease(ESRD),depending on the replacement therapy employed,expending considerable medical resources.Therefore,it is extremely important to study the mechanisms involved in AAN and find effective measures to prevent and treat this disease.Preliminary studies have found that a variety of kidney injuries,including AAN,exhibit abnormal activation of the mammalian target of rapamycin(mTOR)pathway in both the early and advanced disease stages5.mTOR,a serine-threonine protein kinase that belongs to the phosphatidylinositol 3 kinase(PI3K)family,is an effector protein downstream of the PI3K/AKT signaling pathway6 and plays key regulatory roles in the proliferation,survival,differentiation,metabolism,and autophagy of normal and malignant cells.The mTOR signaling pathway plays an important role in the regulation of renal cell fibrosis and apoptosis by activating macrophages,myofibroblasts,and nuclear factor kappa-B(NF-?B),suggesting that blocking or inhibiting the mTOR pathway may delay the progression of renal fibrosis and apoptosis observed in A AN7.Rapamycin is a specific inhibitor of mTOR,a powerful immunosuppressant8.It was found that rapamycin can effectively reduce kidney damage caused by many factors(such as physical obstruction,drugs,and autoimmunity),but its role on AAN has not been reported9-0.Rapamycin can inhibit the cell cycle,block the expression of various cytokines related to cell proliferation,and promote autophagy by inhibiting the mTOR pathway.Autophagy is a process in which a cell engulfs its own cytoplasmic proteins or organelles,followed by their envelopment them into vesicles and fusion of the vesicles with lysosomes to form autophagic lysosomes,resulting in degradation of the encapsulated contents.Autophagy has two-sided effects of "protection" and "injury"on cells.Studies have reported that autophagy is closely related to inflammation,apoptosis,and fibrosis.The activation processes of inflammation,apoptosis,and fibrosis are accompanied by the occurrence and development of AAN,indicating that rapamycin-induced autophagy may participate in the regulation of apoptosis and fibrosis in the progression of AAN,although there are no relevant research reports regarding this phenomenon.This study investigated the therapeutic effect of rapamycin by blocking the mTOR pathway in AAN and the underlying mechanism.In vivo experiments were performed using a murine AAN model.Mice were randomly divided into a control group(Ctrl group),an AAI-treated group(AAI group),and a rapamycin treatment group(RAP group).Renal functions in the mice were evaluated using serum creatinine and serum urea nitrogen levels.Hematoxylin and eosin staining(H&E)and periodic acid Schiff(PAS)staining were used to observe pathological changes in the kidney.The extent of fibrosis,epithelial-mesenchymal transition(EMT),and apoptosis in the three groups was assessed through western blotting,immunohistochemical staining,and TUNEL assays.Autophagy-related proteins were detected to illustrate the possible mechanism underlying the therapeutic effects of rapamycin in AAN.For in vitro experiments,we used renal epithelial cells(HK-2)to construct AAN cell models,which were divided into an untreated control group(Ctrl group),AAI-induced group(AAI group),and rapamycin treatment group(RAP group).Cell Counting Kit-8 and lactate dehydrogenase activity assays were used to evaluate cell proliferation and damage.RT-qPCR,western blotting,and flow cytometry were used to evaluate fibrosis,EMT,and apoptosis,the effect of rapamycin and AA on HK-2 cells were explored.Finally,we detected autophagy using transmission electron microscopy,western blotting,and immunofluorescence.By exploring the mechanisms involved in the therapeutic effects of rapamycin in AAN cell models,we hope to shed light on the treatment of AAN.Purpose:1.To observe changes in the pathological structure of the kidneys of the AAN mouse model and the effects of rapamycin treatment;2.To observe the damage induced by AA to renal tubular epithelial cells and the therapeutic effect of rapamycin on reversing the damage;3.To detect the effects of AA and rapamycin on mouse kidneys and renal tubular epithelial cells(HK-2 cells),especially the expression of cytokines related to epithelial cell fibrosis,EMT,and apoptosis.4.To detect cellular autophagy levels in mice and HK-2 cells injured by AAI to explore the role of autophagy in the murine AAN model and using rapamycin.Methods:1.Animal experiments:? Observation of clinical indicators of AAN in mice in each groupMouse models of AAN were developed and randomly divided into three groups:a normal control group(Ctrl group),an AAI-induced nephropathy group(AAI group),and a rapamycin treatment group(RAP group).After successful modeling,serum creatinine and blood urea nitrogen levels were measured.? Effects of AA and rapamycin on kidney morphology in mice with AAN:H&E and PAS staining of kidney tissues in each group of mouse models was performed to observe gross tissue morphology.? Effects of AA and rapamycin on renal fibrosis,EMT,and apoptosis in AAN mice:The mice were sacrificed after anesthesia,and kidney specimens were collected after 6 weeks.Cell proliferation,EMT,and fibrosis indicators(VEGF,STAT3,STAT6,SMA,PCNA,vimentin,TGF?1,and ECa)were detected by immunohistochemistry.Apoptosis and apoptosis-related proteins(Bcl-2 and Bax)were detected by TUNEL staining and western blotting,respectively.? Effects of AA and rapamycin on autophagy levels in the kidneys of A AN mice:Western blotting was used to detect autophagy markers(P62,Beclin-1,LC3?).2.Cell experiments:? Detection of damage to HK-2 cells by AA:Renal epithelial cell(HK-2)models of AAN were developed and randomly divided into three groups:a normal control group(Ctrl group),an AA-induced nephropathy group group(AAI group),and a rapamycin treatment group(RAP group).After successful modeling,CCK-8 assays were used to detect cellular proliferation levels,and ELISA was used to detect lactate dehydrogenase abundance.? Effects of AA and rapamycin on levels of fibrosis,EMT,and apoptosis of HK-2 cellsRT-qPCR and western blotting were used to detect EMT and fibrosis indicators(VEGF,STAT3,STAT6,SMA,PCNA,vimentin,TGF?1,and ECa)and apoptosis-related proteins(Bcl-2 and Bax),and cell apoptosis was detected by flow cytometry.? Effect of AA and rapamycin on autophagy levels in the HK-2 cell modelWestern blotting was employed to detect autophagy markers(P62,Beclin-1,and LC3?).Immunofluorescence detection of LC3II and transmission electron microscope observations of autophagosomes were conducted.Results:1.Animal experiments:? Serum urea nitrogen and creatinine levels of mice in the AAI group were significantly increased.The above indicators were increased in the RAP mouse group compared with the Ctrl group but less than that in the AAI group.?In the AAI mouse group,dilation of renal tubules,focal or diffuse infiltration of inflammatory cells,necrosis,and enlargement of the renal interstitium were significantly improved by rapamycin treatment.?mRNA and protein expression levels of VEGF,STAT3,STAT6,SMA,PCNA,vimentin,and TGF?1 in the AAI group were higher than those in the control group.After treatment with rapamycin,the levels of these indicators were decreased.The diminished ECa levels in the AAI group were reversed by rapamycin,suggesting that renal EMT and fibrosis were inhibited to some extent in the RAP group.? The levels of apoptosis and of the apoptotic protein Bax in the AAI group were increased,while that of the anti-apoptotic protein Bcl-2 was decreased.After Rapamycin treatment,the level of apoptosis was reduced.Bax levels in the RAP group decreased compared with that in the AAI group,and Bcl-2 abundance increased compared with that in the AAI group.? Protein levels of autophagy markers(P62,Beclin-1,and LC3II)in the AAI group increased significantly,and their expression further increased after RAP treatment.2.Cell experiments:? The viability of HK-2 cells in the AAI group,detected using CCK-8 assays,was significantly lower than that in the Ctrl group,which was significantly reversed by rapamycin treatment.LDH(L-Lactic Dehydrogenase)levels increased in the AAI group and were improved in the RAP cohort.? The transcription and protein levels of VEGF,STAT3,STAT6,SMA,PCNA,vimentin,and TGF?1 in the AAI group were higher than those in the Ctrl group,while the ECa level in the AAI group was lower than that in the Ctrl group.After treatment of cells with rapamycin,the transcription and protein levels of VEGF,STAT3,STAT6,SMA,PCNA,vimentin,and TGF?1 decreased,while ECa levels were elevated.? The level of apoptosis in the AAI group was higher than that in the Ctrl group,whereas the abundance of the apoptotic protein Bax was increased,and levels of the anti-apoptotic protein Bcl-2 were reduced.After rapamycin treatment,the level of apoptosis was reduced,Bax levels decreased,and Bcl-2 levels increased significantly.?In the AAI group,swollen and deformed mitochondria and endoplasmic reticulum as well as numerous autophagosomes were observed in the cytoplasm,using transmission electron microscopy.A large number of autophagosomes containing undigested and damaged organelles with a clear double-layered membrane structure were clearly observed in the RAP group.? The levels of autophagy markers(P62,Beclin-1,and LC3II)in the AAI group increased significantly,and their expression was further increased after RAP treatment.Conclusions:1.AA caused kidney damage by inducing epithelial cell fibrosis and apoptosis.Rapamycin can treat AAN by reversing fibrosis and apoptosis.2.Both AA and rapamycin induced autophagy in renal tubular epithelial cells.Autophagy induced by AA may be protective against damage.Autophagy induced by rapamycin enhances this protective effect by inhibiting renal epithelial cell fibrosis and apoptosis and plays a role in the treatment of AAN.The innovation of this research:Chronic kidney disease caused by consumption of traditional Chinese herbal medicines containing AA is commonly encountered in clinical practice,and the irreversibility of kidney damage has led to several challenges.There is no relevant research on the treatment of AAN by rapamycin,and there are few reports on the effects of autophagy on AAN.In this study,the mTOR inhibitor rapamycin was used in an AAN mouse model and AAI-induced HK-2 cell model.The effects of rapamycin on AAN were studied in vivo and in vitro.The role of this drug in AAN and the exploration of possible mechanisms and related pathways are innovative and would be of great significance in the treatment of AAN and the protection of residual renal function.This research provides a theoretical basis and offers new ideas for the development and clinical application of this drug.