Application Research Of Urine-derived Stem Cells And Their Exosomes In Chemotherapy Drugs-induced Urinary System Injuries

Background and Objective:The clinical application of chemotherapy drugs is often accompanied by a series of adverse reactions,causing multi-organ and multi-system damages,which seriously affects the curative effect and prognosis of chemotherapy.In the urinary system,chemotherapy drugs-induced injuries are mainly manifested as acute kidney injury(AKI)and hemorrhagic cystitis(HC),representative chemotherapy drugs are cisplatin and cyclophosphamide,respectively.At present,the curative effect of clinical therapy for AKI and HC is limited.Stem cell technology represented by the application of mesenchymal stem cells(MSCs)has brought hope for their treatment.However,there are still obstacles in the application of MSCs in the urinary system.Urine-derived stem cells(USCs)are seed cells more suitable for urinary tract diseases than MSCs.USCs and exosomes secreted from USCs(USCs-Exo)have therapeutic effects in a variety of urinary tract diseases,but their application in chemotherapy drugs-induced urinary system injuries such as AKI and HC has not been explored.The purpose of this study is to explore the effects of USCs and USCs-Exo in the treatment of chemotherapy drugs-induced urinary system injuries,and preliminarily explore their mechanisms,so as to provide a new idea and reference for the clinical treatment of such diseases.Methods:Part 1:To explore the therapeutic effect of USCs on AKI induced by cisplatin in rats and to preliminarily explore its mechanism.Sterile urine samples from 6 healthy men were collected for USCs isolation and culture.The growth curve of USCs was measured by CCK-8 experiment.The surface markers of USCs were detected by flow cytometry and immunofluorescence.The multipotential differentiation ability of USCs was tested by differentiation induction experiments.USCs were transfected with green fluorescent protein(GFP)lentivirus for their tracing.Thirty wild-type female Sprague-Dawley rats were randomly divided into control group,model group and treatment group.AKI was induced in rats by single intraperitoneal injection of cisplatin(5 mg/kg),and 2×10~6 USCs was administered by tail vein injection on the 1st day after establishment of the model.On day 2 and day 4,one rat was sacrificed,and the GFP-labeled USCs in renal tissues were detected under confocal microscope.On day 4,all the rats were sacrificed,and the blood was sampled from eye and renal tissues were taken.Serum creatinine(SCr)and blood urea nitrogen(BUN)levels were measured in rats.The changes of renal histological injury in rats were observed by staining with HE and PAS,and the severity of renal histological injury in rats was assessed by tubular injury score,glomerular injury count and PAS positive area ratio.The ultrastructure of rat kidney was observed by transmission electron microscope.Ki67 immunohistochemistry and TUNEL staining were used to observe the changes of proliferation and apoptosis in renal tissues.The expressions of TNF-?,IL-6,NF-?B,BCL-2,BAX and cleaved caspase-3 in renal tissues were detected by Western blot.Cisplatin with gradient concentrations was used to induce the injury of rat renal epithelial cells(NRK-52E),then the cytotoxicity of cisplatin was detected by CCK-8 experiment,cell cycle was detected by flow cytometry,and appropriate concentrations were selected to induce the subsequent cell damage model.After the cell model was constructed and co-cultured with USCs,CCK-8 experiment was used to detect the changes in proliferation activity of damaged cells,and flow cytometry was used to detect changes in cell cycle and apoptosis ratio.Part 2:To explore the therapeutic effect of USCs-Exo on cystitis induced by cyclophosphamide in mice and to preliminarily explore its mechanism.USCs were cultured in the medium with exosome-free fetal bovine serum,supernatant was collected 2 days later,and USCs-Exo was extracted by ultrafast centrifugation method.Then morphology of exosomes was observed by transmission electron microscope.Particle size analysis was used to detect the particle size distribution of exosomes.Western Blot assay was performed to detect exosome marker proteins.Forty-five wild-type female C57BL/6 mice were randomly divided into control group,model group and treatment group,and injected intraperitoneally with 80 mg/kg cyclophosphamide to induce cystitis in mice by 1 injection every other day,4 times in total.On the first day after the establishment of the model,USCs-Exo suspension containing 100 ug total protein was injected into the tail vein as treatment.On the fourth day,all the mice were killed and their bladders were taken.Histological changes of bladder were observed by HE staining,and the thickness of bladder epithelium and the distance of submucosa were measured by software.Mast cell infiltration in bladder tissues was observed by toluidine blue staining.The proliferation of bladder mucosa was observed by immunohistochemistry with Ki67.The apoptosis of bladder mucosa was observed by TUNEL staining.The ultrastructure of the lumen surface epithelium of bladder mucosa was observed by scanning electron microscope.Expressions of TNF-?,IL-6,IL-10,NOX2 and cleaved caspase-3 in bladder tissues were detected by Western blotting.The changes of bladder urination function were detected by urodynamics test.The inflammatory model of human bladder epithelial cells(SV-HUC-1)was induced by 100 ng/mL lipopolysaccharide(LPS),and the USCs-Exo was labeled with PKH67 staining for detection of the absorption of USCs-Exo by LPS-induced SV-HUC-1 cells.CCK-8 assay was used to detect changes in proliferation activity of damaged SV-HUC-1 cells,and scratch assay was used to detect changes in migration ability of damaged SV-HUC-1 cells.RNA was extracted from USCs-Exo and corresponding USCs,and small RNA sequencing was conducted to explore the possible mechanism.Results:1.The isolated USCs grew exponentially and rapidly.USCs were positive for MSCs-like surface markers CD44,CD73,CD105 and CD133,negative for hematopoietic stem cell surface markers CD31,CD34,CD45,positive for embryonic stem cell surface marker SSEA4,positive for pericyte surface markers CD146,PDGFRB and NG2.Additionally,USCs can be induced into smooth muscle and epithelial cells.After USCs treatment,serum BUN and SCr levels were significantly reduced.Tubular injury score,glomerular injury count and PAS positive area ratio were significantly decreased.The ultrastructure of rat kidney was improved obviously.Proliferation of renal tubular epithelial cells was increased while their apoptosis was reduced.GFP-labeled USCs could be found in renal tissues on day 2 and day 4.The expression levels of TNF-?,IL-6,NF-?B,BAX and cleaved caspase-3 were significantly decreased,and the expression level of BCL-2 was significantly increased.Cisplatin with gradient concentrations can lead to decreased proliferation activity and cell cycle arrest in NRK-52E cells.10?M,20?M were chosen for the subsequent induction.After co-culture with USCs,the proliferation activity of induced NRK-52E cells was significantly increased,cell cycle arrest was improved,and cell apoptosis rate was significantly decreased.2.The USCs-Exo extracted by us showed a typical"cup-dish"shape under electron microscope.The particle size was distributed at 80~200 nm.Exosomal marker proteins CD9and CD63 were expressed.After USCs-Exo treatment,the thickness of the bladder mucosa epithelium and the submucosa distance were decreased.Mast cell infiltration was obviously decreased.The proliferation rate and apoptosis rate of mucosal epithelium were both decreased.The ultrastructure of the upper skin of bladder was improved obviously.The expression levels of TNF-?,IL-6,NOX2 and cleaved caspase-3 were decreased,while the expression level of IL-10 was increased.The interval of urination was prolonged and the maximum urination pressure was decreased.The LPS-induced SV-HUC-1 cells could absorb USCs-Exo,and the proliferation activity and migration ability were enhanced after co-culture with USCs-Exo.Small RNA sequencing suggests that microRNAs in USCs-Exo may be responsible for the underlying mechanism of its therapeutic effect.Conclusions:1.USCs can alleviate renal injury in cisplatin-induced AKI rat models in vivo and in vitro via suppressing inflammatory reaction,suppressing the apoptosis of renal tubular epithelial cells,and improving the proliferation of renal tubular epithelial cells.2.The therapeutic effect of USCs on AKI in rat models may be through directly homing and differentiation into renal cells or by paracrine.3.USCs-Exo can alleviate cystitis induced by cyclophosphamide and promote the recovery of urination function via suppressing inflammatory reaction and oxidative stress,inhibiting pathological proliferation and apoptosis of bladder epithelial cells,and promoting migration and differentiation of bladder epithelial cells.4.The therapeutic effect of USCs-Exo on cystitis in mouse models is likely related to the microRNAs contained in them.