Repair Of Gentamicin-induced Acute Kidney Injury By Canine Adipose-derived Mesenchymal Stem Cells And Its Mechanism

Acute Kidney Injury(AKI)in dogs is a clinically frequent disease with a high mortality rate.Conventional therapy has limited effect on dogs with severe conditions.Stem cell therapy is a new treatment method emerging in the past decade.Studies based on humans or mice have shown that Mesenchymal Stem Cells(MSCs)show good therapeutic effects on various tissues including kidneys.However,there are few studies on the treatment of canine AKI with MSCs,and its therapeutic effect and mechanism are not yet clear.To this end,this article treated gentamicin to prepare canine AKI cell models and animal models,and carried out the following studies:1.The effect of canine AD-MSCs and its conditional medium on the proliferation,apoptosis and related gene expression of gentamicin-induced Madin-Darby Canine Kidney(MDCK)cells.In this experiment,MDCK cells were treated with gentamicin sulfate at different concentrations(1,2,4,6,and 8 mmol/L),and the optimal concentration was determined for subsequent experiments.The grouping is as follows:(1)Negative control group:MDCK cells without gentamicin treatment;(2)Model group:MDCK cells treated with gentamicin;(3)AD-MSCs group:MDCK cells treated with gentamicin,and then co-cultured with AD-MSCs;(4)Adipose-derived mesenchymal stem cell conditioned medium(AD-MSC-CM)group:MDCK cells were treated with gentamicin and added to AD-MSC-CM cultivation.At 24 h and 48 h,the cell proliferation activity of each group was measured by cck-8 method,the apoptosis rate was detected by flow cytometry,and the proliferation(PCNA)and apoptosis(Bcl-2and Bax)related gene m RNA expression levels were measured by fluorescent quantitative PCR.The results show that the optimal dose of gentamicin is 5 mmol/L,which can significantly inhibit the proliferation of MDCK cells and promote apoptosis,but canine AD-MSCs and AD-MSC-CM can reverse this effect.The above results indicate that canine AD-MSCs and its conditioned medium have a certain repair effect on gentamicin-induced MDCK cells damage.2.The therapeutic effect of canine AD-MSCs on gentamicin-induced canine AKI.The test was divided into two stages:modeling and treatment.Six healthy Chinese rural dogs(5-6 months old)were randomly divided into a model group and a treatment group,with 3 in each group.During the modeling stage,both groups of dogs were injected subcutaneously with 80,000 IU/kg gentamicin sulfate for 6 days.During the treatment stage,the dogs in the treatment group were intravenously infused with 2×10~6 cells/kg of AD-MSCs on the 7th,10th,and 13th days,and the dogs in the model group were given the same amount of normal saline.The evaluation indicators of modeling and treatment effect are as follows:serum creatinine and urea nitrogen,serum tumor necrosis factor-α(TNF-α),neutrophil gelatinase-associated lipocalin(NGAL),Kidney injury molecule-1(KIM-1),urine routine,anatomical observation,histopathological observation.The results show that compared with before the model,gentamicincan cause a significant increase in canine serum creatinine and urea nitrogen,as well as the inflammatory factor TNF-αand early renal injury factors NGAL and KIM-1 continue to increase,urine routine Abnormalities(such as decreased p H,increased glucose and protein,partial occult blood,decreased urine specific gravity,etc.),and 9 days after drug withdrawal,canine renal cortical blood flow decreased,more renal tubule obstruction was seen under the microscope,and renal tubular epithelial cells generally appeared Transgender and necrosis.Compared with the model group,canine AD-MSCs can improve gentamicin-induced increase in serum creatinine and urea nitrogen,reduce serum TNF-αlevels,restore normal levels of NGAL and KIM,and significantly improve kidney after 9 days of treatment Cortical blood flow reduces renal tubular epithelial cell damage and maintains the structural integrity of the renal tubules.The above results indicate that canine AD-MSCs have a certain therapeutic effect on gentamicin-induced canine AKI.3.Exosome Derived from Adipose-Derived Mesenchymal Stem Cells(AD-MSC-Exo)can repair MDCK cell damage induced by gentamicin and its molecular mechanism.First,AD-MSC-Exo was separated by low-temperature ultra-high-speed centrifugation,and related identification was performed.The fluoroscopic electron microscope showed that the canine AD-MSC-Exo was oval,and a particle size of97.45%from 30 nm to 150 nm,and the average particle size of 62.31 nm.Western blot results showed that canine AD-MSC-Exo expressed CD9,CD63 and CD81surface markers.Subsequently,to explore the effect of AD-MSC-Exo on the proliferation,apoptosis and related gene expression of damaged MDCK cells,the experimental grouping and treatment are as follows:(1)negative control group:MDCK cells without gentamicin treatment;(2)model group:gentamicin-treated MDCK cells;(3)AD-MSC-Exo group:MDCK cells were treated with gentamicin,and then added with100μg/m L AD-MSC-Exo culture..The cck-8 method was used to measure the cell proliferation activity of each group,the flow cytometry was used to detect the apoptosis rate of each group,and the fluorescence quantitative PCR method was used to determine the m RNA expression levels of PCNA,Bcl-2 and Bax genes in each group of cells.The results show that canine AD-MSC-Exo can significantly promote the proliferation of injured MDCK cells and reduce apoptosis,which has a similar effect to canine AD-MSCs,suggesting that canine AD-MSCs can play a repairing role through its Exo secretion.Finally,in order to determine the mechanism of AD-MSC-Exo functioning,high-throughput sequencing of canine AD-MSC-Exo was performed in this experiment,and cfa-mi R-99b with higher expression was screened for further study.Synthesize cfa-mi R-99b mimic,inhibitor and Negetive Control(NC),and transfect the above compounds to MDCK cells treated with gentamicin,respectively.The results showed that:compared with the NC group,cfa-mir-99b mimic can significantly promote the proliferation of MDCK cells and reduce apoptosis,while the inhibitor results are opposite.Bioinformatics predicts 17 target genes of cfa-miR-99b,and the main signaling pathways involved include proliferation-related MAPK signaling pathways.The above results suggest that cfa-mi R-99b is involved in the proliferation-promoting and apoptosis-inhibiting effects of AD-MSC-Exo on gentamicin-treated MDCK cells.The proliferative effects may be related to the MAPK signaling pathway,but further research is still needed.