Effect And Mechanism Of BMSCs On Adriamycin Nephropathy In Rats

Objective:To isolate,culture,identify and transfect rat bone marrow mesenchymal stem cells(BMSCs),and compare the renal homing capacity of transplanted BMSCs by different routes of infusion,to obtain the infusion route with the best homing capacity of BMSCs,and to use the best homing capacity infusion route,i.e.ultrasound-guided transrenal artery infusion transplantation,to investigate the effect and potential mechanism of BMSCs in the treatment of Adriamycin nephropathy(AN)in rats,and to investigate the application of multimodal ultrasound technology in the evaluation of BMSCs for AN in rats.Methods:1.Isolation,culture,identification and lentivirus transfection of fluorescently labeled rat BMSCs.2.Male Sprague-Dawley(SD)rats were injected twice with Adriamycin(ADR)via the tail vein to establish the AN rat model.3.5 groups were set up: BMSCs tail intravenous(IV)injection graft group(IV group),ultrasound-guided Renal parenchyma(RP)infusion transplantation group(RP group),ultrasound-guided Renal artery(RA)infusion transplantation group(RA group),negative control group and blank control group.The amount of green fluorescent protein(GFP)-BMSCs homing in the kidney tissues of the IV,RP and RA groups was measured by immunofluorescence section and reverse transcriptionpolymerase chain reaction on day 1 and 7 days after the infusion of transplantation.Tumor necrosis factor-β 1(TGF-β 1),Receptor-interacting protein kinase 3(RIPK3)and mixed-linkage kinase domain-like protein(MLKL)were measured by Western Blot(WB)in the kidneys of five groups of rats after 7 days.The levels of GFP m RNA and m RNA expression of TGF-β 1,RIPK3 and MLKL in kidney tissues were measured by RT-PCR,which was used to analyze and compare the expression levels of BMSCs in kidney tissues of AN rats by different infusion transplantation routes.The results were used to analyze and compare the homing ability of BMSCs in AN rat kidney tissues by different infusion routes,and to obtain the transplantation route with the best homing effect.4.Using the best homing route,i.e.ultrasound-guided transplantation via renal artery infusion,serum creatinine(SCr)and blood urea nitrogen(BMSCs)were measured in the untreated group,the Control medium(CM)group(1 m L of medium by ultrasound-guided transplantation via renal artery infusion),the ADR group(1 m L of phosphate buffer by ultrasound-guided transplantation via renal artery infusion),and the BMSCs group(1 m L of BMSCs by ultrasound-guided transplantation via renal artery infusion).The levels of serum creatinine(SCr),blood urea nitrogen(BUN)and urinary albumin(ALb)were measured in the rats of the four groups.The m RNA levels of TGF-β,RIPK3 and MLKL were measured by RT-PCR in the renal tissues.Through analysis and comparison,the effect and mechanism of BMSCs intervention in the treatment of AN were investigated.5.In the BMSCs group,the renal pathological changes were significantly improved;the multimodal ultrasound results showed no further reduction in renal volume and parenchymal thickness.The parenchymal echo,resistance index(RI),virtual touch tissue imaging quantification(VTIQ)index and area under the timeintensity curve(AUC)index did not increase further.The sensitivity and accuracy of ultrasonography alone were higher than those of the other tests when used to evaluate the effect of BMSCs on AN in rats,while the sensitivity and accuracy of the combined test were higher than those of the individual tests.Results:1.Successful isolation,culture,identification and transfection of labelled rat BMSCs.2.The rat AN model was successfully established with two tail intravenous injection of Adriamycin.3.The highest GFP m RNA content in the RA group among the three BMSCs infusion transplantation routes.The WB results showed that compared with the ADR group,the transplantation of BMSCs by different infusion routes in all three groups reduced the expression of TGF-β,RIPK3 and MLKL proteins.The above results suggest that the ultrasoundguided transplantation route via renal artery infusion is the most effective way to homologate kidney tissue in AN rats,and the mechanism may be related to RIPK3-related pathways.4.The levels of SCr,BUN and ALb in the kidneys of AN rats treated with ultrasound-guided transrenal arterial infusion of transplanted BMSCs were significantly reduced.The expression of RIPK and MLKL was decreased and renal cell necrosis was reduced in AN rats treated with ultrasound-guided transrenal artery infusion of transplanted BMSCs;the expression of toll-like receptors 4(TLR-4),nuclear factor kappa-B(NF-κB)and TGF-β was reduced,reduced inflammation and fibrosis in the kidney.5.In the BMSCs group,the renal pathological changes were significantly improved;the multimodal ultrasound results showed no further reduction in renal volume and parenchymal thickness.The parenchymal echo,resistance index(RI),virtual touch tissue imaging quantification(VTIQ)index and area under the timeintensity curve(AUC)index did not increase further.The sensitivity and accuracy of ultrasonography alone were higher than those of the other tests when used to evaluate the effect of BMSCs on AN in rats,while the sensitivity and accuracy of the combined test were higher than those of the individual tests.Conclusion:1.BMSCs can be isolated,cultured and transfected successfully.2.After transplantation of GFP-BMSCs by different pathways,ultrasoundguided transrenal artery transplantation of GFP-BMSCs can improve the homing of BMSCs to AN kidney of rats,and the mechanism may be related to RIPK3 pathway.3.Ultrasound-guided transrenal artery transplantation of BMSCs can inhibit renal necrosis,inflammation and fibrosis and improve AN renal injury in rats,the mechanism of which may be related to RIPK3/MLKL,TLR-4/NF-κB and TGF-βpathways.4.Multimodal ultrasound technology has certain application value in evaluating the effect of ultrasound-guided transrenal artery transplantation of BMSCs in the treatment of AN rats.