| Background and purpose:The incidence of chronic kidney disease(CKD)is increasing year by year,all kinds of CKD will progress to formate extensive scar in the renal tissue,which will lead to end-stage renal failure irreversibly,renal interstitial fibrosis(RIF)is widely regarded as the common histologic hallmark of CKD progressing to end-stage renal diseases(ESRD).Connective tissue growth factor(CTGF)was found to be an important downstream mediator of transforming growth factor-?1(TGF-?1),and controls pro-fibrotic activities in RIF,because of its unique function in mediating fibrogenic activity,this novel modulator is considered to be a more suitable therapeutic target than direct inhibition of TGF-?1.Gene therapy holds enormous potential in the treatment of disease,it will become a new effective way to prevent RIF and eventually conquer ESRD,however,there are still a lot of problems in the field of gene therapy that results in a poor effect.Low-frequency ultrasound,in combination with some microbubble formulations,has been explored as a mean for targeted delivery of biomolecules through a mechanism known as ultrasound targeted microbubble delivery(UTMD).In the present study,we attempted to explore the effect of UTMD in mediating and enhancing plasmid transfetion in ameliorating RIF,in order to provide a new safe effective idea and method for preventing the process of RIF.Methods:1.shRNAs targeting CTGF were synthesized,and cloned into tool plasmid and sequenced,the plasmid was conjugated with the cationic microbubbles(Targesphere)through electrostatically coupled.Plasmid and microbubble complexes were fluorescence labeled with the green nucleotide-avid fluorophore YoYo-1,plasmid binding to the microbubble surface was confirmed by epifluorescence microscopy and flow cytometry.2.Rat renal fibroblasts(NRK-49F)cells were cultured,knockdown efficiency of three CTGF-targeted shRNAs was verified with lipofection,the shRNA sequence with the highest silencing efficiency was selected for subsequent use with UTMD.The UTMD mediated plasmid transfection to NRK-49F cells was carried out in three groups.The acoustic intensity was 1.5W/cm2,the irradiation duration was 15s,and the duty cycle was 20%.The expression of green fluorescent protein(GFP)in NRK-49F cells was observed by inverted fluorescence microscope at 48h after transfection,the expression of CTGF mRNA was detected by reverse transcription polymerase chain reaction(RT-PCR)and fluorescence quantitative real-time PCR(FQ-RT-PCR),and the expression of CTGF protein was detected by Western blot.3.A UUO model was developed by ligated the right ureter.Conventional B-mode ultrasound and Color Doppler flow imaging(CDFI)were performed to obtain the baseline echogenicity and blood flow of the kidney.The relative expression CTGF mRNA of sham kidney and the obstructed kidney on day 3,6,9,12,week 2,week 3 and week 4 after UUO was detected by FQ-RT-PCR.The pathological change and collagen staining in obstructive renal tissue were detected by HE staining and Masson staining.The expression of CTGF protein in the obstructive kidney was detected by immunohistochemical.4.The mice were randomly divided into six experimental groups at day 3 after UUO:sham group(Sham),UUO group(UUO),UUO with plasmid group(P),UUO with plasmid and microbubble group(P+MB),UUO with plasmid and ultrasound exposure group(P+US),UUO with plasmid and microbubble and ultrasound exposure group(P+UTMD).Plasmid and microbubble complexes or plasmid alone were administered in 60 ?l as a bolus by retroorbital injection.The sonoporation probe was positioned at the right kidney region,the acoustic intensity was 2 W/cm2,the irradiation duration was 30s,and the duty cycle was 25%.The sonoporator was cycled on and off in 30-s intervals,this process was repeated for a total duration of 5 min,at which time clearance of all circulating microbubbles was observed by contrast ultrasound imaging,a second dose of microbubbles was then administered,and the UTMD treatment repeated for a total of three times.All mice were euthanized 14 d post-treatment,the mRNA and protein of CTGF and TGF-?1 in the kidney was measured by FQ-RT-PCR and Western blot,the expression of CTGF,TGF-?1,?smooth muscle actin(?-SMA)and type ? collagen was evaluated by immunohistochemistry,the pathological changes and collagen staining in renal tissue were detected by HE staining and Masson staining.Results:1.After sequencing,the inserted shRNA coding sequence was proved to be correct and the CTGF-shRNA plasmid was constructed successfully.Successful coupling of the plasmid to the microbubble surface was verified by flow cytometry and epifluorescence microscopy.2.shRNA sequence 1 had the highest silencing efficiency and was selected for subsequent use with UTMD.Green fluorescence could be observed in post-transfectional NRK-49F cells,which convinced that cell transfections were successful.RT-PCR and FQ-RT-PCR showed that the expression of CTGF mRNA was down regulated in P+UTMD group when compared with blank control group(P<0.01),Western blot showed that the expression of CTGF protein was down regulated in P+UTMD group when compared with blank control group(P<0.05),the results showed that the expression of CTGF was inhibited when CTGF-shRNA plasmid was transfected with UTMD.3.The results of HE staining and Masson staining showed that the model of RIF was successfully developed.At day 3 after UUO,on conventional ultrasound imaging,the obstructed kidney was noticeably larger than the sham group kidney,the parenchyma was progressively atrophic and surrounded a dilated collecting system,and slight hydronephrosis appeared in the obstructed kidney of all UUO animals,with the increase of modeling time,the renal parenchyma was atrophic and the hydronephrosis was aggravating gradually.FQ-RT-PCR results showed that the expression of CTGF mRNA in the kidney was upregulated from the early stage of RIF(at day 3),further increased and peaked at day 14,then declined gradually after day 14 and then remained nearly constant.The immunohistochemical results showed that the expression of CTGF in the UUO group was significantly higher than that in the sham group(P<0.05).4.Compared with UUO group,the cohort treated with plasmid-carrying microbubbles and ultrasound exhibited reduced mRNA of CTGF and TGF-?1(p<0.05),as well as reduced protein expression of CTGF and TGF-?1(p<0.01).Furthermore,CTGF gene silencing decreased the interstitial deposition of CTGF,TGF-?1,?-SMA and type ? collagen as assessed in immunohistochemistry,as well as reduced renal fibrosis in pathologic alterations(p<0.01).No significant changes in target mRNA,protein expression or disease pathology were observed in the control cohorts(p>0.05).Conclusions:A single treatment of UTMD is able to deliver sufficient shRNA to inhibit the expression of CTGF and provide a meaningful reduction in renal interstitial fibrosis induced by TGF-?1.This technique provides a noninvasive and non-viral method for efficient and localized application of gene therapy for treatment of RIF. |
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