| BackgroundVesicoureteral reflux(VUR)is an inevitable pathophysiological progression in the patients with neurogenic bladder(NGB).Continuous VUR causes a series of damage,including hydronephrosis,renal failure and even patients’ death,which affects the quality of life of patients.At present,there is no non-invasive clinical biomarkers,which can diagnose or warn the occurrence of VUR.Objectives1.This study aims to identify different protein profiles of urinary exosomes between the VUR and Non-VUR patients,and to explore whether there are potential biomarkers that can diagnose or warn the emergence of VUR.2.This study also hopes to preliminarily explore the influence mechanism of the identified candidate markers and the progression of lower urinary tract damage.Methods1.Urine samples from NGB patients meeting the inclusion criteria(including 25 VUR and 35 Non-VUR patients)were collected.Urine exosome samples were isolated and collected by ultracentrifugation.Transmission electron microscopy(TEM)and nanoparticle tracking analysis(NTA)was used for the morphological identification of exosomes.Exosome markers(Alix and CD63)were identified by Western blotting(WB).2.Spectrometry analysis by Tandem mass tag(TMT)was used to identify the proteome profiles of the urine exosomes between the VUR and Non-VUR groups.Candidate biomarkers were selected by bioinformatics analysis of differential proteins and parallel reaction monitoring(PRM).3.Candidate markers were further validated by the WB and ELISA experiments.Moreover,the data obtained from ELISA were used to generate the receiver operating characteristic curve(ROC)for calculating the diagnostic efficacy.4.A total of 10 surgical-removed bladder tissues(including 5 fibrosis samples and 5 nonfibrotic samples)were collected.The activation status of TGF-β1/Smad2/3 signaling pathway and the expression of candidate markers were both analyzed by WB and immunohistochemistry(IHC).Results1.Clinical data showed that decreased bladder compliance,increased residual bladder urine and intravesical pressure were found in VUR patients compared with Non-VUR patients.The results of exosome identification showed that the patients in the VUR group excreted exosomes with bigger diameters and expressed more exosome markers than those in the Non-VUR group.2.TMT spectrometry analysis found that 99 upregulated proteins and 134 downregulated proteins were identified in the VUR group with 1.5-fold as the threshold of differential expression change when compared with those in the Non-VUR group.After a bioinformatic analysis of the differential proteins,a total of 18 candidates were selected for a further validation by PRM.3.Bioinformatics analysis showed that most of the 18 candidate proteins were related to the biological mechanisms of extracellular vesicles or exosomes.Functional enrichment analysis showed that the 18 candidate proteins were highly correlated with the fibrosis mechanism,the TGF-β1 signaling pathway,and the regeneration function,especially VTN,MBP,COL1A1,and ITIH4.4.Both the PRM and WB results showed that only vitronectin(VTN)and COL1A1 had significant differences between the two groups.The ELISA results showed that only the VTN was significantly different between the VUR and Non-VUR groups.The ROC curve had an AUC of 0.795(95%CI,0.667-0.923)for VTN,with 80%sensitivity at 82.9%specificity.5.The results of WB and IHC in the bladder tissue experiments showed that there was a significant activation of TGF-β1/Smad2/3 signaling pathway and the expression of VTN and COL1A1 were also significantly increased in the process of fibrosis of bladder tissues.ConclusionsThe VTN in the urinary exosomes can be used as a non-invasive biomarker for the diagnosis of VUR emergence and the early warning of the deterioration of the lower urinary tract function.VTN may trigger the activation of the TGF-β1/Smad2/3 signaling pathway,which maybe one of the possible mechanisms of the remodeling and fibrosis in the bladder tissue. |
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