Effect Of Alprostadil On The Expression Of PAI-1 In Urinary Exosomes Of Patients With Diabetic Nephropathy

Object:To observe the changes of renal function and proteinuria after the treatment with alprostadil injection in patients with DN,and to verify the feasibility of differential ultracentrifugation for the extraction of urinary exosomes from DN patients.To Compare the efficacy of the treatment with alprostadil on the expression of PAI-1 in urine exosomes of DN patients.To explore the evidence for alprostadil in treating DN by altering PAI-1 expression level.Method:According to the inclusion criteria,30 patients with DN were randomly divided into the control group and the alprostadil treatment group,15 cases each.The basic treatment measures were the same in both groups,and the alprostadil treatment group was given the basic treatment and alprostadil 10ug/d by injection.Fresh morning urine was collected before and after the treatment,and urine exosomes were extracted by differential ultracentrifugation.Exosome was identified by transmission electron microscopy,nanoparticle tracking analysis(NTA)and western blot.The mRNA of urinary exosomes was further extracted by kit method,and the expression of PAI-1 was detected by real-time quantitative PCR(RT-qPCR).The concentration of PAI-1 protein was detected by Western blot after concentration by BCA method.The laboratory tests and the expression changes of PAI-1 in urinary exosomes were analyzed before and after treatment.Result:1.General information: A total of 30 patients were enrolled in the study and were randomized into 15 patients in the alprostadil treatment group and the control group.There were 17 males(56.7%)and 13 females(43.3%).The average age of the included patients was 60.17±12.56 years old.There were 23(76.7%)patients with a history of hypertension and 10(33.3%)patients with a history of smoking.There were no significant differences in gender,age,history of hypertension,and smoking history between the two groups(p>0.05).2.Laboratory examination: There were no significant differences in the levels of urea nitrogen,serum creatinine,urinary microalbumin,urinary albumin/urinary creatinine,and 24 h urine protein between the alprostadil and the control group(p>0.05)before treatment.After treatment,the levels of urinary albumin,urinary microalbumin/urinary creatinine and 24 h urine protein in the alprostadil group were lower than those in the control group,and the difference was statistically significant(p< 0.05).There were no significant changes in urea nitrogen and serum creatinine between the two groups before treatment(p>0.05).3.The urinary exosomes of DN patients isolated by differential ultracentrifugation are round,"cup-shaped" under the electron microscope,and the shape of the bilayer membrane is about 40-100 nm in diameter.4.The urine exosomes of DN patients isolated by differential ultracentrifugation,detected by NTA,have a particle size of about 100 nm.5.Exosome-labeled proteins TSG101 and CD9 were detected by Western blot.6.There was no significant difference in the expression of PAI-1 mRNA before treatment with prostaglandin and control group,p>0.05.After drug treatment: the expression of PAI-1 mRNA in the control group did not change significantly(p > 0.05).The expression of PAI-1 mRNA in the alprostadil group was lower than that before the treatment,and was significantly lower than the control group.The change was statistically significant.(p <0.05).7.There was no significant difference in PAI-1 protein expression between the two groups before administration(p>0.05).After administration,the expression of PAI-1 protein in the control group was not significantly different from that before administration(p > 0.05).The expression of PAI-1 protein in the alprostadil group was lower than that before administration,and the change was statistically significant(p< 0.05).Conclusion:1.Alprostadil treatment can reduce urinary protein in patients with DN.2.Extraction of urinary exosomes from patients with DN by differential ultracentrifugation is easy and feasible.And the mRNA of PAI-1 gene and its expressed protein exist in the urine exosomes of DN patients.3.Prostaglandin treatment can inhibit the expression of PAI-1 in urinary exosomes of DN patients.Innovation:After most database searches,such as Pubmed,Web of Science,Wanfang database,China Knowledge Network,etc.no reports on the expression of PAI-1 mRNA and protein in urine exosomes of DN patients were found.The reports are mostly limited to animal experiments,and the clinical trials mostly refer to the patient's urine itself.This study confirmed the feasibility of obtaining urine samples from patients with large amounts of DN and the effect of differential ultracentrifugation on the exosome in urinary exosomes of patients with DN and the operability for future clinical application.The presence of PAI-1 mRNA and expressed protein in urinary exosomes of DN patients was confirmed.It was confirmed that alprostadil treatment can inhibit the expression of PAI-1 in urinary exosomes of DN patients.Add new evidence for the potential clinical value of exosomes that may become biomarker for early diagnosis and treatment of DN.