Urinary MicroRNA As Novel Biomarkers For Childhood Nephrotic Syndrome

Nephrotic syndrome(NS)is a common clinical renal disease in children,which mainly manifested as a group of clinical syndromes with massive proteinuria,hypoalbuminemia,hyperlipidemia and edema.The incidence of NS is ranged from 1.15 ～ 16.9 per 100,000 in all aged children.At present,only few reports have investigated the epidemiology of the NS in China.Currently,none specific serum marker is available for NS children,and biopsy is still the “golden standard” for diagnosing and histology classification for NS.Nevertheless,kidney biopsy is an invasive procedure with potential complications,and is generally not suitable for disease serial monitoring.Micro RNAs(mi RNAs)are a class of small single-strand non-coding RNAs with the length about 22 nucleotides,which can regulate gene expression at the post-transcriptional level through combing with the 3’-untranslated region(UTR)of target gene.Numerous studies have demonstrated that mi RNAs are involved in kidney diseases,and mi RNAs in body fluid can serve as useful biomarkers for diagnosis,monitoring and prognosis of NS.However,studies on urinary mi RNA and NS children are rarely reported,and comprehensively and systematic research on this issue is urgently needed currently.In the present study,we firstly performed an epidemiology investigation in children with NS in China to uncovered the characteristics,regularity and influencing factors of the NS.Subsequently,according to the research updates and limitations of the published evidence on urinary mi RNA studies of NS children,we performed a systematic study on urinary mi RNA profile in urine samples that were collected from 126 NS children and 126 matched control children by using high-throughput BGISEQ UMI Small RNA sequencing technology combined with a quantitative reverse-transcription polymerase chain reaction(RT-q PCR)assay verification in individual samples,with the purpose to identifying markedly and steadily increased urinary mi RNAs in NS children compared with controls.In addition,we also evaluated the association of the dysregulated urinary mi RNAs with NS,and their potential as non-invasive biomarkers for NS.Furthermore,we still predicted the target genes as well as the pathway of the altered urinary mi RNAs in NS to clarify the molecular mechanism of the mi RNAs that involved in the pathogenesis of NS.1.Epidemiological investigation in children with nephrotic syndromeIn this study,we performed a retrospective study in 655 NS children who were admitted to the Department of Paediatrics of Jingling Hospital in Nanjing,China,between March 2017 and December 2019 and diagnosed according to the definition of the International Society of Kidney Disease in Children.The clinical features,family history,predisposing factors,laboratory indexes and renal biopsy results of the NS children were collected,and epidemiological investigation was subsequently performed.The results showed that the majority of NS children were boys,and the ration of the incidence between boys and girls was 3.04:1.The initial onset age of NS children was ranging from 1 to 5 years old,with the median onset age of 4-year old.Renal biopsy results revealed that Minimal change disease(MCD)was the mostly common histological type,and then Mesangial proliferative glomerulonephritis(Ms PGN),and followed by Focal segmental golmerulosclerosis(FSGS),Ig A nephropathy(Ig AN),Membranous proliferative glomerulonephritis(MPGN)and Membranous nephritis(MN).Further analyzes found that in most of the boys with NS were histological classified as MCD,while girls with NS were often diagnosed as FSGS.Moreover,the youngest NS children were often histological classified as MCD,and the oldest were often diagnosed as MN.On the other hand,the morbidity of NS was highest in winter,and main predisposing factors of NS were recognized as respiratory tract infection.Of the 655 NS children,only 3.82% children have positive family history,of whose immediate families have related kidney diseases.By analyzing the laboratory indexes of NS children,we found that several indexes showing markedly dysregulation as compared with the reference value,such as hyperlipidemia,hypoalbuminemia,severe proteinuria,immune disorders,anemia.Notably,after dividing the NS children into groups with gender or age,we observed that several laboratory indexes showed markedly difference among groups,even though the exact reason is unknow.Nevertheless,we speculated that these differences may generated by the morbidity,severity of NS or prognosis of the children,and which still need further clarified in the future.2.Study on the urinary micro RNAs as novel biomarkers for children with nephrotic syndromeIn this study,we performed a systematic study on urinary mi RNA profile in urine samples that were collected from 126 NS children who were admitted to the Department of Paediatrics of Jingling Hospital in Nanjing,China,between March 2017 and December 2019 and diagnosed according to the definition of the International Society of Kidney Disease in Children.All of the NS children were excluded with secondary nephrotic syndrome,other kidney diseases,immune diseases,urinary tract abnormalities,and urinary tract infection.In the meantime,126 age and gender matched healthy children were enrolled in Nanjing Children Hospital as normal controls.We firstly analyzed the comprehensive and systematic urinary small RNA profile by using BGISEQ UMI Small RNA technology in two pooled urine samples that respectively consisting of 30 urine samples from NS children and 30 urine samples from control subjects.We found the urinary small RNA profile of NS children was obvious different from that of controls,and numerous urinary mi RNAs and pi RNAs showed abnormal expression patterns in NS children.Of these dysregulated small RNAs,6 urinary mi RNAs were elevated and 137 mi RNAs were declined in NS children,and 5 urinary pi RNAs were increased and 46 pi RNAs were decreased in NS children.These dysregulated urinary mi RNAs and pi RNAs may have the potential as novel non-invasive biomarkers for NS children.We subsequently performed RT-q PCR assays to vitrificated 8 candidate mi RNAs including mi R-25-3p,let-7c-5p,let-7a-5p,let-7i-5p,mi R-126-3p,mi R-30a-5p,mi R-196a-5p and mi R-196b-5p that were selected from sequencing results and literature research in training set(24 NS and 24 controls)and validation set(72 NS and 72 controls).We also predicted the target genes as well as the pathway of the altered urinary mi RNAs in NS to clarify the molecular mechanism of the mi RNAs that involved in the pathogenesis of NS.The results showed that five urinary mi RNAs,including let-7c-5p,let-7i-5p,mi R-126-3p,mi R-30a-5p and mi R-196a-5p were markedly and steadily increased in NS children as compared with controls(all with P < 0.05).ROC curve analysis revealed that the AUCs of these five mi RNAs ranged from 0.712 to 0.938 in the training set,ranged from0.771 to 0.935 in the validation set,and ranged from 0.753 to 0.93 in the combined training and validation sets.Among them,let-7i-5p and mi R-30a-5p presented the best performance for NS diagnosis.Additional risk score analyzes showed the AUCs of the five-mi RNA panel for training set,validation set and combined set were 0.946(95% CI = 0.890 ～ 1.000),0.966(95% CI = 0.942 ～ 0.991)and 0.953(95% CI = 0.926 ～ 0.981),respectively.And which showed an excellent diagnostic performance than any individual mi RNA alone,when at the optimal cutoff point of 11.09,the five-mi RNA panel showed the highest diagnostic effect,with the sensitivity and specificity was 86.5% and 94.8%,respectively.Binary logistic regression analysis revealed that the OR of the five mi RNAs were range from 1.116 to 2.062(all with P < 0.001).Spearman’s rank correlation analyzed demonstrated that urine concentrations of let-7c-5p,let-7i-5p,mi R-30a-5p,mi R-196a-5p and the five-mi RNA panel were significantly negatively associated with serum TP and ALB,while significantly positively associated with sera TC,TG and Urea.In addition,urine concentrations of let-7c-5p,mi R-30a-5p,and mi R-196a-5p were also statistically positively associated with serum Uric acid and Creatinine.Notably,urinary let-7i-5p and mi R-30a-5p levels were also positively correlated with 24 h urine protein.Furthermore,bioinformatic analysis showed that the five altered mi RNAs were closely related with the pathogenesis of NS,for which were participated in regulation of the EMT,epithelial cell proliferation,cell junction,cell cycle,renal system and renal epithelial functions.In summary,we have performed an epidemiology investigation in children with NS in China,and uncovered the characteristics,regularity and influencing factors of the NS.Moreover,we also conducted a global and systematic analysis of the urinary mi RNAs profile in NS children and successfully identified five mi RNA including let-7c-5p,let-7i-5p,mi R-126-3p,mi R-30a-5p and mi R-196a-5p that were markedly and steadily increased in NS children and may serve as novel non-invasive biomarkers for auxiliary diagnosis of NS,and these mi RNAs were also involved in the pathogenesis of NS.The results of the present study may provide novel ideas and theoretical basis for investigation the epidemiology of NS children as well as the development of urinary biomarkers and molecular pathogenesis of NS.