Study Of Urinary MRNA Biomarkers For Chronic Kidney Disease Based On Bioinformatics

ObjectiveChronic kidney disease(CKD)is a major public health problem worldwide.However,there is still a lack of early and accurate diagnostic methods.Recent studies have indicated that analysis of urinary mRNA might be a novel and effective way for early and non-invasive diagnosis of CKD.However,due to the complexity of CKD pathogenesis and the source of urine cells,the accuracy of urine mRNA diagnosis is still not satisfactory.In recent years,bioinformatics has emerged as a powerful tool for biomarker research.Therefore,the purpose of this study was to systematically study the CKD transcriptional characteristics and screen novel urinary mRNA biomarkers of CKD based on a bioinformatics approach.Besides,we also aim to develop a TaqMan one step quantitative PCR kit that can quickly and accurately detect the screened molecules.Method1.We systematically collected over 250 Affymetrix microarray datasets from the glomerular and tubulointerstitial compartments of healthy renal tissues and those with various types of established CKD(diabetic kidney disease,hypertensive nephropathy,and glomerular nephropathy).Then,using stringent bioinformatics analysis,shared differentially expressed genes(DEGs)of CKD were obtained.These shared DEGs were further analyzed by the gene ontology(GO)and pathway enrichment analysis.Finally,the protein-protein interaction networks(PINs)were constructed to further refine our results.2.We first constructed a PCR array consisted of 61 mRNAs involved in the pathogenisis of renal fibrosis.Then,a 2 stage cross-sectional study was performed to verify the diagnostic role of these candidates in renal fibrosis.In stage I,62 cases of CKD patients confirmed by renal biopsy and 14 cases of healthy control(HC)in were selected as the screening group,and in stage II,another 41 CKD patients(also confirmed by renal biopsy)and 9 cases of HC were selected as the verification group.The expression of urinary mRNAs was detected by the targeted fluorescence quantitative PCR array constructed.The balanced iterative random forest algorithm was used to select important urinary mRNAs that could reflect tubular interstitial fibrosis(TIF)and glomerulosclerosis(GS).Then,the correlation between expression of selected mRNAs and renal pathologic/function parameters was analyzed.The diagnostic ability of combined biomarkers based on random forest algorithm and conventional biomarkers of CKD was also compared.3.To select candidate mRNAs,a total of 127 Affymetrix microarray datasets of diabetic kidney tissues and other tissues from humans were compiled and analyzed using an integrative boinformatics approach.Then,using a PCR array,we verified urinary expression of candidate mRNAs with the largest fold changes in a cross-sectional study involving 82 participants.Next,the in situ expression of the selected mRNA was further confirmed using fluorescent in situ hybridization assay.Lastly,its potential use in other types of kidney diseases was also explored by bioinformatics analysis.4.Based on the candidate molecules selected in the second and third parts,the full gene sequences of the genes of interest and internal reference genes were downloaded from NCBI,and specific primers and TaqMan probes were designed accordingly.By preparing primer and probe mixtures,PCR enzyme mixture and MasterMix at appropriate concentrations,and optimizing PCR reaction conditions we were able to develop a TaqMan probe fluorescence quantitative PCR one-step kit that can rapidly detect the above mentioned genes.Finally,a concentration gradient was set up to verify the reproducibility and sensitivity of this kit.Results1.Our analysis identified 176 and 50 shared DEGs in diseased glomeruli and tubules,respectively,including many transcripts that have not been previously reported to be involved in kidney disease.Enrichment analysis also showed that the glomerular and tubulointerstitial compartments underwent a wide range of unique pathological changes during chronic injury.As revealed by the GO enrichment analysis,shared DEGs in glomeruli were significantly enriched in exosomes.By constructing PINs,we identified several hub genes(e.g.,OAS1,JUN,and FOS)and clusters that might play key roles in regulating the development of CKD.2.Sixteen mRNAs were selected as candidates,of which four mRNAs(BBOX1,CCL18,NPHS2 and SLC3A1)were significantly upregulated in the urine of patients with DKD.After multiple comparison correction,urinary BBOX1(uBBOX1)levels remained significantly elevated.The expression of uBBOX1 was also increased in normoalbuminuric diabetes subjects,while remained unchanged in patients with urinary tract infection or bladder cancer.Besides,uBBOX1 levels correlated with glycemic control,albuminuria and urinary tubular injury marker levels,but not with eGFR.Specifically,uBBOX1 had an AUC of 0.805 in discriminating the test group(those with diabetes)and the control group(those without diabetes).Fluorescent in situ hybridization assay further demonstrated that BBOX1 mRNA was predominantly located in renal tubular epithelial cells,while its expression in podocytes and urothelium was weak.Further bioinformatics analysis also suggested that tubular BBOX1 mRNA expression was quite stable in various types of kidney diseases,suggesting that elevated uBBOX1 might also act as an indicator of tubular injury in these diseases.3.By the iterative random forest method,we identifed a four-mRNA signature in urinary sediment,including TGFβ1,MMP9,TIMP2,and VIM,as important features of tubulointerstitial fibrosis(TIF).All four mRNAs significantly correlated with TIF scores and discriminated TIF with high sensitivity,which was further validated in the stage-II study.The combined classifiers showed excellent sensitivity and outperformed serum creatinine and estimated glomerular filtration rate measurements in diagnosing TIF.Another four mRNAs significantly correlated with glomerulosclerosis.4.By optimizing the PCR system components and reaction conditions,our newly developed TaqMan probe fluorescence quantitative PCR one-step kit can achieve effective simultaneous amplification and quantitative detection of six mRNAs including BBOX1,VIM,TGF_β1,MMP9,TIMP2,and B2M(internal reference genes)in urine.Reproducibility and sensitivity tests showed that the coefficient of variation of the kit is less than 0.5,and it has good stability.This kit can detect the expression level of mRNAs of interest from urine sediment mRNA system at concentrations as low as 0.5 ng/10ul and has good sensitivity.The entire amplification and quantification process can be completed within 1 h,greatly reducing detection time as compared to traditional PCR methods.Conclusion1.We systematically constructed the transcriptional features of the CKD kidney tissues with the largest sample size so far.The related DEGs and molecular patterns provide important reference for the further exploration of CKD biomarkers and mechanisms.2.We first established the bioinformatics based analysis of urinary kidney-specific mRNAs in this study.We found that uBBOX1 may be a new biomarker for early noninvasive diagnosis of DKD patients.3.We first used the random forest algorithm to analyze the urinary RNAs of renal fibrosis and constructed random forest classifiers that can better diagnose renal fibrosis than single urinary mRNA or renal function parameter.4.The one step fluorescence quantitative PCR kit using TaqMan probe for detecting the above urine mRNAs was established.It has high specificity and sensitivity,and provides a convenient and effective detection method for large-scale clinical verification and translation in the future.