Urinary mRNA Research Articles

Celastrol attenuates symptoms of preeclampsia in rats by inhibiting matrix metalloproteinase-9

Preeclampsia is reported in pregnant women around the world and often causes maternal/fetal mortality and morbidity. In the current study, we assessed the efficacy of celastrol on a rat preeclampsia model induced by Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME). Pregnant rats were administered L-NAME to establish preeclampsia. A total of 48 animals were randomly assigned into 4 groups (n = 12 each): control, control plus celastrol treatment (control+celastrol), preeclampsia, and preeclampsia plus celastrol. Physiological parameters including total urine protein, urine volume and blood pressure were evaluated. Urinary messenger RNA (mRNA) levels of podocin and nephrin were determined using RT-PCR. Further, levels of serum placenta growth factor (PlGF), matrix metalloproteinase (MMP)-9 and renal renal soluble fms-like tyrosine kinase-1 (sFlt-1) were also measured. In rats with preeclampsia, there were robust increases in total urine protein, urine volume and blood pressure, which were significantly attenuated in rats treated with celastrol. Urinary mRNA levels of podocin and nephrin, as well as PlGF, MMP-9 and sFlt-1, were all reversed in preeclampsia plus celastrol group compared to rats in the preeclampsia group without celastrol treatments. MMP-9 overexpression in rats completely abolished the alleviating effect of celastrol. We hereby presented the first evidence that celastrol attenuated preeclampsia symptoms in an L-NAME-induced rat model of preeclampsia through inhibition of MMP-9 expression, supporting the potential therapeutic value of celastrol in the treatment of preeclampsia.

Urinary cytokines and mRNA expression as biomarkers of disease activity in lupus nephritis.

Introduction Renal involvement is one of the most serious manifestations of systemic lupus erythematosus, but non-invasive assessment of inflammatory response in kidneys is challenging. In this study we aimed to validate markers of active lupus nephritis (LN) using urine immune profiling. Methods Urine and serum cytokines (17-plex array) and urine mRNA expression (∼40 immune and glomerular injury genes) were measured in LN patients with active disease ( n = 17) during remission ( n = 16) and in healthy subjects ( n = 18). Results Urine and serum levels of CCL2, CCL5 and CXCL10 were elevated in active LN as compared with disease remission (best discrimination for urine CXCL10 and CCL2) and correlated with LN activity. In the active disease, urinary cell transcriptome showed marked upregulation of proinflammatory cytokines (e.g. TNF, CCL2, CCL5, CXCL10), and type-1 immunity-related genes (e.g. CD3G, CD4, TBX21, IFNG). An active pattern of gene expression was also observed in four patients in remission, who had moderately increased urinary leucocyte count. Two patients from this group developed renal exacerbation during the following 3 months. Markers of type-17 immune axis (e.g. IL-17A) were not significantly increased in active LN. Conclusions Active LN patients were characterized by marked increase of proinflammatory mediators in the urine. Urine cytokines (CCL2 and CXCL10) and type-1 T-cell-related gene markers in the urine sediment had similar diagnostic performance in detection of active LN.

Urinary mRNA analysis of biomarkers to epithelial mesenchymal transition of renal allograft

Renal allograft loss is most often a chronic process, irrespective of the mechanism at stake. In this prospective study, we studied the expression of epithelial to mesenchymal transition (EMT) markers vimentin and β-catenin by immunohistochemistry in the surveillance biopsy and measured the mRNA encoding vimentin (VIM), CD45, GAPDH and uroplakin 1a (UPK) by quantitative PCR in urinary cells in 75 renal transplant patients. The aim is to establish a simple screening test for chronic renal allograft dysfunction. We found that the value of the mRNA of vimentin and CD45 relative to the uroplakin 1a (UPK) mRNA is correlated with the score in vimentin immunostaining in routine biopsies. These biomarkers could be used as a noninvasive tool to monitor the renal graft fibrogenesis. This test could be used for early detection of fibrotic diseases of the kidney transplant.

Urinary Exosomal mRNA of WT1 as Diagnostic and Prognostic Biomarker for Diabetic Nephropathy.

Diabetic nephropathy (DN) is the major cause of end-stage renal failure and is associated with increased morbidity and mortality as compared to other causes of renal disease. Albuminuria is often the first clinical indicator of the presence of DN. However, albuminuria or proteinuria is a common symptom in patients with various renal disorders. Therefore, specific biomarkers for the diagnosis of DN are required. A primary hallmark of DN is the progressive damage and death of glomerular podocytes, resulting in the leaking of proteins into the urine. Urinary exosomes released by podocytes are microvesicles containing information of the originated cells. Podocyte-derived signal transduction factors (PDSTFs) are good candidates to assess podocyte injuries. The profile of PDSTFs in urinary exosomes from patients with DN is different from that from patients with minimal change nehrotic syndrome. In addition, PDSTFs molecules in exosomes were derived from primary murine podocytes under high glucose conditions. Among PDSTFs in urinary exosomes, Wilms tumor 1 (WT1) levels reflected damage of diabetic glomeruli in the patients. Urinary exosomal WT1 can predict the decline in eGFR for the following several years. In conclusion, urinary exosomal WT1 is a useful biomarker to improve risk stratification in patients with DN. J. Med. Invest. 65:208-215, August, 2018.

Urinary mRNA and lupus disease flare.

In the past 20 years, extraction and quantification of mRNA from urinary sediment has emerged as a novel laboratory technique. The potential application of urinary mRNA quantification to the clinical care of patients with SLE has become an active field of research. However, published studies in this area are limited to small-scale ones. mRNA levels of several of cytokine and transcript factor genes have been found to have the potential for the diagnosis or monitoring of disease activity in patients with lupus nephritis. Our previous studies showed that a high urinary mRNA level of T-bet, the key transcription factor of type 1 T helper cells (Th1), was an independent predictor of lupus flare and probably reflects an intrinsic characteristic of patients with predisposition to Th1 activation. Other studies suggest that monitoring of monocyte chemoattractant protein-1(MCP-1), interleukin (IL)-17, GATA-3 (the key transcription factor of type 2 T helper cells) and forkhead box P3 (FOXP3, the key transcription factor of regulatory T cells) mRNA level in urinary sediment may provide an early clue for detecting disease flare in lupus patients. As a simple and non-invasive method, urinary mRNA level deserves further studies to validate its role in risk stratification and monitoring of patients with lupus nephritis.

Development of a 90-Minute Integrated Noninvasive Urinary Assay for Bladder Cancer Detection

Despite suboptimal sensitivity urine cytology is often performed as an adjunct to cystoscopy for bladder cancer diagnosis. We aimed to develop a noninvasive, fast molecular diagnostic test for bladder cancer detection with better sensitivity than urine cytology while maintaining adequate specificity. Urine specimens were collected at 18 multinational sites from subjects prior to cystoscopy or tumor resection, and from healthy and other control subjects without evidence of bladder cancer. The levels of 10 urinary mRNAs were measured in a training cohort of 483 subjects and regression analysis was used to identify a 5-mRNA model to predict cancer status. The performance of the GeneXpert® Bladder Cancer Assay, an assay labeled for investigational use only to detect the 5 mRNAs ABL1, CRH, IGF2, ANXA10 and UPK1B, was evaluated in an independent test cohort of 450 participants. In the independent test cohort the assay ROC curve AUC was 0.87 (95% CI 0.81-0.92). At an example cutoff point of 0.4 overall sensitivity was 73% while specificity was 90% and 77% in the hematuria and surveillance patient populations, respectively. We developed a 90-minute, urine based test that is simple to perform for the detection of bladder cancer. The test can help guide physician decision making in the management of bladder cancer. Additional evaluation in a prospective study is needed to establish the clinical usefulness of this assay.

Abstract 053: Urinary Extracellular Messenger RNA Transcripts as Biomarkers of Mineralocorticoid Receptor Activity

Objectives: Mineralocorticoid receptor (MR) activation regulates sodium homeostasis & blood pressure. Our objective was to identify a non-invasive biomarker of MR activation in humans. Background: MR is a ligand-activated transcription factor that alters expression of target genes in the distal renal tubule, as shown in cell & animal models. Renal tubular cells secrete extracellular vesicles which carry RNA into urine. Whether human urinary extracellular mRNA reflects changes in gene expression in the renal tubule & thus might provide insight into MR activation is unknown. Methods: Pre-hypertensive participants consumed a low-sodium diet followed by sodium loading. We designed qPCR assays for MR gene targets & control genes not expected to respond to MR activation. Using samples collected during low-sodium diet & after sodium infusion, we assayed plasma renin activity (PRA), serum aldosterone, urinary sodium & urinary MR-responsive & control extracellular mRNA. Results: Eighteen participants’ low-sodium & 17 participants’ sodium-loaded urine samples were available. PRA & serum aldosterone were higher ( P <0.001) & urinary sodium excretion was lower ( P <0.001) during low-sodium diet. Four of 14 target gene qPCR assays (29%) changed after sodium loading, including assays for SCNN1A , SCNN1G & TSC22D3 ( P =0.006 to 0.01), whereas an assay for SGK1 approached significance ( P =0.07). In contrast, only 1 out of 10 control gene assays (for NR3C2 , encoding MR itself) was significantly different after sodium loading ( P =0.003). Log serum aldosterone inversely associated with C t values for 7 of 14 target gene assays (including assays for SCNN1A , SCNN1G , SGK1 & TSC22D3 [r= -0.46 to -0.52, P =0.003 to 0.048]) & for control gene assays for NR3C2 & UMOD . C t values for 6 of 14 target gene qPCR assays associated with log urinary sodium/creatinine ratio including assays for SCNN1A , SCNN1G , SGK1 & TSC22D3 associated with log urinary sodium/creatinine ratio ([r= 0.36 to 0.52, P =0.003 to 0.048]) but not with control gene assays. Conclusions: Perturbations in human endocrine physiology can be detected as changes in urinary extracellular mRNA. Our findings suggest a new strategy to screen for mineralocorticoid excess & to assess pharmacologic or dietary changes in MR activity.

High expression of TIM-3 and KIM-1 in blood and urine of renal allograft rejection patients

BackgroundT cell immunoglobulin and mucin domain 3 (TIM-3) is involved in alloimmune and autoimmune responses, as well as tolerance induction in kidney transplantation. Kidney injury molecule-1 (KIM-1) is highly expressed in epithelial cells of the injured proximal tubule. In this study, we have investigated both urinary and blood TIM-3 mRNA expressions, urinary KIM-1 mRNA expression, and urinary and serum KIM-1 proteins in renal allograft recipients diagnosed with acute allograft rejection (AR) and chronic allograft dysfunction (CAD), as well as those with well-functioning transplants (WFG). MethodsWe divided 85 patients into the following groups: AR (n=24), CAD (n=19), and WFG (n=42). TIM-3 and KIM-1 mRNA expressions were quantified using real-time reverse-transcription TaqMan probe polymerase chain reaction (RT-PCR). An ELISA test was used to measure the amount of KIM-1 protein in serum and urine samples. ResultsAR and CAD patients had significantly greater urinary and blood TIM-3 mRNA expressions, urinary KIM-1 mRNA expression, and urinary and serum KIM-1 proteins compared to WFG patients. Receiver operating characteristic (ROC) analysis showed that these molecules discriminated Allograft rejections from WFG. ConclusionQuantification of TIM-3 and KIM-1 mRNA expressions, along with KIM-1 protein measurements in urine and blood could be employed as promising tools for noninvasive diagnosis of allograft dysfunction.

Both absolute and relative quantification of urinary mRNA are useful for non-invasive diagnosis of acute kidney allograft rejection.

Urinary mRNA analysis with three-gene set (18S rRNA, CD3ε, and IP-10) has been suggested as a non-invasive biomarker of acute rejection (AR) in kidney transplant recipients using quantitative real-time PCR (qPCR). Application of droplet digital PCR (ddPCR), which has been suggested to provide higher sensitivity, accuracy, and absolute quantification without standard curves, could be a useful method for the quantifying low concentration of urinary mRNA. We investigated the urinary expression of these three genes in Korean patients with kidney transplantation and also evaluated the usefulness of ddPCR. 90 urine samples were collected at time of allograft biopsy in kidney recipients (n = 67) and from patients with stable renal function more than 10 years (n = 23). Absolute quantification with both PCR system showed significant higher mRNA levels of CD3ε and IP-10 in AR patients compared with stable transplants (STA), but there was no difference in 18S rRNA expression across the patient groups. To evaluate discrimination between AR and STA, ROC curve analyses of CTOT-4 formula yielded area under the curve values of 0.72 (95% CI 0.60–0.83) and 0.77 (95% CI 0.66–0.88) for qPCR and ddPCR, respectively. However, 18S normalization of absolute quantification and relative quantification with 18S showed better discrimination of AR from STA than those of the absolute method. Our data indicate that ddPCR system without standard curve would be useful to determine the absolute quantification of urinary mRNA from kidney transplant recipients. However, comparative method also could be useful and convenient in both qPCR and ddPCR analysis.

Urinary podocyte-associated mRNA levels correlate with proximal tubule dysfunction in early diabetic nephropathy of type 2 diabetes mellitus

AimThe study assessed mRNA expression of podocyte-associated molecules in urinary sediments of patients with type 2 diabetes mellitus (DM) in relation to urinary podocytes, biomarkers of podocyte injury and of proximal tubule (PT) dysfunction.MethodsA total of 76 patients with type 2 DM and 20 healthy subjects were enrolled in a cross-sectional study, and assessed concerning urinary podocytes, urinary mRNA of podocyte-associated genes, urinary biomarkers of podocyte damage and of PT dysfunction.ResultsWe found significant differences between urinary mRNA of podocyte-associated molecules in relation with albuminuria stage. In multivariable regression analysis, urinary mRNA of nephrin, podocin, alpha-actinin-4, CD2-associated protein, glomerular epithelial protein 1 (GLEPP1), ADAM 10, and NFκB correlated directly with urinary podocytes, albuminuria, urinary alpha1-microglobulin, urinary kidney-injury molecule-1, nephrinuria, urinary vascular endothelial growth factor, urinary advanced glycation end-products (AGE), and indirectly with eGFR (p < 0.0001, R2 = 0.808; p < 0.0001, R2 = 0.825; p < 0.0001, R2 = 0.805; p < 0.0001, R2 = 0.663; p < 0.0001, R2 = 0.726; p < 0.0001, R2 = 0.720; p < 0.0001, R2 = 0.724).ConclusionsIn patients with type 2 DM there is an association between urinary mRNA of podocyte-associated molecules, biomarkers of podocyte damage, and of PT dysfunction. GLEPP1, ADAM10, and NFκB may be considered additional candidate molecules indicative of early diabetic nephropathy. AGE could be involved in this association.

MP33-17 POTENTIAL ROLE OF A NOVEL URINARY BIOMARKER-BASED RISK SCORE TO SELECT PATIENTS FOR MULTIPARAMETRIC MRI FOR PROSTATE CANCER DETECTION.

You have accessJournal of UrologyProstate Cancer: Detection & Screening III1 Apr 2017MP33-17 POTENTIAL ROLE OF A NOVEL URINARY BIOMARKER-BASED RISK SCORE TO SELECT PATIENTS FOR MULTIPARAMETRIC MRI FOR PROSTATE CANCER DETECTION. Rianne J Hendriks, Leander Van Neste, Mulders Marloes, Jack A Schalken, and Inge M Van Oort Rianne J HendriksRianne J Hendriks More articles by this author , Leander Van NesteLeander Van Neste More articles by this author , Mulders MarloesMulders Marloes More articles by this author , Jack A SchalkenJack A Schalken More articles by this author , and Inge M Van OortInge M Van Oort More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.1013AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Prostate cancer (PCa) diagnostics would greatly benefit from more accurate, non-invasive techniques for the detection of clinically significant disease, leading to a reduction of over-diagnosis and over-treatment. Multiparametric MRI (mpMRI) is being used increasingly and has proven to be a valuable addition to the PCa diagnostic pathway. A novel biomarker-based risk score (SelectMDx) assessing urinary HOXC6 and DLX1 mRNA expression levels combined with traditional clinical risk factors, was recently developed to predict high-grade PCa (Gleason score >/=7) upon prostate biopsy and to reduce the number of unnecessary biopsies. The aim of this study was to investigate the correlation between the risk score and mpMRI outcomes. METHODS The patients in this retrospective observational cohort were previously included in the validation study of the SelectMDx risk score, in which urine was collected after digital rectal examination (DRE) from men undergoing prostate biopsies based on an elevated serum PSA level (>/=3.0 ng/ml) and/or suspicious DRE. A subset of patients underwent an mpMRI after prostate biopsies were performed (n=174). The indications for performing MRI were based on persistent clinical suspicion of PCa after negative prostate biopsies or staging after PCa was found upon biopsy. RESULTS 102 of 174 patients (59%) had PCa detected upon prostate biopsy, of which 54 (53%) had high-grade disease and a significantly higher SelectMDx risk score (p<0.001). The median SelectMDx risk score was also significantly higher in patients who had a suspicious lesion on MRI (p<0.001). For 81 mpMRI's the PIRADS classification was reported and there was a positive correlation observed between the risk score and the PIRADS classification (Figure 1). A Kruskal-Wallis test indicated a statistically significant difference in SelectMDx risk scores between the different PIRADS groups (p<0.001). CONCLUSIONS The novel urinary biomarker-based risk score is a promising tool in PCa detection. This study showed promising results regarding the correlation between the SelectMDx risk score with MRI outcomes. This risk score could potentially guide clinicians in selecting patients at risk for significant PCa for mpMRI. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e424 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Rianne J Hendriks More articles by this author Leander Van Neste More articles by this author Mulders Marloes More articles by this author Jack A Schalken More articles by this author Inge M Van Oort More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MP15-15 URINE SAMPLE DERIVED CK20- AND IGF2-EXPRESSION AS BIOMARKER FOR THE DETECTION OF BLADDER CANCER

You have accessJournal of UrologyBladder Cancer: Non-invasive I1 Apr 2017MP15-15 URINE SAMPLE DERIVED CK20- AND IGF2-EXPRESSION AS BIOMARKER FOR THE DETECTION OF BLADDER CANCER Karsten Salomo, Doreen Huebner, Manja U. Boehme, Alexander Herr, Ulrike Heberling, Oliver W. Hakenberg, Daniela Jahn, Marc-Oliver Grimm, Astrid Enkelmann, Daniel Steinbach, Susanne Fuessel, and Manfred P. Wirth Karsten SalomoKarsten Salomo More articles by this author , Doreen HuebnerDoreen Huebner More articles by this author , Manja U. BoehmeManja U. Boehme More articles by this author , Alexander HerrAlexander Herr More articles by this author , Ulrike HeberlingUlrike Heberling More articles by this author , Oliver W. HakenbergOliver W. Hakenberg More articles by this author , Daniela JahnDaniela Jahn More articles by this author , Marc-Oliver GrimmMarc-Oliver Grimm More articles by this author , Astrid EnkelmannAstrid Enkelmann More articles by this author , Daniel SteinbachDaniel Steinbach More articles by this author , Susanne FuesselSusanne Fuessel More articles by this author , and Manfred P. WirthManfred P. Wirth More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.500AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Cytokeratin 20 (CK20) and Insulin-like growth factor 2 (IGF2) were previously proposed to be elevated in patients with bladder cancer (BCa). A two cohort design validation study was used to assess the relevance for BCa detection of both markers in urine samples. The results were compared to voided urine cytology (VUC). METHODS RNA isolation was carried out using 5 ml spontaneous urine samples of 196 and 97* histologically positive BCa patients for the test and validation* cohort, respectively. Additionally patients with benign urological diseases, nephrolithiasis and unconfirmed BCa suspicion as well as healthy control subjects were included for a total of 103/50* control subjects in the test and validation* cohort. Urinary mRNA levels were determined by qPCR and the results were normalized as molecule ratio to RPLP0. Receiver operating characteristic curves and corresponding AUC values were used to evaluate the diagnostic performance of both markers. Optimal cut-off values were determined by Youden Index. RESULTS Urinary mRNA levels were significantly elevated 3.4/11*-fold for CK20 and 188/64*-fold for IGF2 in BCa patients compared to controls in the test and validation* cohort, respectively. IGF2 performed slightly better with an AUC of 0.83 for both cohorts compared to CK20 with an AUC of 0.78 and 0.82* for the test and validation* cohort, respectively. Combined analysis of IGF2 and CK20 with at least one positive marker resulted in a sensitivity (0.78/0.90*) and specificity (0.88/0.84*) similar to that of VUC. The sensitivity of VUC in combination with IGF2 and CK20 was considerably increased (0.95/0.93*) while specificity was slightly reduced (0.72/0.84*) compared to VUC alone in the test and validation* cohort. CONCLUSIONS Expressions of IGF2 and CK20 correlated significantly with BCa with a diagnostic performance similar to VUC. Combined analysis of voided urine cytology together with expression levels of CK20 and IGF2 clearly enhances overall test performance. Therefore, its relevance for BCa diagnosis should be evaluated in a larger cohort. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e176 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Karsten Salomo More articles by this author Doreen Huebner More articles by this author Manja U. Boehme More articles by this author Alexander Herr More articles by this author Ulrike Heberling More articles by this author Oliver W. Hakenberg More articles by this author Daniela Jahn More articles by this author Marc-Oliver Grimm More articles by this author Astrid Enkelmann More articles by this author Daniel Steinbach More articles by this author Susanne Fuessel More articles by this author Manfred P. Wirth More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Hypoxia Inducible Factor 1: A Urinary Biomarker of Kidney Disease.

Identifying noninvasive biomarkers of kidney disease is valuable for diagnostic and therapeutic purposes. Hypoxia inducible factor 1 (HIF‐1) expression is known to be elevated in the kidneys in several renal disease pathologies. We hypothesized that the urinary HIF‐1a mRNA level may be a suitable biomarker for expression of this protein in chronic kidney disease (CKD). We compared HIF‐1a mRNA levels from urine pellets of CKD and healthy subjects. To ensure that urinary HIF‐1a mRNA is of kidney origin, we examined colocalization of HIF‐1a mRNA with two kidney specific markers in urine cells. We found that HIF‐1a mRNA is readily quantifiable in urine pellets and its expression was significantly higher in CKD patients compared with healthy adults. We also showed that the urinary HIF‐1a mRNA comes primarily from cells of renal origin. Our data suggest that urinary HIF‐1a mRNA is a potential biomarker in CKD and can be noninvasively assessed in patients.

Feature selection and classification of urinary mRNA microarray data by iterative random forest to diagnose renal fibrosis: a two-stage study

Renal fibrosis is a common pathological pathway of progressive chronic kidney disease (CKD). However, kidney function parameters are suboptimal for detecting early fibrosis, and therefore, novel biomarkers are urgently needed. We designed a 2-stage study and constructed a targeted microarray to detect urinary mRNAs of CKD patients with renal biopsy and healthy participants. We analysed the microarray data by an iterative random forest method to select candidate biomarkers and produce a more accurate classifier of renal fibrosis. Seventy-six and 49 participants were enrolled into stage I and stage II studies, respectively. By the iterative random forest method, we identified a four-mRNA signature in urinary sediment, including TGFβ1, MMP9, TIMP2, and vimentin, 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. These findings showed that urinary mRNAs can serve as sensitive biomarkers of renal fibrosis, and the random forest classifier containing urinary mRNAs showed favourable performance in diagnosing early renal fibrosis.

Urinary Biomarker Panel to Improve Accuracy in Predicting Prostate Biopsy Result in Chinese Men with PSA 4-10 ng/mL.

This study aims to evaluate the effectiveness and clinical performance of a panel of urinary biomarkers to diagnose prostate cancer (PCa) in Chinese men with PSA levels between 4 and 10 ng/mL. A total of 122 patients with PSA levels between 4 and 10 ng/mL who underwent consecutive prostate biopsy at three hospitals in China were recruited. First-catch urine samples were collected after an attentive prostate massage. Urinary mRNA levels were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The predictive accuracy of these biomarkers and prediction models was assessed by the area under the curve (AUC) of the receiver-operating characteristic (ROC) curve. The diagnostic accuracy of PCA3, PSGR, and MALAT-1 was superior to that of PSA. PCA3 performed best, with an AUC of 0.734 (95% CI: 0.641, 0.828) followed by MALAT-1 with an AUC of 0.727 (95% CI: 0.625, 0.829) and PSGR with an AUC of 0.666 (95% CI: 0.575, 0.749). The diagnostic panel with age, prostate volume, % fPSA, PCA3 score, PSGR score, and MALAT-1 score yielded an AUC of 0.857 (95% CI: 0.780, 0.933). At a threshold probability of 20%, 47.2% of unnecessary biopsies may be avoided whereas only 6.2% of PCa cases may be missed. This urinary panel may improve the current diagnostic modality in Chinese men with PSA levels between 4 and 10 ng/mL.

The effects of sildenafil citrate on urinary podocin and nephrin mRNA expression in an L-NAME model of pre-eclampsia.

We investigated the effects of sildenafil citrate (SC) on podocyturia in N ω-nitro-L-arginine methyl ester hydrochloride (L-NAME) model of pre-eclampsia (PE). One hundred and twenty Sprague-Dawley rats (SDR) were divided into five groups like pregnant control (PC), early-onset PE (EOPE), late-onset PE(LOPE), early and late-onset PE with SC-treated groups [EOPE (SC); LOPE (SC)]. PE was induced in SDR by oral administration of L-NAME in drinking water for 4-8days for EOPE and 8-14day for LOPE. The blood pressure, urine volume and total urine protein were increased in EOPE and LOPE groups when compared to PC, and all the above parameters decreased in EOPE (SC) and LOPE (SC) groups when compared to EOPE and LOPE groups, respectively. The EOPE and LOPE groups showed an increase in urinary nephrin mRNA and podocin mRNA levels compared to PC group. Increases in serum and renal soluble fms-like tyrosine kinase-1 (sFlt-1) expression levels and decreases in renal vascular endothelial growth factor (VEGF) expression and serum placenta growth factor (PlGF) levels were observed in EOPE and LOPE groups when compared to PC group. In addition, decreases in serum and renal sFlt-1 expression levels and increases in renal VEGF expression and serum PlGF levels were observed in EOPE (SC) and LOPE (SC) groups when compared to EOPE and LOPE groups, respectively. The light microscopy showed that the renal tissue of L-NAME-treated rats had extensive glomerular damage, tubular damage and infiltration by mononuclear cells when compared to PC group. Therefore, SC ameliorated podocyturia through its effects on the antiangiogenic/angiogenic status in this animal model.

Urinary MicroRNA as Biomarker in Renal Transplantation.

Urine represents a noninvasive source in which proteins and nucleic acids can be assessed. Such analytes may function as biomarkers to monitor kidney graft pathology at every desired frequency, thereby providing a time window to prevent graft damage by therapeutic intervention. Recently, several proteins have been measured in urine as markers of graft injury. However, the specificity is limited, and measuring urinary proteins generally lacks the potential to predict early kidney graft damage. Currently, urinary mRNA and microRNA are being investigated to evaluate the prognostic value of changes in gene expression during the initial stages of graft damage. At such time point, a change in treatment regimen and dosage is expected to have maximum potency to minimize future decline in graft function. Both mRNA and microRNAs have shown promising results in both detection and prediction of graft injury. An advantage of microRNAs compared to mRNA molecules is their stability, a characteristic that is beneficial when working with urine samples. In this review, we provide the current state of urinary biomarkers in renal transplantation, with a focus on urinary microRNA. In addition, we discuss the methods used to study urinary microRNA expression.

Urinary mRNA for the Diagnosis of Renal Allograft Rejection: The Issue of Normalization.

Urinary messenger RNA (mRNA) quantification is a promising method for noninvasive diagnosis of renal allograft rejection (AR), but the quantification of mRNAs in urine remains challenging due to degradation. RNA normalization may be warranted to overcome these issues, but the strategies of gene normalization have been poorly evaluated. Herein, we address this issue in a case-control study of 108 urine samples collected at time of allograft biopsy in kidney recipients with (n = 52) or without (n = 56) AR by comparing the diagnostic value of IP-10 and CD3ε mRNAs-two biomarkers of AR-after normalization by the total amount of RNA, normalization by one of the three widely used reference RNAs-18S, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Hypoxanthine-guanine phosphoribosyltransferase (HPRT)-or normalization using uroplakin 1A (UPK) mRNA as a possible urine-specific reference mRNA. Our results show that normalization based on the total quantity of RNA is not substantially improved by additional normalization and may even be worsened with some classical reference genes that are overexpressed during rejection. However, considering that normalization by a reference gene is necessary to ensure polymerase chain reaction (PCR) quality and reproducibility and to suppress the effect of RNA degradation, we suggest that GAPDH and UPK1A are preferable to 18S or HPRT RNA.

Nested quantitative PCR approach for urinary cell-free EZH2 mRNA and its potential clinical application in bladder cancer.

EZH2 is overexpressed in bladder cancer (BC) and plays important roles in tumor development and progression. Recent studies show cell free (cf) RNAs released from cancer cells can reflect tissues changes and are stable and detectable in urine. Although conventional quantitative real-time PCR (qPCR) is highly sensitive, low abundances of urinary cf-RNAs usually result in false-negatives. Thus, this study develops a nested qPCR (nqPCR) approach to quantify cf-EZH2 mRNA in urine and further assess its clinical significance for BC. Forty urine samples were first selected to evaluate feasibility of nqPCR. Then, levels of urinary cf-EZH2 mRNA were detected using developed method in an independent cohort of subjects with 91 healthy, 81 cystitis, 169 nonmuscle invasive BC (NMIBC) and 103 muscle-invasive BC (MIBC). In cf-EZH2 mRNA detection, nqPCR method was significantly associated with qPCR, but it could detect more urine samples and increase detection limit three orders of magnitude. Based on nqPCR method, cf-EZH2 mRNA levels have been found to be increased in urine of NMIBC and MIBC patients (p < 0.001). Compared with cytology, cf-EZH2 mRNA showed higher diagnostic ability for MIBC (p < 0.001) while not for NMIBC (p > 0.05). Moreover, it also could distinguish MIBC from NMIBC, with AUC of 0.787. For MIBC patients, high expression of cf-EZH2 mRNA associated with advanced stage and was an independent predictor of reduced disease free survival or overall survival. In conclusion, detection of cf-EZH2 mRNA in urine by nqPCR is a sensitive and noninvasive approach and may be used for diagnosis and prognosis prediction of MIBC.

Urinary mRNA biomarker panel for the detection of urothelial carcinoma

The early detection of bladder cancer is important as the disease has a high rate of recurrence and progression. The development of accurate, non-invasive urinary assays would greatly facilitate detection. In previous studies, we have reported the discovery and initial validation of mRNA biomarkers that may be applicable in this context. In this study, we evaluated the diagnostic performance of proposed molecular signatures in an independent cohort.Forty-four mRNA transcripts were monitored blindly in urine samples obtained from a cohort of 196 subjects with known bladder disease status (89 with active BCa) using quantitative real-time PCR (RT-PCR). Statistical analyses defined associations of individual biomarkers with clinical data and the performance of predictive multivariate models was assessed using ROC curves. The majority of the candidate mRNA targets were confirmed as being associated with the presence of BCa over other clinical variables. Multivariate models identified an optimal 18-gene diagnostic signature that predicted the presence of BCa with a sensitivity of 85% and a specificity of 88% (AUC 0.935). Analysis of mRNA signatures in naturally micturated urine samples can provide valuable information for the evaluation of patients under investigation for BCa. Additional refinement and validation of promising multi-target signatures will support the development of accurate assays for the non-invasive detection and monitoring of BCa.