Urinary mRNA Research Articles

FOXP3 mRNA Profile Prognostic of Acute T Cell-mediated Rejection and Human Kidney Allograft Survival.

T cell-mediated rejection (TCMR) is the most frequent type of acute rejection and is associated with kidney allograft failure. Almost 40% of TCMR episodes are nonresponsive to therapy, and molecular mechanisms for the nonresponsiveness are unknown. Our single-center study identified that urinary cell FOXP3 mRNA abundance predicts TCMR reversibility and allograft survival. We developed PCR assays and measured absolute copy numbers of transcripts for FOXP3, CD25, CD3E, perforin, and 18S rRNA in 3559 urines from 480 kidney allograft recipients prospectively enrolled in the multicenter Clinical Trials in Organ Transplantation-04. In this replication study, we investigated the association between mRNA profile and TCMR diagnosis, TCMR reversibility, and allograft survival. 18S rRNA normalized levels of mRNA for FOXP3 (P = 0.01, Kruskal-Wallis test), CD25 (P = 0.01), CD3E (P < 0.0001), and perforin (P < 0.0001) were diagnostic of TCMR, but only FOXP3 mRNA level predicted TCMR reversibility (ROC AUC = 0.764; 95% confidence interval, 0.611-0.917; P = 0.008). Multivariable logistic regression analyses showed that urinary cell FOXP3 mRNA level predicted reversal, independent of clinical variables. A composite model of clinical variables and FOXP3 mRNA (AUC = 0.889; 95% CI, 0.781-0.997; P < 0.001) outperformed FOXP3 mRNA or clinical variables in predicting TCMR reversibility (P = 0.01, likelihood ratio test). Multivariable Cox proportional hazards regression analyses showed that FOXP3 mRNA level predicts kidney allograft survival (P = 0.047) but not after controlling for TCMR reversal (P = 0.477). Urinary cell level of FOXP3 mRNA is diagnostic of TCMR, predicts TCMR reversibility, and is prognostic of kidney allograft survival via a mechanism involving TCMR reversal.

Outfoxing Rejection: Urinary FOXP3 mRNA, TCMR, and the Fate of Allografts.

Outfoxing Rejection: Urinary FOXP3 mRNA, TCMR, and the Fate of Allografts.

Evaluation of urinary FOXP3 mRNA as a biomarker of lupus nephritis in Egyptian patients with systemic lupus erythematosus.

Lupus nephritis (LN) is one of the most serious complications of SLE. Tregs (Regulatory T lymphocytes) are thought to play a part in the pathogenesis of SLE. According to recent research, Foxp3, a Treg identification marker, plays a significant role in the pathogenesis of SLE. This study aimed to compare the urinary Foxp3 mRNA levels of patients with active and inactive forms of LN and healthy control subjects to see whether it played a role in disease activity. We measured FOXP3 messenger RNA (mRNA) expression in the urine of 50 people with active LN, 50 people with inactive lupus, and 50 healthy people. We found that the expression level of FOXP3 was significantly higher in urine from patients with active LN than from subjects with inactive lupus and healthy controls (22.93 ± 4.13 vs 5.66 ± 0.47 vs 0.57 ± 0.15copy; P < 0.001).Urinary FOXP3 mRNA level significantly correlated with SLEDAI (0.000057) In the active group, urinary FOXP3 mRNA level also significantly correlated with histological activity index (< 0.00001). We concluded that urinary FOXP3 mRNA is elevated in patients with active LN and that it is linked to the SLEDAI and the severity of the disease. FOXP3 mRNA in urine sediment may be used as a non-invasive biomarker for evaluating the severity of LN and risk stratification.

Urinary Exosomal CA9 mRNA as a Novel Liquid Biopsy for Molecular Diagnosis of Bladder Cancer.

ObjectiveThe objective of this study was to assess the possibility of using urinary exosomal CA9 mRNA as a novel liquid biopsy for the molecular diagnosis of bladder cancer.Patients and MethodsA total of 168 bladder cancer patients and 90 control subjects were included in the study. An isolation kit was used to isolate urinary exosomes. Transmission electron microscopy (TEM) was used to examine the presence of exosomes. Flow cytometry was used to examine the exosomal marker CD63. The expression level of exosomal CA9 mRNA was detected by RT-qPCR. The diagnostic performance of urinary urinary exosomal CA9 mRNA was evaluated.ResultsTEM confirmed the enriched exosomes from urinary bladder patients. Flow cytometry indicated a strong positive expression of exosome marker CD63. Successful extraction of RNA was performed from exosome samples. The level of urinary exosomal CA9 mRNA was significantly higher in bladder cancer group than in control group (p<0.001). The area under the ROC curve was 0.837 (95% CI: 0.743–0.859) with a sensitivity of 85.18% and a specificity of 83.15% for the diagnosis of bladder cancer.ConclusionWe found that the urinary exosomes were abundant in the urine of bladder cancer patients. CA9 mRNA could be detectable in urinary exosomes. The urinary exosomal CA9 mRNA may present a new liquid biopsy for the diagnosis of bladder cancer.

Enhanced expression of ncc1 and clc2c in the kidney and urinary bladder accompanies freshwater acclimation in Mozambique tilapia

Euryhaline fishes maintain hydromineral balance in a broad range of environmental salinities via the activities of multiple osmoregulatory organs, namely the gill, gastrointestinal tract, skin, kidney, and urinary bladder. Teleosts residing in freshwater (FW) environments are faced with the diffusive loss of ions and the osmotic gain of water, and, therefore, the kidney and urinary bladder reabsorb Na+ and Cl− to support the production of dilute urine. Nonetheless, the regulated pathways for Na+ and Cl− transport by euryhaline fishes, especially in the urinary bladder, have not been fully resolved. Here, we first investigated the ultrastructure of epithelial cells within the urinary bladder of FW-acclimated Mozambique tilapia (Oreochromis mossambicus) by electron microscopy. We then investigated whether tilapia employ Na+/Cl− cotransporter 1 (Ncc1) and Clc family Cl− channel 2c (Clc2c) for the reabsorption of Na+ and Cl− by the kidney and urinary bladder. We hypothesized that levels of their associated gene transcripts vary inversely with environmental salinity. In whole kidney and urinary bladder homogenates, ncc1 and clc2c mRNA levels were markedly higher in steady-state FW- versus SW (seawater)-acclimated tilapia. Following transfer from SW to FW, ncc1 and clc2c in both the kidney and urinary bladder were elevated within 48 h. A concomitant increase in branchial ncc2, and decreases in Na+/K+/2Cl−cotransporter 1a (nkcc1a) and cystic fibrosis transmembrane regulator 1 (cftr1) levels indicated a transition from Na+ and Cl− secretion to absorption by the gills in parallel with the identified renal and urinary bladder responses to FW transfer. Our findings suggest that Ncc1 and Clc2c contribute to the functional plasticity of the kidney and urinary bladder in tilapia.

Non-Invasive Diagnosis for Acute Rejection Using Urinary mRNA Signature Reflecting Allograft Status in Kidney Transplantation.

Urine has been regarded as a good resource based on the assumption that urine can directly reflect the state of the allograft or ongoing injury in kidney transplantation. Previous studies, suggesting the usefulness of urinary mRNA as a biomarker of acute rejection, imply that urinary mRNA mirrors the transcriptional activity of the kidneys. We selected 14 data-driven candidate genes through a meta-analysis and measured the candidate genes using quantitative PCR without pre-amplification in the cross-sectional specimens from Korean kidney transplant patients. Expression of 9/14 genes (CXCL9, CD3ϵ, IP-10, LCK, C1QB, PSMB9, Tim-3, Foxp3, and FAM26F) was significantly different between acute rejection and stable graft function with normal pathology and long-term graft survival in 103 training samples. CXCL9 was also distinctly expressed in allografts with acute rejection in in situ hybridization analysis. This result, consistent with the qPCR result, implies that urinary mRNA could reflect the magnitude of allograft injury. We developed an AR prediction model with the urinary mRNAs by a binary logistic regression and the AUC of the model was 0.89 in the training set. The model was validated in 391 independent samples, and the AUC value yielded 0.84 with a fixed manner. In addition, the decision curve analysis indicated a range of reasonable threshold probabilities for biopsy. Therefore, we suggest the urine mRNA signature could be used as a non-invasive monitoring tool of acute rejection for clinical application and could help determine whether to perform a biopsy in a recipient with increased creatinine.

FC 091URINARY CCL5 MRNA, A POTENTIAL BIOMARKER FOR PROGRESSION OF TYPE 2 DIABETIC NEPHROPATHY

Abstract Background and Aims In recent years, it has been found that targeted mRNA detection of sediment cells in urine can be used as novel biomarkers for the diagnosis of diabetic nephropathy (DN). However, its value in predicting the progression of DN is not clear. The purpose of this study is to seek for urinary mRNA markers that evaluate the prognosis of DN through systems biological screening, clinical verification and prospective studies. Method GEO database and “Nephroseq” platform were searched, and the transcriptome data of DN glomeruli and tubules and their clinical information were obtained. Weighted gene co-expression network analysis (WGCNA) combined with gene ontology (GO) annotation and KEGG pathway enrichment analysis were used to screen the hub genes negatively related to eGFR, and the hub genes were used as candidate markers for mRNA detection in urine sediment in DN patients. A total of 91 patients with DN diagnosed by renal biopsy were included, and 60 patients with type 2 diabetes and 61 healthy people were selected as the control groups. The mRNA expression of candidate molecules was detected by Taqman probe quantitative PCR, and the correlation between mRNA expression and eGFR and urinary protein levels were analyzed. Patients with DN were followed up for a median time of 21 months, and the primary end point was defied as end stage renal disease or eGFR decreasing by more than 50%. Multivariate Cox regression was used to evaluate the value of mRNA in predicting DN progression. Results GSE30528 and GSE30529 datasets were selected for analysis, including mRNA expression data of 9 cases of DN and 13 cases of normal glomeruli; and 10 cases of DN and 12 cases of normal tubules respectively. The clinical data of the patients in this study, including gender, race, age and eGFR, were searched on the Nephroseq platform. The gene modules negatively related to eGFR were screened by WGCNA. GO and KEGG analysis showed that the main function of the gene modules in both datasets were related to the activation of inflammatory cells and chemokines pathway. Through the screening of hub genes and the comparison of expression levels, CCL5, CXCL1, CXCL6 and CXCL12 were finally obtained as candidate genes. Quantitative PCR showed that the levels of CCL5 and CXCL1 was significantly increased in DN group, CCL5 was negatively correlated with eGFR and positively correlated with urinary protein level, while CXCL1 was negatively correlated with eGFR, but had no significant correlation with urinary protein level. Multivariate Cox regression showed that eGFR, urinary protein level, degree of renal fibrosis and urinary CCL5 were independent risk factors of primary end point. Conclusion The activation of chemokine signal pathway in renal tissue is involved in the progression of DN. Urinary CCL5 mRNA can independently predict the prognosis of DN and may be served as a novel biomarker for the progression of DN.

SelectMDx and Multiparametric Magnetic Resonance Imaging of the Prostate for Men Undergoing Primary Prostate Biopsy: A Prospective Assessment in a Multi-Institutional Study.

Simple SummaryProstate-specific antigen and digital rectal examination, used to guide prostate biopsy, often result in overdiagnosis of indolent prostate cancer (PCa) while missing clinically significant PCa (csPCa). The aim of this study was to evaluate the diagnostic accuracy of SelectMDx and its association with multiparametric magnetic resonance imaging (mpMRI) in predicting PCa in prostate biopsies. SelectMDx was revealed to be a good predictor of PCa, while with regards to csPCa detection, it was demonstrated to be less effective, showing results similar to mpMRI. The best diagnostic strategy to avoid unnecessary biopsy is performing SelectMDx after an initial negative mpMRI. Biopsy could be proposed for all cases of positive mpMRI and to those with a negative mpMRI followed by a positive SelectMDx.Prostate-specific antigen (PSA) testing as the sole indication for prostate biopsy lacks specificity, resulting in overdiagnosis of indolent prostate cancer (PCa) and missing clinically significant PCa (csPCa). SelectMDx is a biomarker-based risk score to assess urinary HOXC6 and DLX1 mRNA expression combined with traditional clinical risk factors. The aim of this prospective multi-institutional study was to evaluate the diagnostic accuracy of SelectMDx and its association with multiparametric magnetic resonance (mpMRI) when predicting PCa in prostate biopsies. Overall, 310 consecutive subjects were included. All patients underwent mpMRI and SelectMDx prior to prostate biopsy. SelectMDx and mpMRI showed sensitivity and specificity of 86.5% vs. 51.9%, and 73.8% vs. 88.3%, respectively, in predicting PCa at biopsy, and 87.1% vs. 61.3%, and 63.7% vs. 83.9%, respectively, in predicting csPCa at biopsy. SelectMDx was revealed to be a good predictor of PCa, while with regards to csPCa detection, it was demonstrated to be less effective, showing results similar to mpMRI. With analysis of strategies assessed to define the best diagnostic strategy to avoid unnecessary biopsy, SelectMDx appeared to be a reliable pathway after an initial negative mpMRI. Thus, biopsy could be proposed for all cases of mpMRI PI-RADS 4–5 score, and to those with Prostate Imaging-Reporting and Data System (PI-RADS) 1–3 score followed by a positive SelectMDx.

Can repeat biopsy be skipped after initial complete resection of T1 bladder cancer? The role of a novel urinary mRNA biomarker

ObjectivesTo prospectively investigate the role of a urinary mRNA biomarker (Xpert Test) after initial complete resection of T1 bladder cancer (BC) for the prediction of positive repeat biopsy for malignancy. MethodsPatients who underwent TURBT for NMIBC between September 2018 and April 2020 were included. Patients with benign pathology, incomplete resection, concomitant CIS/upper tract urothelial tumor or muscle invasive BC, were excluded. 2 to 6 weeks after primary TURBT, voided urine sample was retrieved for Xpert analysis and patients were scheduled for repeat biopsy. The primary outcome was to determine the role of positive Xpert test to predict positive repeat biopsy for malignancy. ResultsDuring the study period, 254 patients met the study inclusion criteria of which 61 (24%) patients had recurrent NMIBC. Complete resection was censured by the presence of detrusor muscle in the specimen with documented T1 disease in all study participants. Xpert test was positive in 128 patients; of whom 85 (66.4%) showed positive repeat biopsy (HR=6.2, 95%CI=3.46-9.4, P = 0.002). The sensitivity, specificity, positive and negative predictive values of Xpert test for repeat biopsy were 85.9% (95%CI: 82-89), 72.3% (95%CI: 68-76), 66.4% (95%CI: 62-71) and 88.9% (95%CI: 85-94), respectively. On median (range) follow up of 12(3-25) months, tumor recurrence was encountered in 84 (35%) patients. On multivariate Cox regression analysis, Xpert test was significantly associated with tumor recurrence (HR= 9.7, 95%CI=5-18, P <0.001). ConclusionsPositive Xpert test after primary complete resection of T1 BC is significantly associated with positive repeat biopsy for malignancy. In addition, Xpert test is an independent predictor of early tumor recurrence. Xpert test might be applied after initial complete resection of NMIBC to minimize unnecessary repeat biopsy with potential saving of healthcare costs and reduction in patient morbidity.

Bladder cancer risk stratification using a urinary mRNA biomarker panel – A path towards cystoscopy triaging

ObjectivesThe risk of bladder cancer (BCa) diagnosis and recurrence necessitates cystoscopy. Improved risk stratification may inform personalized triage and surveillance strategies. We aim to develop a urinary mRNA biomarker panel for risk stratification in patients undergoing BCa screening and surveillance. Methods and MaterialsUrine samples were collected from patients undergoing cystoscopy for BCa screening or surveillance. In patients who underwent transurethral resection of bladder tumor, urine samples were categorized based on tumor histopathology, size, and focality. Subjects with intermediate and high-risk BCa based on American Urological Association (AUA) guideline for non-muscle invasive bladder cancer were classified as “increased-risk”; those with no cancer and AUA low-risk BCa were classified as “low-risk”. Urine was evaluated for ROBO1, WNT5A, CDC42BPB, ABL1, CRH, IGF2, ANXA10, and UPK1B expression. A diagnostic model to detect “increased-risk” BCa was created using forward logistic regression analysis of cycle threshold values. Model validation was performed with ten-fold cross-validation. Sensitivity and specificity for detection of “increased-risk” BCa was determined and net benefit analysis performed. ResultsUrine samples (n = 257) were collected from 177 patients (95 screening, 76 surveillance, 6 both). There were 65 diagnoses of BCa (12 low, 22 intermediate, 31 high risk). ROBO1, CRH, and IGF2 expression correlated with “increased-risk” disease yielding sensitivity of 92.5% (95% CI, 84.9%–98.1%) and specificity of 73.5% (95% CI, 67.7–79.9%). The overall calculated standardized net benefit of the model was 0.81 (95%CI, 0.71–0.90). ConclusionsA 3-marker urinary mRNA panel allows for non-invasive identification of “increased-risk” BCa and with further validation may prove to be a tool to reduce the need for cystoscopies in low-risk patients.

Discovery and Validation of a Urinary Exosome mRNA Signature for the Diagnosis of Human Kidney Transplant Rejection.

Developing a noninvasive clinical test to accurately diagnose kidney allograft rejection is critical to improve allograft outcomes. Urinary exosomes, tiny vesicles released into the urine that carry parent cells' proteins and nucleic acids, reflect the biologic function of the parent cells within the kidney, including immune cells. Their stability in urine makes them a potentially powerful tool for liquid biopsy and a noninvasive diagnostic biomarker for kidney-transplant rejection. Using 192 of 220 urine samples with matched biopsy samples from 175 patients who underwent a clinically indicated kidney-transplant biopsy, we isolated urinary exosomal mRNAs and developed rejection signatures on the basis of differential gene expression. We used crossvalidation to assess the performance of the signatures on multiple data subsets. An exosomal mRNA signature discriminated between biopsy samples from patients with all-cause rejection and those with no rejection, yielding an area under the curve (AUC) of 0.93 (95% CI, 0.87 to 0.98), which is significantly better than the current standard of care (increase in eGFR AUC of 0.57; 95% CI, 0.49 to 0.65). The exosome-based signature's negative predictive value was 93.3% and its positive predictive value was 86.2%. Using the same approach, we identified an additional gene signature that discriminated patients with T cell-mediated rejection from those with antibody-mediated rejection (with an AUC of 0.87; 95% CI, 0.76 to 0.97). This signature's negative predictive value was 90.6% and its positive predictive value was 77.8%. Our findings show that mRNA signatures derived from urinary exosomes represent a powerful and noninvasive tool to screen for kidney allograft rejection. This finding has the potential to assist clinicians in therapeutic decision making.

Transition of ANXA10 expression is a useful diagnostic and prognostic marker in upper tract urothelial carcinoma

Little is known about the diagnostic and prognostic markers of upper urinary tract urothelial cancer (UTUC) because of its rarity. ANXA10, a calcium dependent phospholipid binding protein, is differentially expressed in several cancers. It has been reported that ANXA10 expression is a urinary mRNA biomarker for detection of bladder urothelial cancer (BUC), and loss of ANXA10 expression is associated with BUC progression. To clarify the significance of ANXA10 in UTUC, we studied ANXA10 expression with immunohistochemistry (IHC). We analyzed ANXA10 by IHC in 117 respective cases of UTUC treated by radical nephroureterectomy without neoadjuvant chemotherapy. ANXA10 positivity (ANXA10+) was defined as >10 % of nuclear staining. ANXA10 expression was weak in normal urothelium of upper tract and was positive in 39/117 (33%) of UTUC. ANXA10+ was more frequently in tumors with pure UC (36%, p<0.05), papillary morphology (50%, p<0.01), histological grade 1/2 (G1/2: 57%, p<0.01) and pTa/is/1 stage (55%, p<0.01) than in those with histological variants (0%), nodular morphology (9%), G3 (16%) and pT2/3/4 (13%), respectively. Patients with ANXA10+ showed better cancer specific survival (CSS) than those with ANXA10- (P<0.05). With IHC, ANXA10+ was detected more in TP53 negative cases (44%, P<0.01) and Uroplakin 3 (UPK3) positive cases (44%, p= 0.089) than in TP53 positive cases (6%) and UPK3 negative cases (28%), respectively. In TCGA dataset of MIBC, higher ANXA10 expression was correlated with papillary morphology, lower grade/stage, luminal papillary subtype and wild type of TP53. ANXA10 expression increases during carcinogenesis, and is observed more frequently in papillary UC with lower grade and stage. However, its expression decreases during cancer progression. Transition of ANXA10 expression in UTUC mimicking BC is clinically useful for decision-making.

Urinary podocyte mRNAs precede microalbuminuria as a progression risk marker in human type 2 diabetic nephropathy

Earlier detection of progression risk in diabetic nephropathy will allow earlier intervention to reduce progression. The hypothesis that urinary pellet podocyte mRNA is a more sensitive progression risk marker than microalbuminuria was tested. A cross sectional cohort of 165 type 2 diabetics and 41 age and sex-matched controls were enrolled. Podocyte stress (Urinary pellet podocin:nephrin mRNA ratio), podocyte detachment (Urinary pellet podocin mRNA:creatinine ratio: UPPod:CR) and a tubular marker (Urinary pellet aquaporin 2:creatinine ratio) were measured in macro-albuminuric, micro-albuminuric and norm-albuminuric groups. eGFR was reassessed after 4 years in 124 available diabetic subjects. Urinary pellet podocyte and tubular mRNA markers were increased in all diabetic groups in cross-sectional analysis. After 4 years of follow-up univariable and multivariate model analysis showed that the only urinary markers significantly related to eGFR slope were UPPod:CR (P < 0.01) and albuminuria (P < 0.01). AUC analysis using K-fold cross validation to predict eGFR loss of ≥ 3 ml/min/1.73m2/year showed that UPPod:CR and albuminuria each improved the AUC similarly such that combined with clinical variables they gave an AUC = 0.70. Podocyte markers and albuminuria had overlapping AUC contributions, as expected if podocyte depletion causes albuminuria. In the norm-albuminuria cohort (n = 75) baseline UPPod:CR was associated with development of albuminuria (P = 0.007) and, in the tertile with both normal kidney function (eGFR 84 ± 11.7 ml/min/1.73m2) and norm-albuminuria at baseline, UPPod:CR was associated with eGFR loss rate (P = 0.003). In type 2 diabetics with micro- or macro-albuminuria UPPod:CR and albuminuria were equally good at predicting eGFR loss. For norm-albuminuric type 2 diabetics UPPod:CR predicted both albuminuria and eGFR loss.

Investigations on Urinary mRNA Biomarkers That Reflect Pathologic Features of Patients with Diabetic Kidney Diseases

Investigations on Urinary mRNA Biomarkers That Reflect Pathologic Features of Patients with Diabetic Kidney Diseases

Your blood pressure might be normal, but what about your podocytes?

Associations among hypertension, podocyte depletion, and chronic kidney disease are well-established, but whether mean arterial pressure (MAP) in the normal range influences podocyte depletion has not been previously examined. In this issue, Naik etal. use non-invasive urinary mRNA analysis to demonstrate that higher podocyte stress and detachment are associated with higher MAP in the normal range. The relationship between blood pressure and podocyte health suddenly got much more interesting.

Urinary exosomal mRNA detection using novel isothermal gene amplification method based on three-way junction

Exosomal messenger RNA (mRNA) has emerged as a valuable biomarker for liquid biopsy-based disease diagnosis and prognosis due to its stability in body fluids and its biological regulatory function. Here, we report a rapid one-step isothermal gene amplification reaction based on three-way junction (3WJ) formation and the successful detection of urinary exosomal mRNA from tumor-bearing mice. The 3WJ structure can be formed by the association of 3WJ probes (3WJ-template and 3WJ-primer) in the presence of target RNA. After 3WJ structure formation, the 3WJ primer is repeatedly extended and cleaved by a combination of DNA polymerase and nicking endonuclease, producing multiple signal primers. Subsequently, the signal primers promote a specially designed network reaction pathway to produce G-quadruplex probes under isothermal conditions. Finally, G-quadruplex structure produces highly enhanced fluorescence signal upon binding to thioflavin T. This method provides a detection limit of 1.23 pM (24.6 amol) with high selectivity for the target RNA. More importantly, this method can be useful for the sensing of various kinds of mRNA, including breast cancer cellular mRNA, breast cancer exosomal mRNA, and even urinary exosomal mRNA from breast cancer mice. We anticipate that the developed RNA detection assay can be used for various biomedical applications, such as disease diagnosis, prognosis, and treatment monitoring.

Urinary Cell Transcriptome Profiling and Identification of ITM2A, SLAMF6, and IKZF3 as Biomarkers of Acute Rejection in Human Kidney Allografts.

Background.Identification of a shared gene expression pattern between T cell–mediated rejection (TCMR) and antibody-mediated rejection (AMR) in human kidney allografts may help prioritize targets for the treatment of both types of acute rejection.Methods.We performed RNA sequencing and bioinformatics of genome-wide transcriptome profiles of urinary cells to identify novel mRNAs shared between TCMR and AMR and of mechanistic relevance. Customized RT-QPCR assays were then used to validate their abundance in urinary cells. Urinary cell transcriptome profiles and mRNA abundance were assessed in 22 urine samples matched to 22 TCMR biopsies, 7 samples matched to 7 AMR biopsies, and 24 samples matched to 24 No Rejection (NR) biopsies and correlated with biopsy diagnosis.Results.RNA sequencing data and bioinformatics identified 127 genes in urine to be shared between TCMR and AMR. We selected 3 novel mRNAs—ITM2A, SLAMF6, and IKZF3—for absolute quantification and validation by customized RT-QPCR assays. The abundance of all 3 mRNAs was significantly higher in urine matched to TCMR or AMR than in urine matched to NR biopsies. Receiver-operating-characteristic curve analysis showed that all 3 mRNAs distinguished TCMR or AMR from NR. Their abundance was similar in patients with TCMR and those with AMR.Conclusions.State-of-the-art antirejection therapies are mostly effective to treat TCMR but not AMR. Our identification of mRNAs shared between TCMR and AMR and contributing to T cell–B cell interactions may help prioritize therapeutic targets for the simultaneous treatment of TCMR and AMR.

49 - Urinary mRNA using droplet digital PCR for prostate cancer screening in a Korean population

49 - Urinary mRNA using droplet digital PCR for prostate cancer screening in a Korean population

Ameliorative Effects of Bredemolic Acid on Markers Associated with Renal Dysfunction in a Diet-Induced Prediabetic Rat Model.

Recently, studies have shown that renal dysfunction is associated not only with overt diabetes but also with the preceding stage known as prediabetes. Diet and pharmacological interventions are the therapeutic approaches to managing prediabetes, but the compliance in combining the two interventions is low. Hence, the efficacy of pharmacological intervention is reduced without diet modification. In our previous study, we established that bredemolic acid (BA) ameliorated glucose homeostasis via increased GLUT 4 expression in the skeletal muscle of prediabetic rats in the absence of diet intervention. However, the effects of bredemolic acid on renal function in prediabetic condition are unknown. Therefore, this study was aimed at investigating the ameliorative effects of bredemolic acid on renal dysfunction in a diet-induced prediabetic rat model. Thirty-six Sprague-Dawley male rats (150–180 g) were divided into two groups: the nonprediabetic (n = 6) and prediabetic (n = 30) groups which were fed normal diet (ND) and high-fat high-carbohydrate (HFHC) diet, respectively, for 20 weeks. After the 20th week, the prediabetic groups were subdivided into prediabetic control (PD) and 4 other prediabetic groups which were treated with either BA (80 mg/kg) or metformin (MET, 500 mg/kg) for further 12 weeks (21st to 32nd). Plasma, urine, and kidney samples were collected for biochemical analysis. The untreated prediabetic (PD) rats presented increased fluid intake and urine output; increased creatinine, urea, and uric acid plasma concentrations; albuminuria; proteinuria; sodium retention; potassium loss; increased aldosterone and kidney injury molecule (KIM-1) concentration; and increased urinary podocin mRNA expression. However, BA administration attenuated the renal markers and oxidative stress and decreased the urinary podocin mRNA expression. In conclusion, BA administration, regardless of diet modification, attenuates renal dysfunction in an experimentally induced prediabetic state.

Podocyte-associated messenger ribonucleic acids profile in urine of patients with idiopathic nephrotic syndrome and lupus nephritis.

Background: Podocytes associated proteins are essential in the maintenance of a healthy glomerular filtration barrier, a spectrum of different glomerular diseases occurs due to podocyte abnormalities. Urinary podocytes mRNAs are a more accurate tool for monitoring the progression of different glomerular diseases than proteinuria.Methods: Quantification of podocyte mRNAs in urinary sediment by real time polymerase chain reaction (PCR) of idiopathic nephrotic syndrome, lupus nephritis patients (LN) and the healthy controls, each group contains 15 individuals to be correlated with proteinuria level and eGFR (by MDRD equation).Results: the urinary podocyte mRNAs in both idiopathic nephrotic syndrome and LN groups differed significantly in comparison to that of controls, there was a significant correlation when comparing renal function tests and estimated glomerular filtration rate (by MDRD equation) between nephrotic, LN groups and control group, the value of podocin showed significant correlation in active LN subgroup with its corresponding value in the non-active LN subgroup.Conclusions: This study has revealed that urine pellet podocyte mRNAs can be used as a tool for monitoring the progression of idiopathic nephrotic syndrome and lupus nephritis patients and that urinary podocin can be used as a marker for lupus nephritis activity.