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Contains fulltext : 235565.pdf (Publisher’s version ) (Open Access), There is an unmet need for noninvasive tools for diagnosis of rejection after kidney transplantation. The aim of this study was to determine the discriminative value of a combined cellular and molecular biomarker platform in urine for the detection of rejection. METHODS: First, microRNA (miR) molecules were screened in transplant biopsies and urine sediments of patients with acute rejection and patients without rejection and stable graft function. Second, the expression of 15 selected miRs was quantified in an independent set of 115 urine sediments of patients with rejection and 55 urine sediments of patients without histological signs of rejection on protocol biopsy. Additionally, CXCL-9 and CXCL-10 protein levels were quantified in the urine supernatant. RESULTS: Levels of miR-155-5p (5.7-fold), miR-126-3p (4.2-fold), miR-21-5p (3.7-fold), miR-25-3p (2.5-fold), and miR-615-3p (0.4-fold) were significantly different between rejection and no-rejection urine sediments. CXCL-9 and CXCL-10 levels were significantly elevated in urine from recipients with rejection. In a multivariable model (sensitivity: 89.1%, specificity: 75.6%, area under the curve: 0.94, P < 0.001), miR-155-5p, miR-615-3p, and CXCL-9 levels were independent predictors of rejection. Stratified 10-fold cross validation of the model resulted in an area under the curve of 0.92. CONCLUSIONS: A combined urinary microRNA and chemokine profile discriminates kidney transplant rejection from stable graft conditions.

Contains fulltext : 174152.pdf (publisher's version ) (Open Access), Animal studies have shown that the calcineurin inhibitors (CNIs) cyclosporine and tacrolimus can activate the thiazide-sensitive NaCl cotransporter (NCC). A common side effect of CNIs is hypertension. Renal salt transporters such as NCC are excreted in urinary extracellular vesicles (uEVs) after internalization into multivesicular bodies. Human studies indicate that CNIs also increase NCC abundance in uEVs, but results are conflicting and no relationship with NCC function has been shown. Therefore, we investigated the effects of CsA and Tac on the abundance of both total NCC (tNCC) and phosphorylated NCC at Thr60 phosphorylation site (pNCC) in uEVs, and assessed whether NCC abundance in uEVs predicts the blood pressure response to thiazide diuretics. Our results show that in kidney transplant recipients treated with cyclosporine (n = 9) or tacrolimus (n = 23), the abundance of both tNCC and pNCC in uEVs is 4-5 fold higher than in CNI-free kidney transplant recipients (n = 13) or healthy volunteers (n = 6). In hypertensive kidney transplant recipients, higher abundances of tNCC and pNCC prior to treatment with thiazides predicted the blood pressure response to thiazides. During thiazide treatment, the abundance of pNCC in uEVs increased in responders (n = 10), but markedly decreased in non-responders (n = 8). Thus, our results show that CNIs increase the abundance of both tNCC and pNCC in uEVs, and these increases correlate with the blood pressure response to thiazides. This implies that assessment of NCC in uEVs could represent an alternate method to guide anti-hypertensive therapy in kidney transplant recipients.

Contains fulltext : 174177.pdf (Publisher’s version ) (Open Access), 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.

Item does not contain fulltext, BACKGROUND: Levels of urinary biomarkers, such as kidney injury molecule 1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL), can determine the risk of renal damage and predict the outcome of multiple kidney diseases, including transplant rejection. Studies of urinary biomarkers frequently use urine that has been stored frozen before analysis without taking into account the potential of biomarker degradation during storage. STUDY DESIGN: Observational study. SETTING & PARTICIPANTS: 39 kidney transplant patients visiting an outpatient clinic. PREDICTOR: Urine storage conditions (4 degrees C and -80 degrees C with or without protease inhibitors) and centrifugation after thawing of samples. OUTCOMES: Urinary concentrations of NGAL and KIM-1. MEASUREMENTS: NGAL and KIM-1 were measured with commercially available enzyme-linked immunosorbent assay kits. RESULTS: Urinary NGAL and KIM-1 concentrations were stable in urine up to 48 hours when stored at 4 degrees C and up to 6 months when stored at -80 degrees C, independent of the addition of protease inhibitors. A centrifugation step prior to biomarker measurement did not change urinary biomarker concentrations of urine stored at -80 degrees C. LIMITATIONS: Only KIM-1 and NGAL were measured, which possibly limits the relevance of the findings when making determinations about other urinary biomarkers. CONCLUSIONS: This study recommends that urine samples collected for the assessment of KIM-1 or NGAL are immediately cooled to 4 degrees C and subsequently frozen at -80 degrees C within 2 days. At -80 degrees C, both KIM-1 and NGAL concentrations are stable for at least 6 months.

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