*Purpose: A major issue in kidney transplantation is the lack of validated non-invasive biomarkers for monitoring graft function, current biochemical markers (i.e., creatinine) have poor sensitivity and specificity and usually detect graft damage late on the injury process, and the shortcoming associated with currently used gold standard (graft biopsy). Consequently, most study designs in kidney transplantation include poor study end-points, jeopardizing patient classification and overall identification of new markers. In this study we used a combination of current used study endpoints along with molecular approaches to identify cell-free urine miRNA markers and extracellular vesicle (EVs) phenotyping as tools for monitoring graft function.

*Methods: From a prospectively collected large biorepository comprising 296 deceased donor primary renal transplant patients, 12 months urine samples from 65 patients with progression to graft dysfunction (P) and normal graft function (NP) were tested for cell free miRNA. 84 different miRNAs related to fibrosis and inflammation pathways were profiled. Normalized cell free miRNA data was used for unsupervised cluster analysis. MiRNA and clinical parameters of the clusters were compared. The phenotyping of the extracellular vesicles (EVs) from samples representing each cluster were done and compared.

*Results: Unlike the dichotomous categorization of samples based on eGFR trend, the normalized cell free molecular data-based classification of patients showed more than 2 cluster groups. Though the extreme groups predominantly consisted either P or NP-to allograft dysfunction following their eGFR trend there were a set of samples that were misclassified as NP while the molecular profiles were similar to P to allograft dysfunction. Three miRNAs (miR-204-5p, miR-10b-5p, miR-744-5p) were differentially expressed in the P group. mRNA target analysis for these miRNA revealed increase in growth factors like CTGF, IGF2, TGFB1, BMP4 all involved in overall sclerosis , increased IL3 signaling genes involved in hematopoiesis and B cell development. Furthermore, there were genes that enhance mesenchymal transitions like SMAD family genes, WNT5a, DVL3. The immune phenotyping of EVs (mainly epithelial tubular cell markers) showed the origin of these cell free urine miRNA.

*Conclusions: The current use of gold standards of renal function should be challenged. Hereby, a cell free urine miRNA panel was identified as an accurate and non-invasive graft monitoring tool for renal graft function.

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