MicroRNAs in urine are suitable targets for non-invasive detection of acute rejection (AR), since they represent relatively stable analytes. Indeed, we found that PCR signals for several microRNAs in both sedimentary cells and isolated exosomes remained stable in urine collected freshly from 4 renal transplant patients at the bedside, when these urine samples had been incubated for up to 24 hours at room temperature.

Next, within the transplant period 2007-2014 we set up a discovery-validation strategy to identify microRNAs that are associated with incidence of AR. First, 742 microRNAs were profiled by qPCR (Exiqon LNA miRNome PCR panels) in urinary sediment of 8 recipients with AR and in 8 recipients with stable graft function. As validation, 10 microRNAs were analyzed in urinary sediments of 140 recipients with AR: morphologically indicative of borderline (n=5), cellular (n=67), vascular (n=37) or humoral rejection (n=31). Biopsies were negative for SV40. The group was compared with urinary sediment from 64 recipients, who had a surveillance biopsy taken showing no morphologic alterations indicative of rejection. RNA was extracted from the sediments, and a spike-in was added to the RNA to check efficiency of the cDNA reaction. MicroRNA levels were corrected for 3 reference microRNAs.

In the discovery set, 32 microRNAs were differentially expressed (P<0.05) between groups, of which 20 were significantly lower in the AR group. Several microRNAs matched with what was found in previous profiling studies in transplant biopsies and urines. In the validation set, most pronounced differences in the AR group compared to controls (all P<0.00001) were found for miR-25-3p (1.9-fold), miR-126-3p (2.9-fold), miR-142-5p (0.40-fold), miR-155-5p (3.1-fold), and miR-615-3p (0.23-fold). The latter four together distinguished AR from controls in a multivariate logistic regression model with a sensitivity of 90.0% and a specificity of 81.0%. Humoral and cellular rejections differed mostly in hematopoietic-cell-related miRs 126-3p (P=0.032) and 155-5p (P=0.016), but the difference was of lower significance after correction for multiple comparisons.

Measurement of microRNAs in urinary sediment of kidney transplant patients may help to non-invasively identify acute graft rejection.

CITATION INFORMATION: Gielis E, Anholts J, de Fijter H, Bajema I, Claas F, Eikmans M. MicroRNAs in Urinary Sediments as Non-Invasive Tool to Detect Acute Rejection After Kidney Transplantation. Am J Transplant. 2016;16 (suppl 3).

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