NIV Congres

RNA assessment in urine sediments from kidney transplant recipients may represent a non-invasive tool for detecting acute rejection. MicroRNAs are small, non-coding RNA molecules that negatively regulate mRNA expression, and which have gained interest for their role in rejection of kidney transplants.

Urinary sediment cells are prone to generate low-quantity and -quality RNA, the latter being a result of degradation of the RNA. Therefore, we first tested microRNA stability of artificially degraded RNA from isolated leukocytes and found that microRNA molecules remained stable, regardless of the extent of RNA degradation. Furthermore, microRNA expression could be reproducibly quantified in extremely low amounts of RNA (10 pg per reaction) with the help of locked nucleic acid (LNA)-enhanced PCR primers. In a pilot study, we aimed to discover in an unbiased manner urinary microRNAs that best distinguish conditions of acute rejection. Expression of all known human microRNAs (Exiqon, PCR panel, n=742) was profiled in RNA from urinary sediment samples of 6 kidney transplant recipients with biopsy-supported acute rejection (80.0 ± 46.9 days posttransplant; 4 with T-cell mediated rejection, 2 with vascular rejection) and of 6 patients without rejection (84.0 ± 18.8 days posttransplant). For each patient, microRNA signals were standardized to the average signal of five reference microRNAs. For the 499 microRNAs detected, the level of 15 microRNAs significantly discriminated acute rejections from controls (Mann-Whitney rank test, P<0.05). MicroRNAs 451 and 25 represented the most promising discriminative analytes, since their expression levels were high in the sediments and were at least 13-fold increased (P≤0.017) during acute rejection compared to the control group. 

In conclusion, we identified microRNAs in the urinary sediment, of which the expression levels are associated with acute rejection. Predictive and diagnostic value of these levels for acute rejection will be verified in a larger patient cohort.