Introduction and Aims: Kidney transplantation is the best available treatment for patients with end stage chronic kidney disease.The diagnosis of the pathology of the kidney graft relies on the determination of the creatinine, estimated glomerular filtration rate and proteinuria. These parameters are altered after the onset of renal injury and they are not able to discern between various pathologies. The kidney biopsy, an invasive and not extent of complications procedure, remains essential for the diagnosis of the renal pathology. Hence the importance of finding parameters that allow an early diagnosis of the pathology involved in graft dysfunction. Urine is ideal for the study of possible biomarkers, its collection is easy, non-invasive and repeatable.Our aim was to analyse the miRNA content of urinary extracellular vesicles of the transplanted patients and define differential patterns of expression of miRNA specific to the different pathologies of the transplanted kidney.
Methods: Kidney transplanted patients with a kidney biopsy performed for clinical indication were selected. The biopsies were assessed according to Banff classification. From all biopsies those exhibiting acute cellular rejection (ACR), interstitial fibrosis and tubular atrophy (IFTA) and calcineurin inhibitors toxicity
(CIT) were selected. Kidney transplanted patients with normal function (defined as eGFR CKD-EPI >90ml/min/1,72m2) and without proteinuria were defined as control group. First morning void urine was collected from patients. After low speed centrifugation to eliminate cell debris, Urine EVs were obtained by Size exclusion chromatography after urine concentration. EV-contained fractions selected as CD63 and CD9 positive fractions were pooled. Then, total RNA was extracted using mirCURY kit and high-throughput sequencing was done. Taqman qPCR were performed using the Taqman Advance miRNA assays for the following miRNAs (hsa-miR-1; has-miR-2, hsa-miR-3; hsa-miR-4, hsa-miR-5, hsa-miR-6; hsa-miR-7; hsa-miR-8; hsa-miR-9, has-miR-10). hsa-miR-10a-5p was used as internal control and Total RNA kidney (Thermosfisher) was used as positive control. Ct values of triplicates were averaged and used for ΔCt calculations. Principal component analysis (PCA), Receiver Operating Characteristic (ROC) analysis and Mann-Whitney test were done using GraphPrims v5 and SPSS v15.
P<0.05 were set as significant.
Results: A total of 8 control samples and 14 pathological samples (3 IFTA, 6 ACR and 5 CIT) were processed by next generation sequencing. A total of 1500 miRNAs were identified, then after normalization and principal component analysis 12 miRNAs were selected to be tested in a new cohort. A new control group(N=8) and patients (IFTA: n=9; ACR: n=9; CIT: n=6) were selected as mentioned before. Then, uEVs and total RNA were isolated as described above. We found that eight miRNAs had different expression patterns in the urine of patients with any kind of graft pathology compared to controls.
Conclusion: miRNA profiling in urine has potential as a novel method for detecting pathology in kidney transplant.