P1747GLOBAL MIRNOME PROFILE REVEALS INVOLMENT OF MITOGEN ACTIVATED PROTEIN KINASE (MAPK) PATHWAY IN PATIENTS WITH ANTIBODY MEDIATED REJECTION (CABMR) OF RENAL TRANSPLANT
Authors: Prasad Narayan , Singh Sushma , Singh Mantabya , Agarwal Vikas , SINGH HARSHIT 
Published: June 2020

Abstract

Background and Aims
MicroRNAs (miRNAs) are non-coding RNA that play pivotal role in modulating expression of multiple target genes at post-transcriptional level and have potential to modulate physiological & pathological processes thus can be used as potential therapeutic targets. The impact of miRNA regulations have been shown in various kidney disorders but its involvement in allograft function and immunity has not been investigated thoroughly thus we studied the miRNAs expression involved in CABMR.
The impact of miRNA regulation involved in allograft function and immunity has not been investigated thoroughly thus we studied the miRNAs expression involved in CABMR.

Method
Patients with CABMR (Banff’s classification- 2017) were included. 25 blood donors (M=18) with a mean age of 52.4Y were served as controls. Samples were processed for RNA isolation. RNA integrity was checked by running over 1% agarose gel and quantity and qualities were checked using nanodrop with 260/230 and 260/280 ≥ 1.8. Massive parallel sequencing was performed by HiSeq 3000 sequencing system (Illumina Inc.,USA). The data obtained from the sequencing run were processed and filtered using the miRNA analysis app (Illumina Inc., USA) and analysis were performed using the BaseSpace Sequence Hub genomics computing environment (Illumina Inc., USA). Dysregulated miRNAs were detected by quantitative miRNA stem loop RT-PCR technology (TaqMan MicroRNA Assays, Applied Biosystems, CA) as per manufactures protocol. Expression was measured by calculating fold change. Targets of dysregulated miRNAs were predicted by computational analysis using bioinformatics tools like miRTarBase and Target Scan Human as well as using databases. Pathway analysis of predicted targets were performed using DIANA - mirPath online analysis tool and Kyoto Encyclopedia Genes and Genomes (KEGG) database. Evaluation of MAPK signaling pathway was done by Immuno histochemistry (IHC).

Results
Total 46 patients (Male=40 with mean age of 46.82 Y) were enrolled for the study with post- transplant duration of 33.72 months. The demographic details with mean values were: S Creatinine = 1.68 mg/dl, BUN = 28.26 mg/dl, Tac level = 5.98 ng/ml, S Uric Acid = 6.95 mg/dl, S Albumin = 3.46 g/dl, Na+/K+ = 128.9/4.16 mmol/L, S Phosphorus = 3.18 mg/dl, S Alkaline phosphate = 83.22 u/L. Induction regimen (Basiliximab=40, ATG= 6) Baseline Immunosuppression MMF+Pred (Tacrolimus/Cyclosporin) = 43/3. C4d were positive in 23 and DSA+ in 3 patients.
Comparative miRnome analysis revealed that a total of 123 miRNAs showed differential level of expression in CABMR patients (n=3) as compared to the control (n=1) out of which 93 were upregulated while 40 were downregulated. Conserved level of differential expression shows upregulation of miR-9 (p≤0.0001), miR-21 (p=0.0024), miR-29a (p=0.0001), miR-126 (p=0.0076), miR-210 (p=0.0001) and miR-223 (p=0.0006) while hsa-miR-221 was downregulated.
Pathway analysis shows involvement of MAPK signaling pathways, Transforming growth factor-β (TGF-β) superfamily signaling pathways, Wnt signaling pathways, ErbB signaling pathways. IHC of MAPK signaling shows significantly positive cytoplasmic tubular intensity as well as inflammatory cells intensity.

Conclusion
Altered miRNA expression profiling was found in CABMR compared to healthy controls. MAPK signaling pathways is involved in the pathogenesis of CABMR MicroRNAs are crucial regulators of cell function. They are easy to detect and represent potentially good targets for novel therapies.