Background: BK polyomavirus assoiciated nephropathy (BKVAN) is one of the major complications in renal transplants, and is associated with significant fibrosis It is important to study the mechanism of BKV pathogenesis which will help in determining biomarkers for early diagnosis of BKVAN and effective treatments to prevent graft failure.

Objective: To determine the key epigenetic mechanism BKV uses to induce fibrosis and necroptosis, and to assess DNA methyl transferase 1 (DNMT1) as a potential therapeutic target.

Methodology: Human Proximal Tubular Epithelial Cells (HPTCs) and CCD1105 were infected with BKV and RNA/DNA was isolated. Hypermethylation of DNA was assessed by Bisulfite Sequencing (BS) and Methylation Specific PCR (MSP), and gene expression was studied with Real-time PCR. Flow-cytometry assays and Western blots were done to study necroptosis.

Results: MSP showed hypermethylation in the promoters of E-cadherin (CDH1) and Collagen IV (COLIVA1) genes following BKV infection. As expected with increases in DNA methylation, both COL IV and CDH1 gene expression was downregulated (p-value<0.029), however, Collagen I was increased. The loss of E-cadherin, a decrease in the basal membrane collagen component and increases in collagen I all favor the process of epithelial to mesenchymal transition (EMT). BKV increased the expression of DNMT1, suggesting a mechanism of increased DNA methylation. Treating BKV infected cells with DNMT1 inhibitor RG108 increased transcription of COLIVA (mean factor of 40.368).

BKV infection increased the expression of Receptor-interacting serine/threonine-protein kinase 3 (RIPK3), phospho-Mixed Lineage Kinase Like pseudokinase (pMLKL) and High Mobility Group Protein B1 (HMGB1). Treatment of BKV infected cells with RG108 reduced the expression of these genes, suggesting that BKV may induce necroptosis epigenetically in renal tubular cells.

BS and MSP demonstrated that BKV hypermethylates the RB1 gene promoter, and treatment with RG108 showed a significant decrease in BKV replication (p-value<0.037).

Conclusion: Our results suggest that BKV epigenetically induces EMT and necroptosis. RG108 inhibits DNMT1, prevents these epigenetic events and blocks BKV replication. Hence, hypermethylated genes could act as potential biomarkers for early diagnosis and studies of targeting DNA methyltransferase to reduce BKV induced fibrosis are warranted.

CITATION INFORMATION: Borkar M, Srivastava V, Cunnington R, Sharma T, Tibbles L. Potential Biomarkers and Treatment for BK Virus Associated Nephropathy. Am J Transplant. 2016;16 (suppl 3).

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