Effect of anti-HLA antibodies on mRNA and miRNA expression in HK-2 cells: a preliminary study

Purpose: This study aimed to address the limited understanding of complement-associated molecular mechanisms in antibody-mediated rejection (ABMR) by evaluating ABMR-related mRNA and miRNA expression in an in vitro HK-2 cell model exposed to donor-specific anti-HLA antibodies.Materials and Methods: We used the HK-2 cell line derived from normal kidney epithelial cells. An end-stage renal disease patient with HLA class I donor specific antibody, who was registered on the kidney waiting list for a positive serum [DSA(+)] and a healthy male without blood transfusion [negative control DSA (-)] were included in the study. The model includes both complement-dependent cytotoxicity (CDC) with and without complement in cell culture. Then, immune response–related mRNA and miRNA expression levels were evaluated by qRT-PCR.Results: mRNA and miRNA expression levels were analyzed by qRT-PCR, revealing pronounced dysregulation in the DSA(+) CDC C(+) group. Let-7c-5p and miR-155 were markedly upregulated (~10³–10⁴-fold), whereas miR-21, miR-217, miR-145, miR-125a-5p, and miR-885 were downregulated (~10⁻²–10⁻⁴-fold). At the mRNA level, strong upregulation of TGF-β (~50-fold), TNF-α (~10-fold), and caspase-3 (~10-fold) was observed, while C1qB was markedly downregulated (~0.1-fold). Additionally, differences in the expression of TNF-α, C1qB and several miRNAs (miR-142-5p, miR-155, miR-125a-5p, miR-21, miR-29b-3p, miR-145, and miR-217) were detected between CDC complement C(−) and C(+) conditions.Conclusion: This study presents the initial characterization of an in vitro ABMR model using kidney tubule epithelial cells and donor specific anti-HLA–A2 (+) serum. Furthermore, this is the first allorecognition model that assesses both C1q mRNA and certain miRNA expressions linked to antibody mediated rejection simultaneously.