*Purpose: Chronic antibody-mediated rejection (CAMR) is an important contributor to late kidney allograft loss, frequently accompanied by interstitial fibrosis and tubular atrophy. Donor-specific antibodies (DSA) that target the major histocompatibility complex (MHC) typically mediate the allograft injury. Yet the link between DSA and graft fibrosis remains incompletely understood. We hypothesized that fibroblasts residing in the kidney could interact with anti-MHC antibodies (Ab), and develop an activated phenotype capable of mediating renal fibrosis.

*Methods: Mouse renal embryonic fibroblasts (MEFs; ATCC: CCL-163; H-2^d) were cultured in the presence and absence of anti-MHC Class I H-2^d Ab. Gene expression of activation markers α-smooth muscle actin (αSMA), fibronectin 1 (FN1) and S100A4 were analyzed by real-time PCR. Cell activation was also evaluated by western blot analysis of phosphorylated mTOR signaling pathway molecules AKT and ERK.

*Results: Not unexpectedly, quiescent MEFs expressed high levels of MHC class I (MHC-I). Exposure of MEFs for 48h with anti-MHC-I Ab led to significant increase in cell proliferation compared to isotype control (78.39-fold±17.89, p=0.01). This was accompanied by a significant upregulation in expression of the profibrotic genes α-SMA (3.81-fold±0.15, p=0.01), S100A4 (2.02-fold±0.05, p=0.01), and FN1 (1.95-fold±0.07, p=0.01), consistent with an activated myofibroblast phenotype. Compared to isotype control Ab treatment, we detected increased phosphorylation of AKT at Ser473 (38.5-fold; p=0.02) and Thr308 (19.3-fold; p=0.02), but no significant change was observed for ERK, p70S6K or S6RP. Blockade of PI3K mitigated anti-MHC-I-mediated AKT phosphorylation and cell proliferation. Rapamycin treatment reduced anti-MHC-I Ab-mediated expression of mTOR, AKT, and the pro-survival signal Bcl-XL. Current studies are underway to investigate the effect of MHC class II stimulation on resident renal fibroblast function and phenotype.

*Conclusions: These studies highlight a novel mechanism of fibroblast activation and provide key insights of the potent ability of anti-MHC-I Abs to mediate this process in vitro through AKT phosphorylation. Further understanding of this mechanism may shed light on novel interventional and preventative strategies to mitigate DSA-mediated allograft fibrosis in the setting of CAMR.

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