Chronic antibody-mediated (CAMR) rejection remains a major obstacle in the long term survival of kidney transplants, and is associated in part with interstitial fibrosis (IF) and tubular atrophy (TA). However, the link between anti-donor HLA antibody and graft fibrosis is incompletely understood. We hypothesize that fibroblasts in the kidney may interact with anti-HLA antibodies, developing an activated phenotype capable of mediating renal fibrosis. To this end, mouse kidneys from H-2^b mice were transplanted into MHC incompatible H-2^d recipients (allografts) or their H-2^b littermates (isografts). Primary kidney fibroblasts (H-2^d) were cultured in the presence of anti-Class I H-2^d antibody or isotype control. Proliferation was measured by BrDU incorporation. Gene expression of activation markers α-smooth muscle actin (αSMA), FSP-1, fibronectin 1(FN1), and collagen1α (Col-1α) were analyzed by real-time PCR. AKT and ERK phosphorylation was measured by western blot. At 12 weeks, compared to isografts, mouse kidney allografts developed reduced renal function and histological features of cellular and antibody mediated injury with IF/TA, and circulating anti-MHC Class I antibody. Intragraft mRNA expression was significantly elevated for α-SMA (3.3±1.3-fold, p=0.04), FSP-1 (5.1±1.2-fold, p=0.007) and Col1α (6.9±1.9-fold; p=0.02) compared to isografts. Exposure of primary renal fibroblasts for 48h with anti-H-2^d antibody led to a significant increase in cell proliferation (26.9±6.7%; p=0.04). This was accompanied by a marked and significant increase in fibroblast mRNA expression (Table I), demonstrating an activated myofibroblast phenotype. At 30min exposure with 1¯o;g/ml anti-H-2^d antibody, compared to isotype control, there was dramatic phosphorylation of AKT at ser473 (38.5 ±0.4-fold; p=0.015) and thr308 (19.3 ±0.01-fold; p=0.02) but not ERK. These data demonstrate for the first time the potent effects of anti-MHC class I antibody on renal fibroblasts, with the induction of both proliferation and activation, via AKT phosphorylation. Further understanding of this mechanism may shed light on novel interventions to mitigate allograft fibrosis in the setting of CAMR.

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