Erythropoiesis-Stimulating Agent Protects Against Kidney Fibrosis by Inhibiting G2/M Cell Cycle Arrest

Abstract

Background: G2/M cell cycle arrest of proximal tubular epithelial cells following acute kidney injury results in maladaptive repair and promotes chronic kidney disease. We investigated whether erythropoiesis-stimulating agents (ESA) regulate G2/M arrest and mitigate kidney fibrosis. Methods: Human kidney 2 (HK-2) cells were stimulated with TGF-β or paclitaxel, treated with darbepoetin alfa (DARB) at 0.5 ug/mL or 5 ug/mL, and cell cycles were analyzed using flow cytometry. In vivo experiments involved intraperitoneal administration of DARB (0.5 or 5 ug/kg) to the unilateral ureteral obstruction (UUO) mouse model on post-operative days three and seven. Kidney fibrosis and cell cycle regulatory proteins were analyzed using immunohistochemistry, RT-PCR, and immunoblotting. The effect of DARB on kidney fibrosis was compared with that of a p53 inhibitor. Results: In HK-2 cells treated with TGF-β or paclitaxel, G2/M cell cycle regulatory proteins were upregulated; however, this effect was reversed by DARB treatment. Immunostaining for p53 and Ki-67 indicated that the proliferative and fibrotic activities observed in TGF-β-treated HK-2 cells were mitigated by DARB treatment. Histological analysis of UUO mice using F4/80 staining and TUNEL assay showed that DARB treatment reduced inflammatory cell infiltration and apoptotic cell accumulation. Additionally, fibrotic changes assessed by Masson's trichrome, Sirius red, and PAS staining confirmed the antifibrotic effects of DARB treatment in UUO mice, independent of changes in hemoglobin levels, suggesting a mechanism distinct from its hematopoietic effects. DARB reduced fibrosis-related markers by suppressing G2/M cell cycle regulatory markers and inhibited the JNK and p38-MAPK signaling pathways, which play key roles in kidney fibrosis in TGF-β-treated HK-2 cells and UUO mice. Finally, DARB treatment demonstrated an anti-fibrotic effect in HK-2 cells stimulated with TGF-β or paclitaxel, comparable to that of a p53 inhibitor. Conclusions: DARB treatment decreased G2/M cell phase arrest and attenuated kidney fibrosis, suggesting a new renoprotective mechanism for ESA.