Abstract:

Objective ·To investigate the mechanism by which wingless-type MMTV integration site family member 5A (WNT5A) promotes the transition from ischemia-reperfusion injury (IRI)-induced acute kidney injury (AKI) to chronic kidney disease (CKD). Methods ·An IRI-induced AKI-to-CKD transition model was established in wild-type (WT) mice. Renal histopathological injury was assessed by hematoxylin-eosin (H-E) staining on the 3rd and the 14th day after IRI. Fibrosis was evaluated by Masson staining and immunohistochemistry for collagen type Ⅰ α1 chain (COL1A1). Wnt5a expression was quantified by RNA sequencing (RNA-seq), real-time quantitative PCR (qPCR), and Western blotting. Wnt5a heterozygous knockout (Wnt5a^+/- ) mice were used to establish the IRI-induced AKI-to-CKD transition model. Renal function was evaluated by serum creatinine (Scr) and blood urea nitrogen (BUN), and renal injury and fibrosis were assessed as described above. In vitro, a transforming growth factor-β (TGF-β)-induced fibrotic model was established in HK-2 cells (a human renal cortex proximal tubular epithelial cell line) with WNT5A overexpression. Differentially expressed genes identified by RNA-seq were subjected to Gene Ontology (GO) biological process enrichment analysis to identify key targets. Regulation of forkhead box protein P1 (FOXP1) by WNT5A was validated by qPCR and Western blotting. FOXP1 was overexpressed, or silenced in the context of WNT5A overexpression in HK-2 cells. Expression levels of notch receptor 2 (NOTCH2) pathway components, including NOTCH2, hairy/enhancer-of-split related with YRPW motif 1(HEY1), and hairy and enhancer of split 1 (HES1), as well as fibrosis markers, including COL1A1 and vimentin (VIM), were analyzed by qPCR and Western blotting. Results ·Wnt5a expression was increased in the renal tissue on the 3rd day after IRI, accompanied by marked tubular dilation and inflammatory cell infiltration. On the 14th day, Wnt5a expression was further increased, along with significant upregulation of fibrotic markers. Compared with WT mice, Wnt5a^+/- mice exhibited reduced Scr and BUN levels on the 14th day after IRI, indicating improved renal function. FOXP1 expression was decreased, and tubular injury and renal fibrosis were attenuated in Wnt5a^+/- mice. Mechanistically, WNT5A overexpression markedly upregulated FOXP1 in TGF-β-treated HK-2 cells. RNA-seq and GO analysis indicated significant enrichment of fibrosis-related processes and activation of the NOTCH signaling pathway. FOXP1 overexpression increased the mRNA and protein levels of NOTCH2 and its downstream targets HEY1 and HES1, along with elevated COL1A1 and VIM expression. In contrast, silencing FOXP1 in WNT5A-overexpressing cells reversed the upregulation of NOTCH2 pathway components and fibrosis markers. Conclusion ·WNT5A upregulates the transcription factor FOXP1, which activates the NOTCH2 signaling pathway, thereby accelerating the IRI-induced AKI-to-CKD transition, and promoting renal fibrosis progression.

Key words: acute kidney injury (AKI), chronic kidney disease (CKD), renal fibrosis, wingless-type MMTV integration site family member 5A (WNT5A), forkhead box protein P1 (FOXP1), notch receptor 2 (NOTCH2)