Renal tubular epithelial cell related partial epithelial-mesenchymal transition in AAⅠ induced renal fibrosis via Wnt7b/β-catenin signaling

Abstract

Introduction: This study investigates the pathological progressions in kidneys affected by aristolochic acid nephropathy (AAN) and explores the molecular mechanisms underlying the fibrotic process, specifically focusing on the Wnt7b/β-catenin signaling pathway.

Methods: Both mice and human kidney-2 (HK-2) cells were treated with aristolochic acid I (AAI). In mice, we monitored blood urea nitrogen (BUN), serum creatinine (Scr), kidney injury molecule-1 (KIM-1), pathological modifications of renal tubular epithelial cells (RTECs), and fibrosis degrees during acute/chronic disease phases. Wnt7b/β-catenin expression was evaluated through transcriptome analysis and laboratory assays (immunohistochemistry, Western blotting, immunoelectron microscopy) in acute AAN and cultured cells. Concurrent assays measured representative proteins: Aquaporin 1 (AQP1), Topoisomerase IIα (TOP2A), Vascular Cell Adhesion Molecule-1 (VCAM-1), and α-smooth muscle actin (α-SMA) in chronic AAN RTECs.

Results: AAI increased Scr, BUN, and KIM-1 levels by causing RTEC necrotic shedding in acute stages and promoted renal interstitial fibrosis chronically. Elevated Wnt7b pathway proteins enhanced damaged RTEC regeneration, with regenerated cells expressing mesenchymal proteins VCAM-1 and α-SMA.

Discussion: The Wnt7b/β-catenin signaling pathway connects acute tubule damage to fibrosis, explaining AAN's pathological continuum. These findings clarify how acute injury progresses to chronic fibrosis in AAN.

Keywords: Wnt7b/β-catenin; aristolochic acid nephropathy; damage and repair; epithelial-mesenchymal transition; renal tubular epithelial cell.

FIGURE 1

Renal pathological manifestations in acute AAN. (A) Survival curves. (B) Representative images of HE staining. Yellow arrows point to the rupture of RTEC villi. Blue arrows indicate cell edema. Red arrows show cell necrosis. Black arrows mark the naked basement membrane (BM naked), which is formed following the necrosis and shedding of epithelial cells. (C) Levels of BUN and Scr. (D) Representative images of Masson staining. (E) Representative images of KIM-1. (F) Injured Proportions of total tubules, PCT, and DCT. (G) Analysis of renal interstitial fibrosis. (H) Analysis of KIM-1 positive areas. Notes: Data were presented as mean ± SD with at least three independent experiments. **p < 0.01, ***p < 0.005, ****p < 0.001 vs. AAN-Con.

FIGURE 2

Analysis of the transcriptome database in AAN. (A) Expression levels of genes related to the Wnt signaling pathway. (B) Expression levels of EMT related genes. (C) Based on single-cell sequencing data from Chen (Chen et al., 2022), the levels of Wnt signaling pathway-related genes was increased in the proximal tubule cells.

FIGURE 3

Expression Profiles of Wnt7b, β-catenin, and MMP7 Proteins. (A) Representative images obtained from IHC staining. (B,C) WB images with β-actin and AQP1 as references, respectively. (D) OD analysis of proteins in IHC. (E,F) Quantitative analysis of proteins with β-actin and AQP1 as references, respectively. Data are presented as mean ± SD based on at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001 vs. AAN-Con.

FIGURE 4

Ultrastructural localization of Wnt7b and β-catenin. Red arrows and circles highlight the presence of Wnt7b (tagged with 12 nm gold nanoparticles). Black circles denote the location of β-catenin (labeled with 4 nm gold nanoparticles). Blue arrows point to mitochondria.

FIGURE 5

Response of HK-2 cells to AAI exposure. (A) Morphological changes of HK-2 cells. (B) Cell viability of HK-2 cells. (C) Levels of Wnt7b, β-catenin and MMP7 protein. (D) Analysis of Wnt7b, β-catenin and MMP7 in HK-2 cells. Data are represented as mean ± SD with at least three independent experiments. *p < 0.05, ***p < 0.005, ****p < 0.001vs. Con.

FIGURE 6

PCTECs regeneration and expression levels of AQP1, TOP2A, VCAM-1 and α-SMA. (A) Representative images of HE staining. Blue arrows indicate regenerated flat cells, red arrows indicate regenerated columnar cells. (B) Proportion of seemingly normal tubules (sn-PCTs) (C) Representative images of AQP1, TOP2A, VCAM-1 and α-SMA assayed by IHC staining. (D) OD analysis of proteins in IHC. **p < 0.01, ****p < 0.001 vs. AAN-Con; ^#### p < 0.001 vs. AAN-8d.

FIGURE 7

Mechanism of Wnt7b promoting renal fibrosis in AAN. When PCTECs are exposed to AAⅠ, the expression level of the Wnt7b protein rises. Subsequently, it is secreted extracellularly and activated by porcupine O-acyltransferase (PORCN). Once activated, Wnt7b attaches to receptors on the cell membrane, thus triggering the Wnt/β-catenin signaling pathway. As a result, downstream proteins of this pathway are synthesized, ultimately promoting the epithelial-mesenchymal transition of PCTECs, which is associated with renal fibrosis.