Figure 8. Impact of AAV-directed ALDH2 gene on Acr-PC expression, fibrosis markers, inflammatory cytokines, and kidney damage markers in Aldh2*1 and Aldh2*2/*2 mice after UUO.

Wild-type (WT) mice (n = 5) and Aldh2*2/*2 mice (n = 5) underwent subcapsular (SC) injections of 2 × 10¹¹ genome copies of AAV8-ALDH2-EGFP for 28 days prior to UUO surgery. Mice were sacrificed 7 days after surgery. (A) Fluorescence images of ALDH2-EGFP in cryopreserved kidney and liver tissue sections were used to evaluate EGFP fluorescence signaling. Robust ALDH2-EGFP signaling was observed in the injected kidney, with some signal detected in the contralateral uninjected kidney. Low ALDH2-EGFP signaling was detected in the liver. Scale bar = 20 μm. (B) The first and second panels depict H&E and periodic acid–Schiff (PAS) staining, respectively, to assess morphological changes in kidney tissues. The third panel shows Sirius red staining to evaluate the kidney fibrosis area on day 7 after UUO. The fourth panel displays the immunohistochemistry of Acr-PCs in kidney tissues. Scale bar: 50 μm. (C) Western blot analysis of Acr-PCs in kidney tissues of mice is illustrated with quantification. (D) Western blot analysis of collagen 1, α–smooth muscle actin (α-SMA), and ALDH2 in kidney tissues of mice is illustrated with quantification. mRNA expression of (E) Col1a1, Acta2, (F) Havcr1, Lcn2, and (G) Il6 and Il1b in kidney tissues was assessed through quantitative reverse transcription PCR analysis. Data are presented as mean ± SD. Statistical significance was determined using Mann-Whitney U or Kruskal-Wallis tests, and 2-tailed P values are indicated. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the WT group. AAV, adeno-associated virus; ALDH2, aldehyde dehydrogenase 2; Acr-PCs, acrolein-protein conjugates; Acta2, actin alpha 2; Col1a1, collagen type I alpha 1 chain; Havcr1, hepatitis A virus cellular receptor 1 homolog; Il6, interleukin-6, Il1b, interleukin-1β; Lcn2, lipocalin-2.