Knockdown of ANGPTL2 Protects Renal Tubular Epithelial Cells Against Hypoxia/Reoxygenation-Induced Injury via Suppressing TLR4/NF-κB Signaling Pathway and Activating Nrf2/HO-1 Signaling Pathway

Figures

• 

  Figure 1.. Increased expression levels of ANGPTL2 in HK-2 cells exposed to H/R. HK-2 cells were exposed to H/R, and then the mRNA and protein levels of ANGPTL2 were measured using qualitative real-time-PCR (A) and western blot (B). n = 5. *P < 0.05. ANGPTL2: angiopoietin-like protein 2; H/R: hypoxia/reoxygenation.

• 

  Figure 2.. Improved cell viability of HK-2 cells after transfection with si-ANGPTL2. (A) Western blot was performed to detect the protein levels of ANGPTL2 after transfection with si-ANGPTL2 or si-NC in H/R-induced HK-2 cells. (B) Methylthiotetrazole assay was performed to examine the cell viability of HK-2 cells. n = 6. *P < 0.05 versus control, # P < 0.05 versus si-NC + H/R. ANGPTL2: angiopoietin-like protein 2; H/R: hypoxia/reoxygenation.

• 

  Figure 3.. Decreased oxidative stress in HK-2 cells transfected with si-ANGPTL2. After transfection with si-ANGPTL2 or si-NC, the ROS production (A), MDA level (B), and SOD activity (C) were detected for the assessment of oxidative stress in HK-2 cells. n = 5. *P < 0.05 versus control, # P < 0.05 versus si-NC + H/R. ANGPTL2: angiopoietin-like protein 2; H/R: hypoxia/reoxygenation; MDA: malondialdehyde; ROS: reactive oxygen species; SOD: superoxide dismutase.

• 

  Figure 4.. Decreased inflammatory response in HK-2 cells transfected with si-ANGPTL2. The secretion levels of proinflammatory cytokines, including TNF-α (A), IL-6 (B), and IL-1β (C) in culture supernatants were determined to assess inflammation in HK-2 cells. n = 6. *P < 0.05 versus control, # P < 0.05 versus si-NC + H/R. ANGPTL2: angiopoietin-like protein 2; H/R: hypoxia/reoxygenation; IL: interleukin; TNF-α: tumor necrosis factor-alpha.

• 

  Figure 5.. Decreased cell apoptosis in HK-2 cells transfected with si-ANGPTL2. (A) Western blot was performed to detect the protein levels of bax and bcl-2 after transfection with si-ANGPTL2 or si-NC. (B) Caspase-3 activity in HK-2 cells was determined. n = 4. *P < 0.05 versus control, # P < 0.05 versus si-NC + H/R. ANGPTL2: angiopoietin-like protein 2; H/R: hypoxia/reoxygenation.

• 

  Figure 6.. Enhanced oxidative stress, inflammatory response, and apoptosis in HK-2 cells transfected with pcDNA3.1-ANGPTL2. (A and B) ROS and MDA productions were detected for the assessment of oxidative stress in HK-2 cells. (C–E) Levels of TNF-α, IL-6, and IL-1β in culture supernatants were determined to assess inflammation in HK-2 cells. (F) Caspase-3 activity in HK-2 cells was determined. n = 4. *P < 0.05 versus control, # P < 0.05 versus pcDNA3.1 + H/R. ANGPTL2: angiopoietin-like protein 2; H/R: hypoxia/reoxygenation; IL: interleukin; MDA: malondialdehyde; ROS: reactive oxygen species; TNF-α: tumor necrosis factor-alpha.

• 

  Figure 7.. Induction of Nrf2/HO-1 pathway in HK-2 cells by transfection with si-ANGPTL2. Western blot was performed to detect the expression levels of nuclear Nrf2 and HO-1 in HK-2 cells after transfection with si-ANGPTL2 or si-NC. n = 3. *P < 0.05 versus control, # P < 0.05 versus si-NC + H/R. ANGPTL2: angiopoietin-like protein 2; H/R: hypoxia/reoxygenation.

• 

  Figure 8.. Prevention of the anti-oxidative and anti-apoptotic activities of ANGPTL2 knockdown by transfection with si-Nrf2. HK-2 cells were transfected with si-ANGPTL2 and/or si-Nrf2 under H/R condition. (A) Western blot were performed to detect the protein level of Nrf2. (B) Quantification analysis of Nrf2. (C and D) ROS production and caspase-3 activity in HK-2 cells. n = 4. *P < 0.05 versus control, # P < 0.05 versus si-NC + H/R, & P < 0.05 versus si-ANGPTL2 + H/R. ANGPTL2: angiopoietin-like protein 2; H/R: hypoxia/reoxygenation; ROS: reactive oxygen species.

• 

  Figure 9.. Inhibition of TLR4/NF-κB activation in HK-2 cells by transfection with si-ANGPTL2. Western blot was performed to detect the expression levels of TLR4, p65, and p-p65 in HK-2 cells after transfection with si-ANGPTL2 or si-NC. n = 4. *P < 0.05 versus control, # P < 0.05 versus si-NC + H/R. ANGPTL2: angiopoietin-like protein 2; H/R: hypoxia/reoxygenation; NF-κB: nuclear factor kappa-light-chain-enhancer of activated B cells; TLR4: toll-like receptor 4.

• 

  Figure 10.. Prevention of the anti-inflammatory effect of ANGPTL2 knockdown by transfection with pcDNA3.1-TLR4. HK-2 cells were transfected with si-ANGPTL2 and/or TLR4 under H/R condition. (A) Western blot was performed to detect the protein levels of TLR4. (B–D) Levels of TNF-α, IL-6, and IL-1β in culture supernatants were determined. n = 3. *P < 0.05 versus control, # P < 0.05 versus si-NC + H/R, & P < 0.05 versus si-ANGPTL2 + H/R. ANGPTL2: angiopoietin-like protein 2; H/R: hypoxia/reoxygenation; IL: interleukin; TNF- α: tumor necrosis factor-alpha.