Mouse Renal Glomerular Endothelial Cells

Role of Ferroptosis in LPS-Induced AKI under In Vitro Conditions

Sepsis-associated acute kidney injury (SA-AKI) results in significant morbidity and mortality, and ferroptosis may play a role in its pathogenesis. Sepsis leads to the overproduction of reactive oxygen species (ROS), causing cell damage and organ dysfunction, notably affecting the kidneys.

Zhang's team utilized male C57BL/6 mice induced with sepsis through cecal ligation and puncture (CLP) and examines the effects of GYY4137 on ferroptosis and AKI. Results showed that BUN and Cre levels and the degree of ferroptosis were highest at 24 h in the above experiment. Then, they tested the role of GYY4137 in ferroptosis modulation in in vitro assessments using mouse renal glomerular endothelial cells exposed to LPS. A CCK-8 assay revealed that 100 µg/mL LPS treatment significantly reduced MRGECs viability at 24 hours, likely due to ferroptosis (Fig. 1A). However, this reduced viability improved with the application of the ferroptosis inhibitor Fer-1 at 10 µM (Fig. 1B). Analysis of ferroptosis-related protein expression in MRGECs treated with varying LPS concentrations (0, 0.1, 1, 10, and 100 µg/mL) for 24 hours confirmed intracellular ferroptosis level increased with LPS concentration, peaking at 100 µg/mL (Fig. 1C–F). Fer-1 effectively countered ferroptosis in cells treated with 100 µg/mL LPS by increasing Gpx4 protein levels and decreasing TF and FTH-1 protein levels, compared to cells not treated with Fer-1 (Fig. 1G–J).

Fig. 1. Exacerbation of ferroptosis and AKI in LPS-treated MRGECs (Zhang L, Rao J, et al., 2023).

Allograft Inflammatory Factor-1 Enhances Inflammation and Oxidative Stress via the NF-κB Pathway in Diabetic Kidney Disease

DKD, the primary cause of end-stage renal disease worldwide, is associated with diabetic prevalence and characterized by endothelial cell dysfunction and inflammation. AIF-1, a protein known for its role in inflammation, may contribute to endothelial dysfunction in DKD through the NF-κB pathway. Fu's team hypothesized that AIF-1 promotes glomerular endothelial cell inflammation and oxidative stress via the NF-κB pathway in DKD, and tested their hypothesis in vivo and in vitro.

Mouse renal glomerular endothelial cells (MRGECs) were cultured for in vitro assessments. AIF-1 was overexpressed using lentivirus, confirmed by Q-PCR (Fig. 2B). ELISA results revealed that high glucose (30 mM) significantly increased IL-6, TNF-α, and ROS levels, further elevated in AIF-1 overexpressing cells (Fig. 2C-E). Conversely, T-SOD expression decreased (Fig. 2F). These findings indicate AIF-1 enhances oxidative stress and inflammation under high glucose in MRGECs. Western blot and immunofluorescence showed high glucose induced IκBa phosphorylation and NF-κBp65 nuclear translocation, both enhanced by AIF-1 overexpression (Fig. 2G-I). Overall, AIF-1 enhances NF-κB pathway activity at high glucose levels in MRGECs. Western blot showed that NF-κB inhibitor BAY 11–7082 reduced p-IkBa and nuclear NF-κBp65 levels (Fig. 3A and B). ELISA results demonstrated that BAY 11–7082 reversed the elevated IL-6 and TNF-α levels caused by AIF-1 overexpression (Fig. 3C and D). ROS levels followed a similar pattern (Fig. 3E), while T-SOD levels were inversely related (Fig. 3F). These findings indicate that NF-κB activation enhances AIF-1-driven inflammation in MRGECs.

Fig. 2. AIF-1 elevated high glucose-induced inflammation and oxidative stress and activated the NF-κB pathway in MRGECs (Fu Y, Wang X, et al., 2022).

Fig. 3. NF-κB pathway activation contributed to AIF-1-induced inflammation in MRGECs (Fu Y, Wang X, et al., 2022).