Mechanism of calcium oxalate crystal-induced ferroptosis in human renal tubular epithelial cell
10.3760/cma.j.cn112330-20211007-0052
- VernacularTitle:草酸钙晶体诱导人肾小管上皮细胞铁死亡的机制
- Author:
Kai ZHOU
1
;
Jiawen ZHAO
;
Chengyang LI
Author Information
1. 金华市人民医院肾内科，金华 321000
- Keywords:
Reactive oxygen species;
Ferroptosis;
Cell damage;
Calcium oxalate crystals;
Human renal tubular epithelial cells
- From:
Chinese Journal of Urology
2023;44(8):622-629
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the role of ferroptosis in calcium oxalate (Calcium Oxalate, CaOx) crystal-induced injury of human renal tubular epithelial cells (HK-2 cells).Methods:From March 2021 to September 2021, I used calcium oxalate crystal suspension to intervene HK-2 cells to build a HK-2-CaOx reaction model. Set the concentration gradient group and time gradient of calcium oxalate crystal intervention in HK-2 cells: 7 groups of calcium oxalate crystals with different concentrations (0, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0 mmol/L) were used to intervene HK-2 cells24 hours, the HK-2 cell protein was extracted after the intervention; HK-2 cells were intervened with calcium oxalate crystals at optimum concentration, and extract proteins at different time points (0, 3, 6, 9, 12, 24, 48 h) after intervention, the expression of intracellular ferroptosis marker protein glutathione peroxidase 4 (GPX4) was detected by Western blot. Intervention of HK-2 cells with ferroptosis inducer Erastin and ferroptosis inhibitor ethyl 3-amino-4-cyclohexylaminobenzoate (Ferrostatin-1, Fer-1) to regulate intracellular ferroptosis Level. HK-2 cells were divided into 4 groups: normal control group (NC; no intervention treatment, cultured in complete medium only); calcium oxalate crystal stimulation group (CaOx; cultured in complete medium containing 4.0 mmol/L CaOx crystals); calcium oxalate crystals + erastine treatment group (CaOx+ Erastin; cultured in complete medium containing 10.0 μmol/L erastine and 4.0 mmol/L calcium oxalate crystals); calcium oxalate crystals + Fer-1 Treatment group (CaOx+ Fer-1; cultured in complete medium containing 1.0 μmol/L Fer-1 and 4.0 mmol/L calcium oxalate crystals). After 24 hours, the expression of ferroptosis-related protein GPX4, long-chain fatty acyl-CoA synthase 4 (ACSL4) and solute carrier family 7 member 11 (SLC7A11) in HK-2 cells was analyzed by western blot and immunofluorescence techniques; the content of glutathione in HK-2 cells was detected; DCFH-DA fluorescence staining was used to observe the expression of reactive oxygen species (ROS) in HK-2 cells. The adhesion of calcium oxalate in HK-2 cells in each group was observed by light microscope, and the nuclear damage of HK-2 cells was detected by DAPI staining.Results:The expression levels of GPX4 in cells in the concentration gradient of 0, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0 mmol/L were5.67±1.05, 5.60±0.02, 4.99±0.94, 4.82±0.93, 4.50±0.70, 4.14± 0.53, 0.97±0.53. The expression difference of GPX4 between the 4.0 mmol/L group and the 0 mmol/L group was statistically significant ( P=0.026). 4.0 mmol/L was selected as the optimal concentration to intervene the cells. The expression levels of GPX4 in the time gradient (0, 3, 6, 9, 12, 24, 48 h) cells were 11.73±1.29, 11.68±1.32, 11.72±1.30, 10.97±1.28, 10.63±1.21, 8.79±1.10, 8.03±1.06. The expression difference of GPX4 between the 24h intervention group and the 0h intervention group was statistically significant( P=0.090), so 24h was chosen as the optimal intervention time for calcium oxalate crystals. Compared with the NC group, the CaOx+ Erastin group had higher expression of ACSL4 (9.71±0.68 vs. 3.96±0.17, P<0.01); SLC7A11 (5.76±1.31 vs. 9.18±1.54, P=0.001) and GPX4 (3.61±0.25 vs. 9.26±0.13, P<0.01) the expression level decreased. Compared with the CaOx group, the CaOx+ Fer-1 group had higher protein expression levels of GPX4 (7.52±0.23 vs. 3.61±0.25, P<0.01), SLC7A11 (7.85±1.34 vs. 5.76±1.31, P=0.012), ACSL4 (5.84 ±0.62 vs. 9.71±0.68, P=0.002) protein expression was significantly decreased. Compared with CaOx group, CaOx+ Erastin group had significantly lower protein expression of GPX4 (2.71±0.18 vs. 3.61±0.25, P=0.001), SLC7A11 (3.82±1.60 vs. 5.75±1.31, P=0.017), ACSL4(11.15±0.44 vs.9.71±0.68, P<0.01) protein expression increased. The results of glutathione determination showed that compared with the NC group, the glutathione content in the CaOx group was significantly reduced [(53.38±3.53) mmol/L vs. (81.88±4.02) mmol/L, P<0.01]. Compared with the CaOx group, the CaOx+ Fer-1 group had significantly higher glutathione content [(68.26±4.55)mmol/L vs. (53.38±3.53)mmol/L, P=0.001]. Compared with the CaOx group, the glutathione content was decreased [(38.22±2.95)mmol/L vs.(53.38±3.53)mmol/L, P=0.01]. The results of DCFH-DA fluorescence staining showed that compared with the NC group (63.36±5.17 vs. 22.72±3.73, P<0.01), the CaOx group had a significantly higher fluorescence intensity, Compared with the CaOx group (45.32±4.33 vs. 63.36±5.17, P=0.002), the fluorescence intensity of cells in the CaOx+ Fer-1 group was significantly weakened, Compared with the CaOx group (82.38±6.25 vs.63.36±5.17, P=0.002), the fluorescence intensity of the cells in the CaOx+ Erastin group was significantly increased. The results of immunofluorescence showed that the CaOx group was significantly weakened compared with the NC group (31.63±2.86 vs. 50.36±4.23, P<0.01), and the CaOx+ Fer-1 group was significantly weakened compared with the CaOx group (39.89±3.35 vs. 31.63±2.86), P=0.038), the fluorescence intensity of cells in the CaOx+ Fer-1 group was significantly enhanced, the CaOx+ Erastin group was compared with the CaOx group (23.36±3.74 vs. 31.63±2.86, P=0.022), the cell fluorescence in the CaOx+ Erastin group was The intensity is significantly reduced. DAPI staining to calculate the damage ratio of each group of nuclei: NC group (2.85%), CaOx group (11.96%), CaOx+ Fer-1 group (8.76%), CaOx+ Erastin group (16.27%). Conclusion:CaOx crystals can induce ferroptosis in HK-2 cells by increasing the level of oxidative stress in HK-2 cells.
