Cu-bearing stainless steel reduces cytotoxicity and crystals adhesion after ureteral epithelial cells exposing to calcium oxalate monohydrate

Abstract

Calcium oxalate monohydrate (COM), which is the main component of encrustation, may result in cell membrane injury. In addition, cellular damage is suggested to be the primary event attributing to COM crystal binding. To study the interaction between cells and crystals after incubating with a Cu-bearing stainless steel (316L-Cu SS), MTS and flow cytometric analyses were used to assess the cellular responses. The results confirmed that 316L-Cu SS could inhibit cytotoxicity and cellular apoptosis of ureteral epithelial cells (UECs) after COM treatment. Furthermore, molecular expressions of Cu/Zn superoxide dismutase (CuZnSOD), which were evaluated by western blot analysis and real-time quantitative PCR (qPCR), indicated that 316L-Cu SS could inhibit the oxidative stress attributing to up-regulating of CuZnSOD. Moreover, the crystal adhesion cytokine CD44 was examined with western blot and qPCR, and the corresponding hyaluronic (HA) secreted into the medium was measured by enzyme-linked immunosorbent assay (ELISA). All results were confirmed that the expressions of cells cultured with 316L-Cu SS were down-regulated, demonstrating the inhibitory performance of 316L-Cu SS against crystal adhesion.

Figure 1

Viability of UECs incubated with different extracts plus various concentrations of oxalate (0, 2, 5 or 10 mM) and COM (200 μg/ml) for 4 h, 12 h and 24 h, respectively. The cells viability was determined using a MTS kit according to the product protocol. The data shown are the mean and standard deviation (SD) (N = 5). *Indicates a significant difference at p < 0.05 (*p < 0.05), and ^#indicates no significant difference at p > 0.05 (^#p > 0.05) compared to 316L SS incubated for the same time.

Figure 2

Flow cytometric analyses of the apoptosis of UECs. Cells were seeded on the samples and exposed to oxalate (2 mM) plus COM (200 μg/ml).

Figure 3

316L-Cu SS activated the expression of CuZnSOD of UECs. (a) Expression of CuZnSOD and (b) qPCR analysis of the CuZnSOD-related gene expression for UECs. Cells were seeded on the surfaces of 316L-Cu SS and 316L SS. DMEM containing oxalate (2 mM) and COM (200 μg/ml) was used to induce cell injury. After incubating for 24 h, the cells were harvested and used for western blot analysis. Anti-GAPDH antibody was used to evaluate equal protein loading on the blot. The density of the bands and relative mRNA levels were expressed as the fold difference from the housekeeping protein and gene, respectively. Then, the levels were calculated relative to the control, which was given the same treatment. Values are presented as the mean ± SD (N = 3). A significant difference at p < 0.05 is shown with different letters *p < 0.05. Intact western blot results can be seen in Supplementary information.

Figure 4

Concentrations of ROS after different treatments of UECs for 24 h. Data are presented the as mean ± SD. A significant difference at p < 0.05 is shown with different letters.

Figure 5

316L-Cu SS attenuates the expression of CD44. (a) Western blot analysis and (b) relative mRNA expression gene transcription of CD44. A significant difference at p < 0.05 is shown with different letters *p < 0.05. Intact western blot results can be seen in Supplementary information.

Figure 6

HA secretion into the culture medium after incubating for 24 h. A significant difference at p < 0.05 is shown with different letters.