3.1. Klotho alleviated kidney injury in CI-AKI mice.
CI-AKI mice models were established by following steps (Fig. 1A). According to the HE staining results, CM-challenged mice exhibited remarkable pathological changes, including cell swelling, cell necrosis, severe dilatation, epithelial cell exfoliation, and inflammatory cell infiltration in renal tubules. The animals in the CM + klotho group showed significantly lower renal tubular injury scores (P < 0.001) than those in the CM group (Fig. 1B). The levels of BUN and SCr were significantly (P < 0.001) increased in the CM group compared with the control group. Administration of klotho significantly decreased the levels of BUN and SCr (P < 0.001) as compared with CM group (Fig. 1C and D).
3.2. Effects of Various Concentrations of CM on Oxidative Stress and inflammation in vitro.
In order to explore the role of ioversol in HK-2 cells and determine the optimal intervention concentration, we cultured HK-2 cells and treated with different doses of CM for 2 h (Fig. 2A). Compared to control group (0 mgI/mL of CM), cell viability was measured by CCK-8 assay and was found to decrease in a dose-dependent manner (Fig. 2B). The CM concentration for peak LDH releasing was at 100 mgI/ mL (Fig. 2C). In the CM concentration of 100 mgI/mL, HK-2 cells had the lowest SOD activity and the highest MDA and ROS levels (Fig. 2D, E and F). Simultaneously, it was found that the expression of inflammatory cytokines (IL-6 and TNF-α) in cell supernatants was dramatically increased in the concentration of 100 mgI/mL CM (Fig. 2G and H). We speculated that most HK-2 cells died when exposed to 150 mgI/mL CM. Therefore, CM at 100 mgI/mL was shown to be the significant injury, oxidative stress and inflammation.
3.3. CM exposure stimulated NF-κB/NLRP3 signaling activation in HK-2 cells.
Pyroptosis was another type of programmed cell death, different from apoptosis, and had been proven to be involved in AKI [20]. Here, the cultured HK-2 cells were treated with different concentrations of CM (0, 25, 50, 100 or 150 mg/L) for 2 h. It was shown that 100 mgI/mL ioversol treated HK-2 cells significantly increased PI/DAPI staining-positive cells (Fig. 3A). The expression level of IL-18 and IL-1β in the supernatant of HK-2 cells were highest in 100 mgI/mL ioversol (Fig. 3B and C). Moreover, the expression of pyroptosis-related proteins (NLRP3, caspase-1, GSDMD, and cleaved-GSDMD) were up-regulated after treating with 100 mgI/mL CM (Fig. 3D). We then found that NF-κB, an important activator of NLRP3 inflammasome, was significantly activated in HK-2 cells following CM treatment. With the concentration of 150 mgI/mL CM, we believed that a large number of HK-2 cells death or loss and led to undetectable. In conclusion, the mechanism of NF-κB/NLRP3-mediated pyroptosis might be involved in the injury of CM-treated HK-2 cells.
3.4. The effect of different doses of klotho on HK-2 cells.
HK-2 cells were treated with klotho for 24 h. There was no significant change in cell viability and LDH release (Fig. 4A and B). Therefore, as an exogenous substance, klotho did not cause damage to HK-2 cells.
3.5. The effect of different doses of klotho on CM-induced HK-2 cells.
Subsequently, we evaluated the effect of different doses of klotho on CM-induced HK-2 cells. After 24 h treatment, klotho significantly protected cells from CM-induced injury. Cell viability gradually recovered and LDH decreased compared to the CM group. Statistically, a significant inhibitory effect of klotho on cytotoxicity commenced at 0.2 µg/mL (Fig. 4C and D).
3.6. Klotho prevented CM-induced oxidative stress and inflammation in HK-2 cells.
According to the above experiments, HK-2 cells were treated with the optimal drug concentration (Fig. 5A). The administration of klotho showed a significant increase in cell viability in HK-2 cells when compared with the CM group (Fig. 5B) CM exposure increased LDH leakage in HK-2 cells, whereas klotho treatment decreased the level of LDH leakage in those cells (Fig. 5C). Oxidative stress usually involved in the damage of CI-AKI. Klotho successfully reduced ROS production, MDA level, and SOD activity along with the antioxidant defence restoration in CM-induced HK-2 cells (Fig. 5D, E and F). Investigating the effect of inflammatory cytokines in CM-induced HK-2 cells, we found that klotho treatment successfully reduced upregulation of TNF-α and IL-1β in CM group (Figs. 5G and H).
3.7. Klotho alleviated NF-κB/NLRP3-mediated pyroptosis in HK-2 cells.
CM exposure brought about significant pyroptosis. Fluorescence study showed elevated in PI/DAPI rate in CM-treated HK-2 cells. However, klotho treatment significantly mitigated PI/DAPI rate (Fig. 6A). Klotho decreased the secretion of inflammation cytokines IL-18 and IL-1β in CM-induced HK-2 cells (Fig. 6B and C). In CM + klotho group, there was upregulated expression of phosphorylated NF-κB and pyroptosis-related proteins (NLRP3, caspase-1, GSDMD and cleaved-GSDMD) (Fig. 6D). Therefore, Klotho supplementation significantly reduced pyroptosis reaction.