Klotho alleviates contrast-induced acute kidney injury by suppressing oxidative stress, inflammation, and NF-KappaB/NLRP3-mediated pyroptosis

Abstract

Background: Contrast-induced acute kidney injury (CI-AKI) is a common complication following percutaneous coronary intervention in coronary artery disease (CAD) patients with >30% incidence. Klotho is a multifunctional protein that inhibits oxidative stress and inflammation, but its role in CI-AKI is poorly understood. The present study aimed to explore the effects of klotho in CI-AKI.

Methods: Six-week-old mice and HK-2 were divided into the control, contrast medium (CM), CM + klotho, and klotho groups. H&E staining evaluated kidney injury. Scr and BUN showed renal function. DHE probe and ELISA kit detected the levels of reactive oxygen species (ROS) in kidney tissue, superoxide dismutase (SOD), and malondialdehyde (MDA) in serum. Western blot detected the expressions of NF-κB and phosphorylated NF-κB (p-NF-κB) and pyroptosis-related protein levels of NLRP3, caspase-1, GSDMD, and cleaved-GSDMD in the kidney of CI-AKI mice. CCK-8 and lactate dehydrogenase (LDH) activity assays determined cell viability and damage. Fluorescent probe dichloro-dihydro-fluorescein diacetate (DCFH-DA) and enzyme-linked immunosorbent assay (ELISA) tested oxidative stress-related indicators. These included intracellular reactive oxygen species (ROS), superoxidase dismutase (SOD), and malondialdehyde (MDA). IL-6, TNF-α, IL-1β, and IL-18 in the cell supernatant were tested by ELISA assay and used to reflect inflammation responses. Propidium iodide (PI) staining showed the cell death of HK-2. The expressions of NF-κB, p-NF-κB and pyroptosis-related protein levels of NLRP3, caspase-1, GSDMD, and cleaved-GSDMD were detected by Western blot.

Results: Exogenous klotho administration reduced kidney histopathological alterations and improved renal function in vivo. The levels of reactive oxygen species (ROS) in renal tissue, superoxide dismutase (SOD), and malondialdehyde (MDA) in serum decreased after the klotho intervention. The expression levels of p-NF-κB and pyroptosis-related proteins, including NLRP3, caspase-1, GSDMD, and cleaved-GSDMD, were decreased in CI-AKI mice after the klotho intervention. In vitro, klotho significantly inhibited CM-induced oxidative stress and the production of IL-6 and TNF-α. Moreover, it was found that klotho inhibited the activation of p-NF-κB and down-regulated pyroptosis-related protein (NLRP3, caspase-1, GSDMD, and cleaved-GSDMD).

Conclusion: Klotho has a protective effect on CI-AKI via suppressing oxidative stress, inflammation, and NF-κB/NLRP3-mediated pyroptosis that contributes to the potential therapy of CI-AKI.

Keywords: Contrast-induced acute kidney injury; Inflammation; Klotho; NF-κB/NLRP3-mediated pyroptosis; Oxidative stress.