Ethyl Pyruvate Attenuates CaCl2-Induced Tubular Epithelial Cell Injury by Inhibiting Autophagy and Inflammatory Responses

Abstract

Background/aims: Nephrolithiasis is one of the most prevalent diseases of the urinary system. Approximately 80% of human kidney stones are composed of calcium oxalate (CaOx), and hypercalciuria is one of the most common metabolic disorders. Emerging evidence indicates that autophagy and inflammatory responses are related to the formation of CaOx nephrolithiasis. However, the roles of autophagy and inflammation in patients with hypercalciuria remain unclear. Ethyl pyruvate (EP) displays protective effects in experimental models of many illnesses. In this study, we investigated the protective effects of EP in vitro through its inhibition of autophagy and inflammatory responses after CaCl2-induced tubular epithelial cell injury.

Methods: First, we cultured human tubular epithelial (HK-2) cells in the presence of various concentrations of CaCl2 (0, 0.1, 0.25, 0.5, 1.0, 1.5, and 2.0 mg/ml) for 12 h and EP (0, 1.0, 2.5, 5.0, and 10.0 mM) for 2 h to select the optimum concentration using the Cell Counting Kit-8 assay and lactate dehydrogenase (LDH) assay. Cells in culture were stimulated with CaCl2 (1.0 mg/ml, 12 h) with or without EP pretreatment (2.5 mM, 2 h). After the exposure, we detected the expression of inflammation-related proteins using an enzyme-linked immunosorbent assay and Western blot analysis. Finally, the levels of autophagy-related proteins were determined through Western blot analysis, and the number of GFP-LC3 dots and autophagic vacuoles was detected under confocal microscopy.

Results: With the use of the Cell Counting Kit-8 assay and the LDH assay, we identified the optimum concentration for CaCl2 (1.0 mg/ml) treatment and EP pretreatment (2.5 mM). Our research indicated that CaCl2 can induce autophagy and inflammatory responses in HK-2 cells. Furthermore, treatment with EP prior to CaCl2 stimulation attenuated HK-2 cell injury by inhibiting autophagy and inflammation.

Conclusion: Our results provide evidence that EP attenuates CaCl2-induced injury of HK-2 cells by downregulating the expression of inflammation and autophagy proteins that may be associated with the inhibition of the high-mobility group box-1 (HMGB1)/toll-like receptor 4 (TLR4)/NF-κB pathway and the competitive interaction with Beclin-1 of HMGB1.

Keywords: Autophagy; Ethyl pyruvate; Hypercalciuria; Inflammation; Nephrolithiasis; Tubular epithelial cell.