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.

Atorvastatin inhibits renal inflammatory response induced by calcium oxalate crystals via inhibiting the activation of TLR4/NF‐κB and NLRP3 inflammasome

“…In this study, we found that cell‐crystal reaction led to increased production of ROS and MDA and a decreased level of SOD, which contributed to the activation of oxidative stress. Consistent with our previous results, CaOx crystals significantly increased the secretion of LDH, the adherent and endocytosis of CaOx crystals in HK‐2 cells, and increased the levels of CRE and BUN in rat serum and the deposition of crystals in rat kidney . Yu et al reported that CaOx crystals increased the production of ROS and induced the damage to HK‐2 cells via mediating the AKT/P38/MAPK pathway.…”

“…Consistent with our previous results, CaOx crystals significantly increased the secretion of LDH, the adherent and endocytosis of CaOx crystals in HK-2 cells, and increased the levels of CRE and BUN in rat serum and the deposition of crystals in rat kidney. 20,22 Yu et al 23 reported that CaOx crystals increased the production of ROS and induced the damage to HK-2 cells via mediating the AKT/P38/MAPK pathway. The above data indicated that oxidative stress played a critical role in cell damage and intrarenal crystal formation.…”

Ethyl pyruvate inhibits LPS induced IPEC-J2 inflammation and apoptosis through p38 and ERK1/2 pathways

“…It is derived from pyruvate, an important metabolite in the tricarboxylic acid cycle. EP repairs tissue or cell damage and enhances immunity by anti-oxidation and reducing the secretion and expression of inflammatory factors induced by LPS such as IL-1β, IL-6 and IL-8 [11][12][13]. EP also has a therapeutic effect of repairing intestinal barrier on enteritis induced by external stimuli [14].…”

Role of ROS-Induced NLRP3 Inflammasome Activation in the Formation of Calcium Oxalate Nephrolithiasis

“…Wang et al (48) reported that in patients with calcium-containing kidney stones, an increased expression of HMGB1 and MCP-1 was found in their urine. The results of in vitro studies show that high calcium ions stimulate RTECs to activate the HMGB1-RAGE/TLR4-NF-kB signaling pathway and stimulate the secretion of inflammatory factors TNF-a, IL-1b, and IL-6 (49). Moreover, previous studies have also indicated that there is a close relationship between the secretion of HMGB1 and the activation of NLRP3 inflammasomes (50,51).…”