Calcium oxalate crystals stimulate expression of high mobility group box-1 protein in human macrophages

Huang Peng; Deng Yao-liang; Li Cheng-yang; Tao Zhi-wei; Wang Xiang; Feng You-cai; Wu Bo

doi:10.3969/j.issn.2095-4344.2015.11.014

Abstract：BACKGROUND:There are a large amount of infiltrated monocytes and macrophages around the renal interstitial crystals in a kidney model, indicating that macrophages may be involved in the process of crystal deposition in the kidney. As macrophages are important human innate immune cels, a large number of infiltrated monocytes and macrophages in the kidney wil produce some inflammatory injuries and damage renal tubular epithelial cels, which ultimately lead to the formation of stones. OBJECTIVE: To study the expression of after high mobility group box-1 protein (HMGB1) in macrophages stimulated by calcium oxalate monohydrate crystals. METHODS:Western blot assay was used to detect total protein and HMGB1 levels in the cytoplasm of macrophages under 100 mg/L calcium oxalate monohydrate stimulation for 0, 6, 12, 24, 36 hours. Meanwhile, <br> real-time quantitative PCR was adopted to determine the mRNA expression of HMGB1. Tumor necrosis factor α and interleukin-6 levels in the cultured cel supernatant at 0, 1, 2 and 4 hours after calcium oxalate monohydrate stimulation were measured using ELISA. RESULTS AND CONCLUSION: Under the calcium oxalate monohydrate stimulation, the level of HMGB1 in the cytoplasm of macrophages was lower within 0-6 hours, but gradualy increased within 12-36 hours; the level of HMGB1 in the total protein of macrophages was not high within 0-6 hours, began to increase at 12 hours, and then kept at a higher level within 24-36 hours. RT-PCR results showed that under the calcium oxalate monohydrate stimulation, the mRNA level of HMGB1 in the cultured cels had no changes within 0-12 hours, but significantly increased within 18-24 hours. ELISA results showed that thelevels of tumor necrosis factor α and interleukin-6 in the cultured cel supernatant subject to calcium oxalate monohydrate were increased at 2 hours and peaked at 4 hours. These findings indicate that calcium oxalate monohydrate can increase the HMGB1 expression in macrophages at mRNA and protein levels, and also induce the increase in tumor necrosis factor α and interleukin-6 expression in macrophages. Moreover, the expression of HMGB1 is later than the release of tumor necrosis factor α and interleukin-6.