Abstract
The objective of the study is to clarify the mechanism of p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway in the change of crystal adhesion in rat renal tubular epithelial cells (NRK-52E) induced by calcium oxalate monohydrate (COM) crystals. NRK-52E cells were divided into COM crystal-treated group and control group according to whether the cell culture medium contains different concentrations of COM crystals. The concentrations of lactate dehydrogenase in the both group medium were determined after being cultured for 24 h. Protein and RNA were extracted from both cell groups after being cultured at different time points. SB239063, an inhibitor of the activation of p38 MAPK, was pretreated for 2 h before incubation with COM crystals. Western blotting and RT-qPCR were performed to confirm the expression levels of relative genes. All the experimental results were summarized and analyzed by SPSS 20.0 statistical analysis software. COM crystals (146 µg/cm2) could induce the expression levels of NLRP3, caspase-1 and interleukin-1β (IL-1β) significantly increased in NRK-52E cells. Compared with the control group cells, the transcription and translation levels of p38 MAPK-related molecule (such as p-p38) and adhesion molecules (such as osteopontin, hyaluronic acid and CD44) were significantly increased in COM crystal-treated cells and can be inhibited by SB239063 and NLRP3 gene silencing. This study demonstrated that the p38 MAPK signaling pathway mediated the COM crystal-induced crystal adhesion change in NRK-52E cells and required the involvement of NLRP3 inflammasome.
Highlights
Renal stones are one of the most common diseases in urology; about 5% of American males will be involved in their lifetime, and the percentage is still increasing [1]
By measuring the expression level of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) in calcium oxalate monohydrate (COM)-treated NRK-52E cells, we found that COM crystals (146.0 μg/cm2) could induce the transcription and translation level of NLRP3 significantly increased (Fig. 1b, c, e)
IL-1β has been illustrated to be activated by the activation of NLRP3 inflammasome [19] and we found the expression level of it significantly increased in COM crystal-treated cells (Fig. 1b)
Summary
Renal stones are one of the most common diseases in urology; about 5% of American males will be involved in their lifetime, and the percentage is still increasing [1]. Calcium oxalate is one of the most common components of kidney stones and involved in the formation of 80% renal stones [2]. Studies had demonstrated that the damage of renal tubular epithelial and the change of crystal adhesion in renal tubular epithelial cells were the key chains in the formation of calcium oxalate stones [3, 4]. The underlying mechanisms participating in the change of crystal adhesion in renal tubular epithelial cells were still unclear. Mulay et al demonstrated that renal inflammation induced by calcium oxalate crystals was mediated by NLRP3 inflammasome [9]. The role of the NLRP3 inflammasome in the adhesion of COM crystals in the renal tubular epithelium was still unclear and need further researches to verify
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