Xiao Chengcheng, Zhao Haijun, Zhu Hai, Zhang Yingyu, Su Qiuju, Zhao Feng, Wang Renhe
Department of Urology, Qingdao Municipal Hospital, Qingdao University, Qingdao, China.
Department of Traditional Chinese Medicine, Qingdao Municipal Hospital, Qingdao University, Qingdao, China.
Front Physiol. 2020 Aug 13;11:906. doi: 10.3389/fphys.2020.00906. eCollection 2020.
Renal ischemia-reperfusion injury (IRI) is a major cause of acute kidney injury (AKI). As a transcription factor, the Transcript induced in spermiogenesis 40 (Tisp40) has been found to be involved in renal IRI. However, the role of Tisp40 in tubular epithelial cell (TEC) pyroptosis of renal IRI remains unknown. In this study, we investigated effects of Tisp40 on Gasdermin D (GSDMD)-mediated TEC pyroptosis in renal IRI and underlying molecular mechanisms in I/R-induced kidney by hematoxylin and eosin (HE) staining, Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay,immunohistochemistry (IHC), reverse transcription-quantitative PCR (RT-qPCR) and western blot analysis and oxygen-glucose deprivation/reoxygenation (OGD/R)-stimulated TCMK-1 cells by lactate dehydrogenase (LDH) release assay, CCK-8 assay,enzyme-linked immunosorbent assay (ELISA), flow cytometric analysis, immunofluorescence staining,RT-qPCRand western blot analysis . We found that the levels of Tisp40 and GSDMD-N expression increased gradually, and peaked at 30 min ischemia/24 h reperfusion and 24 h OGD/R/6 h reoxygenation , simultaneously, the levels of TEC pyroptosis and renal injury were correspondingly increased. The data of Pearson's correlation analysis showed that the expression of Tisp40 and GSDMD-N was positively correlated. Furthermore, Tisp40 overexpression aggravated TEC pyroptosis rate and increased the expressions of related proteins, including GSDMD-N, NLRP3, caspase-1, IL-1β, and IL-18 in the OGD/R-stimulated TCMK-1 cell line, whereas the opposite occurred in cells treated with small interfeing RNA (siRNA) targeting Tisp40. Tisp40-deficient mice showed attenuated renal IRI and pyroptosis compared with wild-type mice. In addition, Tisp40 knockout remarkably decreased the levels of GSDMD-N, IL-1β, IL-18, NLRP3, and caspase-1 expression, and alleviated renal pyroptosis induced by I/R. Importantly, Tisp40 overexpression significantly increased TECs pyroptosis via p-p65 activation, however, the effects of Tisp40 overexpression were partially blocked by parthenolide (PTL). Collectively, our findings provide insight into the mechanism of how Tisp40 regulated GSDMD-mediated pyroptosis in renal IRI.
肾缺血再灌注损伤（IRI）是急性肾损伤（AKI）的主要原因。作为一种转录因子，已发现精子发生诱导转录本40（Tisp40）参与肾IRI。然而，Tisp40在肾IRI肾小管上皮细胞（TEC）焦亡中的作用尚不清楚。在本研究中，我们通过苏木精和伊红（HE）染色、末端脱氧核苷酸转移酶dUTP缺口末端标记（TUNEL）检测、免疫组织化学（IHC）、逆转录定量PCR（RT-qPCR）和蛋白质印迹分析，以及通过乳酸脱氢酶（LDH）释放检测、CCK-8检测、酶联免疫吸附测定（ELISA）、流式细胞术分析、免疫荧光染色、RT-qPCR和蛋白质印迹分析对氧糖剥夺/复氧（OGD/R）刺激的TCMK-1细胞，研究了Tisp40对肾IRI中Gasdermin D（GSDMD）介导的TEC焦亡的影响以及I/R诱导的肾脏中的潜在分子机制。我们发现，Tisp40和GSDMD-N的表达水平逐渐升高，并在缺血30分钟/再灌注24小时以及OGD/R 24小时/复氧6小时时达到峰值，同时，TEC焦亡水平和肾损伤相应增加。Pearson相关性分析数据表明，Tisp40与GSDMD-N的表达呈正相关。此外，Tisp40过表达加重了OGD/R刺激的TCMK-1细胞系中的TEC焦亡率，并增加了包括GSDMD-N、NLRP3、半胱天冬酶-1、白细胞介素-1β和白细胞介素-18在内的相关蛋白的表达，而在用靶向Tisp40的小干扰RNA（siRNA）处理的细胞中则出现相反的情况。与野生型小鼠相比，Tisp40缺陷小鼠的肾IRI和焦亡减轻。此外，Tisp40基因敲除显著降低了GSDMD-N、白细胞介素-1β、白细胞介素-18、NLRP3和半胱天冬酶-1的表达水平，并减轻了I/R诱导的肾焦亡。重要的是，Tisp40过表达通过p-p65激活显著增加了TEC的焦亡，然而Tisp40过表达的作用被小白菊内酯（PTL）部分阻断。总的来说，我们的研究结果为Tisp40如何调节肾IRI中GSDMD介导的焦亡机制提供了见解。
