摘 要 目的:探讨Piezo 1在脓毒症相关急性肾损伤(SA-AKI)的发病机制中的作用。方法:利用脂多糖(LPS)处理C57BL/6小鼠和人近端肾小管上皮细胞(HK-2细胞)构建SA-AKI模型,Western Blot和免疫荧光检测Piezo 1的表达。肽毒素铲形机械毒素4(GsMTx4)干预SA-AKI小鼠,HE和PAS染色检测小鼠肾组织病理改变。用Piezo 1 siRNA或Yoda 1干预LPS诱导的HK-2细胞,用Western Blot、实时荧光定量PCR(RT-qPCR)检测肾小管损伤标志物、炎症因子和凋亡标志蛋白的表达;ELISA法检测细胞上清炎症因子水平;流式细胞术检测细胞凋亡率。Piezo 1 siRNA处理后,用Fluo-4 AM钙离子(Ca2+)荧光探针检测细胞内Ca2+含量;Western Blot检测钙蛋白酶2(Calpain 2)、整合素β1(Integrin β1)的表达。PD151746干预后检测Integrin β1的表达。Yoda1和PD151746同时干预LPS诱导的细胞,ELISA法检测细胞上清炎症因子水平和流式细胞术检测细胞凋亡率。结果:Piezo 1在SA-AKI小鼠肾小管和LPS处理的HK-2细胞中表达均增加。GsMTx4抑制Piezo 1可减轻SA-AKI小鼠肾小管损伤。沉默Piezo 1可减少LPS诱导的HK-2细胞损伤、炎症和细胞凋亡,然而,Yoda 1进一步加重上述损伤。通过沉默Piezo 1,可减少LPS诱导的Ca2+细胞内流和Calpain 2、Integrin β1的表达。PD151746抑制Calpain 2可减少Integrin β1的表达,并抑制Yoda 1引起的HK-2细胞炎症和细胞凋亡。结论:Piezo 1在SA-AKI肾小管上皮细胞中表达上调,可能是通过激活Ca2+/Calpain 2/Integrin β1通路引起炎症和细胞凋亡,参与SA-AKI的发生发展。

关键词 Piezo 1 脓毒症相关急性肾损伤 炎症 凋亡

1School of Medicine, South China University of Technology, Guangzhou 510006, China

2Department of Nephrology, Guangdong Provincial People's Hospital (Guangdong Academy of Medical Sciences), Southern Medical University, Guangzhou 510080, China

Corresponding author：LIU Shuangxin (E-mail：13543456446@163.com)

ABSTRACT 

Objective：To investigate the role and possible mechanism of Piezo 1 in LPS-induced sepsis-associated acute kidney injury (SA-AKI). 

Methodology：Establish in vivo and in vitro models of SA-AKI in mice and human proximal renal tubular epithelial cells (HK-2 cells) by LPS, and immunofluorescence and Western blot were used to detect Piezo 1 expression. GsMTx4 was injected into SA-AKI mice, and the renal pathology and renal function were detected. Piezo 1 siRNA or Yoda 1 were used to intervene in LPS-induced HK-2 cells, then the expressions of renal tubular injury markers, inflammatory factors and apoptosis-related marker proteins were detected by Western blot and RT-qPCR, inflammatory factors were detected by ELISA and apoptosis rate was detected by flow cytometry. After treated with Piezo 1 siRNA, the intracellular Ca2+ concentration was detected with a Fluo-4 AM calcium ion fluorescent probe and the expressions of Calpain 2 and Integrin β1 were detected. Integrin β1 expression was detected after PD151746 intervention. After Yoda1 and PD151746 simultaneously intervened in LPS-induced cells, inflammatory factors were detected by ELISA and apoptosis rate was detected by flow cytometry. 

Results：Piezo 1 expression was upregulated in the kidneys of mice with SA-AKI and HK-2 cells induced by LPS. GsMTx4 ameliorated renal pathology and renal function damage in SA-AKI mice. Silencing Piezo 1 reduced LPS-induced HK-2 cell damage, inflammation, and apoptosis; however, the above damage was further exacerbated after Yoda 1 intervention. Mechanistically, Piezo 1 knockdown reduced LPS-induced intracellular Ca2+ and the expression of Calpain 2 and Integrin β1. PD151746 reduced the expression of integrin β1 and inhibited inflammation and apoptosis caused by Yoda 1. 

Conclusion：Piezo 1 expression is upregulated in renal tubular epithelial cells with SA-AKI, possibly causes inflammation and apoptosis by activating the Ca2+/Calpain 2/Integrin β1 pathway, and which is involved in the progression of SA-AKI.

Key words Piezo 1   sepsis-associated acute kidney injury   inflammation   apoptosis

Piezo 1是一种机械敏感阳离子通道,促进钙离子(Ca2+)进入细胞,修改细胞内钙信号,使细胞将机械信号转化为生物信号[6]。Piezo 1在肾脏生理和病理中发挥重要作用,可参与肾小管、肾小球及肾血管等功能调节[7-8]。Calpain 2是一种钙依赖蛋白酶,可以通过细胞内Ca2+的增加来激活;细胞内钙信号和Calpain 2均可激活整合素(Integrin)的胞内结构域,促进Integrin的聚集[9-10]。如Piezo 1通过Ca2+/Calpain 2通路介导神经元氧-葡萄糖剥夺/再氧凋亡[11]。Piezo 1激动剂(Yoda 1)可促进人近端小管上皮细胞(HK-2细胞)Ca2+流入增加,诱导Calpain 2的激活和Integrin β1的上调,导致肾间质纤维化[12]。新近研究发现,LPS可使小鼠脑星形胶质细胞中的Piezo 1敏感激活,抑制Piezo 1可改善脂多糖(LPS)诱导的神经炎症[13]。本研究旨在探讨Piezo 1在SA-AKI的作用和可能的分子机制。

方法

动物实验  本实验经南方医科大学附属广东省人民医院研究伦理委员会批准(编号KY-Q-2022-064-03)。将18只雄性小鼠(体重22～26 g,10周龄)随机平均分为3组,每组6只。(1)LPS组:腹腔注射LPS 10 mg/kg构建SA-AKI小鼠模型;(2)LPS+GsMTx4组:先注射LPS 10 mg/kg,15 min后再注射GsMTx4 10 mg/kg,其中GsMTx4为Piezo 1的抑制剂;(3)正常对照组:腹腔注射等量生理盐水,24 h后采集血清和肾组织标本。血清肌酐、尿素氮检测和HE、PAS病理染色均由武汉赛维尔公司完成。

细胞培养  人近端肾小管上皮细胞(HK-2细胞)在含有 10% FBS的DMEM/F12 培养基培养。分别加入于不同浓度(0.1 μg/mL、1 μg/mL和10 μg/mL)的LPS刺激,创建SA-AKI细胞模型,后续实验以1 μg/mL LPS刺激细胞。

细胞转染与药物刺激  Piezo 1 siRNA用于沉默Piezo 1,使用脂质体(Lipofectamine) 2 000加50 nM Piezo 1 siRNA(stB0009164A: GGGACTGCCTCATTCT GTA)转染HK-2细胞6 h。Yoda1是 Piezo 1的激动剂,刺激浓度为5 μM;PD151746是Calpain2抑制剂,刺激浓度为20 μM,处理时间均为48 h。

免疫荧光  将固定包埋好的切片(或培养好的细胞)在 4%多聚甲醛固定15 min;5%牛血清白蛋白封闭30 min;在4 ℃孵育Piezo 1抗体过夜;次日避光孵育抗555荧光二抗1 h;避光孵育莲花凝集素(LTL)30 min;最后共聚焦采集荧光图像。

实时荧光定量PCR(RT-qPCR)  使用TRIzol从细胞中提取总RNA,用Evo M-MLV RT Premix逆转录试剂盒将测好浓度的mRNA逆转录为cDNA,SYBR Green Premix试剂盒对cDNA进行扩增。引物序列见表1。

GsMTx4干预SA-AKI小鼠后,组织免疫荧光结果显示,与LPS组相比,GsMTx4使小鼠肾小管Piezo 1的表达明显下降,提示Piezo 1低表达小鼠模型构建成功(图1D)。GsMTx4抑制Piezo 1可降低SA-AKI小鼠血清肌酐和尿素氮水平,还能减轻肾小管病理损伤(图1E、F)。

Control组:正常对照组;LPS:脂多糖;LTL:莲花凝集素;DAPI:4',6-二脒基-2-苯基吲哚;GsMTx4:肽毒素铲形机械毒素4;A、E:小鼠血清肌酐、尿素氮水平;B、F:小鼠肾组织HE和PAS染色(×200,比例尺=50 μm);C、D:小鼠肾小管Piezo 1的组织免疫荧光图(×200,比例尺=20 μm);Piezo 1为红色,近端小管刷状缘被LTL标记为绿色,细胞核被DAPI标记为蓝色

Control组:正常对照组;LPS:脂多糖;LTL:莲花凝集素;DAPI:4',6-二脒基-2-苯基吲哚;HK-2细胞:人近端肾小管上皮细胞;A:Piezo 1的蛋白水平;B:HK-2细胞Piezo 1的细胞免疫荧光图(×600,比例尺=10 μm);Piezo 1为红色,近端小管刷状缘被LTL标记为绿色,细胞核被DAPI标记为蓝色;NS:两组间差异无统计学意义

图3 沉默Piezo 1对HK-2细胞小管损伤标志物的影响

Control组:正常对照组;LPS:脂多糖;Scramble:siRNA的阴性对照;Piezo 1 siRNA:Piezo 1小干扰RNA;Yoda 1:Piezo 1激动剂;KIM-1:肾损伤分子1;NGAL:中性粒细胞明胶酶相关脂质运载蛋白;HK-2:人近端肾小管上皮细胞;A:KIM-1和NGAL的mRNA水平;B、C:NGAL的蛋白水平;*：两组比较,P<0.05

图4 沉默Piezo 1对HK-2细胞炎症因子的影响

Control组:正常对照组;LPS:脂多糖;Scramble:siRNA的阴性对照;Piezo 1 siRNA:Piezo 1小干扰RNA;HK-2细胞:人近端肾小管上皮细胞;Yoda 1:Piezo 1激动剂;IL:白细胞介素;TNF-α:肿瘤坏死因子α;A:IL-1β、IL-6和TNF-α的mRNA水平;B、C、E:IL-1β、TNF-α和IL-6的蛋白水平;D、F:细胞上清IL-1β和TNF-α的水平;*：两组比较,P<0.05

图5 沉默Piezo 1对HK-2细胞凋亡的影响

Control组:正常对照组;LPS:脂多糖;Scramble:siRNA的阴性对照;Piezo 1 siRNA;Piezo 1的小干扰RNA;HK-2细胞:人近端肾小管上皮细胞;Yoda 1:Piezo 1激动剂;Bcl-2:凋亡抑制因子;Bax:兔抗人单克隆抗体; Cleaved caspase-3:活化的半胱氨酸蛋白酶3;A:Bax/Bcl-2的mRNA比值水平;B、C:Bax/Bcl-2和Cleaved caspase-3/caspase-3的蛋白比值水平;D、E:流式细胞凋亡率;*：两组比较,P<0.05

抑制Calpain 2可抑HK-2细胞炎症和细胞凋亡  如图6C所示, PD151746抑制Calpain 2可有效地抑制Integrin β1的表达。此外,Yoda 1可使HK-2细胞上清IL-1β和TNF-α 的水平和细胞凋亡率明显上升,但这个结果可被PD151746抑制(图6D、E)。

图6 Piezo 1通过Ca2+/Calpain2/Integrin β1通路参与肾小管上皮细胞炎症和凋亡

Control组:正常对照组;LPS:脂多糖;Scramble:siRNA的阴性对照;Piezo 1 siRNA:Piezo 1小干扰RNA;Yoda 1:Piezo 1激动剂;PD151746:Calpain 2抑制剂;Calpain 2:钙蛋白酶2;Integrin β1:整合素β1;IL-1β:白细胞介素1β;TNF-α:肿瘤坏死因子α;A:Ca2+含量的荧光图像;Fluo-4 AM探针标记的Ca2+为绿色,细胞核为蓝色(×400,比例尺=20 μm);B、C:Calpain 2和Integrin β1的蛋白水平;D:细胞上清IL-1β和TNF-α的水平;E:流式细胞凋亡率;*：两组比较,P<0.05

综上所述,Piezo 1在SA-AKI 肾小管上皮细胞表达增加,可能是通过Ca2+/Calpain 2/Integrin β1通路引起肾小管上皮细胞炎症和细胞凋亡,参与SA-AKI 的发生发展。