背景:伴随着NOD样受体蛋白3(NLRP3)炎性体的激活,异常连接蛋白43(Cx43)半通道介导的ATP释放位于炎性小体组装和炎症的上游,并导致糖尿病的多种继发性并发症和相关的心脏代谢合并症.证据表明,Cx43半通道活性与糖尿病肾脏炎症之间可能存在联系。研究了在糖尿病肾病(DKD)模型中阻断肾小管Cx43半通道介导的ATP释放在引发/激活NLRP3炎性体中的后果。我们检查了炎症的下游标志物以及肾小管分泌组对巨噬细胞募集和激活的促炎和化学引诱作用。
方法:分析来自Nephroseq资料库的人类转录组数据,将基因表达与DKD中的肾功能相关。将原代人肾近曲小管上皮细胞(RPTEC)和单核细胞衍生的巨噬细胞(MDMs)在高糖和炎性细胞因子中培养作为DKD模型,以评估Cx43半通道活性,NLRP3炎性体激活和上皮-巨噬细胞旁分泌介导的串扰。Tonabersat评估了Cx43半通道的作用。
结果:DKD患者肾活检的转录组学分析显示,Cx43和NLRP3表达增加与肾小球滤过率(GFR)下降和蛋白尿增加相关。体外,Tonabersat阻断了Cx43半通道介导的ATP释放的葡萄糖/细胞因子依赖性增加,并降低了RPTEC中炎症标志物和NLRP3炎性体活化的表达。我们观察到一种相互关系,其中NLRP3活性加剧了Cx43表达的增加和半通道介导的ATP释放,由核因子κB(NFκB)介导的引发和Cx43半通道开放驱动的事件,更改被Tonabersat阻止。来自用高糖/细胞因子处理的RPTEC的条件培养基(CM)增加了MDM中炎性标志物的表达,用Tonabersat预处理巨噬细胞时效果降低。使用来自Tonabersat处理的RPTEC的条件培养基的共培养抑制了巨噬细胞炎性标志物的表达并减少了巨噬细胞的迁移。
结论:使用DKD模型,我们首次报道高糖和炎性细胞因子引发异常的Cx43半通道活性,引发NLRP3诱导的RPTEC炎症和上皮-巨噬细胞串扰的事件。回顾以前在糖尿病视网膜病变中报道的观察结果,这些数据表明Cx43半通道阻滞剂(即,Tonabersat)可以减轻糖尿病继发性并发症中观察到的多系统损害。
BACKGROUND: Accompanied by activation of the NOD-like receptor protein 3 (NLRP3) inflammasome, aberrant connexin 43 (Cx43) hemichannel-mediated ATP release is situated upstream of inflammasome assembly and inflammation and contributes to multiple secondary complications of diabetes and associated cardiometabolic comorbidities. Evidence suggests there may be a link between Cx43 hemichannel activity and inflammation in the diabetic kidney. The consequences of blocking tubular Cx43 hemichannel-mediated ATP release in priming/activation of the NLRP3 inflammasome in a model of diabetic kidney disease (DKD) was investigated. We examined downstream markers of inflammation and the proinflammatory and chemoattractant role of the tubular secretome on macrophage recruitment and activation.
METHODS: Analysis of human transcriptomic data from the Nephroseq repository correlated gene expression to renal function in DKD. Primary human renal proximal tubule epithelial cells (RPTECs) and monocyte-derived macrophages (MDMs) were cultured in high glucose and inflammatory cytokines as a model of DKD to assess Cx43 hemichannel activity, NLRP3 inflammasome activation and epithelial-to-macrophage paracrine-mediated crosstalk. Tonabersat assessed a role for Cx43 hemichannels.
RESULTS: Transcriptomic analysis from renal biopsies of patients with DKD showed that increased Cx43 and NLRP3 expression correlated with declining glomerular filtration rate (GFR) and increased proteinuria. In vitro, Tonabersat blocked glucose/cytokine-dependant increases in Cx43 hemichannel-mediated ATP release and reduced expression of inflammatory markers and NLRP3 inflammasome activation in RPTECs. We observed a reciprocal relationship in which NLRP3 activity exacerbated increased Cx43 expression and hemichannel-mediated ATP release, events driven by nuclear factor kappa-B (NFκB)-mediated priming and Cx43 hemichannel opening, changes blocked by Tonabersat. Conditioned media (CM) from RPTECs treated with high glucose/cytokines increased expression of inflammatory markers in MDMs, an effect reduced when macrophages were pre-treated with Tonabersat. Co-culture using conditioned media from Tonabersat-treated RPTECs dampened macrophage inflammatory marker expression and reduced macrophage migration.
CONCLUSIONS: Using a model of DKD, we report for the first time that high glucose and inflammatory cytokines trigger aberrant Cx43 hemichannel activity, events that instigate NLRP3-induced inflammation in RPTECs and epithelial-to-macrophage crosstalk. Recapitulating observations previously reported in diabetic retinopathy, these data suggest that Cx43 hemichannel blockers (i.e., Tonabersat) may dampen multi-system damage observed in secondary complications of diabetes.