Tubulointerstitial fibrosis represents the key underlying pathology of Chronic Kidney Disease (CKD), yet treatment options remain limited. In this study, we investigated the role of connexin43 (Cx43) hemichannel-mediated adenosine triphosphate (ATP) release in purinergic-mediated disassembly of adherens and tight junction complexes in early tubular injury. Human primary proximal tubule epithelial cells (hPTECs) and clonal tubular epithelial cells (HK2) were treated with Transforming Growth Factor Beta1 (TGF-β1) ± apyrase, or ATPγS for 48 h. For inhibitor studies, cells were co-incubated with Cx43 mimetic Peptide 5, or purinergic receptor antagonists Suramin, A438079 or A804598. Immunoblotting, single-cell force spectroscopy and trans-epithelial electrical resistance assessed protein expression, cell-cell adhesion and paracellular permeability. Carboxyfluorescein uptake and biosensing measured hemichannel activity and real-time ATP release, whilst a heterozygous Cx43+/− mouse model with unilateral ureteral obstruction (UUO) assessed the role of Cx43 in vivo. Immunohistochemistry of biopsy material from patients with diabetic nephropathy confirmed increased expression of purinergic receptor P2X7. TGF-β1 increased Cx43 mediated hemichannel activity and ATP release in hPTECs and HK2 cells. The cytokine reduced maximum unbinding forces and reduced cell-cell adhesion, which translated to increased paracellular permeability. Changes were reversed when cells were co-incubated with either Peptide 5 or P2-purinoceptor inhibitors. Cx43+/− mice did not exhibit protein changes associated with early tubular injury in a UUO model of fibrosis. Data suggest that Cx43 mediated ATP release represents an initial trigger in early tubular injury via its actions on the adherens and tight junction complex. Since Cx43 is highly expressed in nephropathy, it represents a novel target for intervention of tubulointerstitial fibrosis in CKD. In proximal tubular epithelial cells (PTECs), tight junction proteins, including zona occuludens-1 (ZO-1), contribute to epithelial integrity, whilst the adherens junction protein epithelial (E)-cadherin (ECAD) maintains cell-cell coupling, facilitating connexin 43 (Cx43) gap junction-mediated intercellular communication (GJIC) and the direct transfer of small molecules and ions between cells. In disease, such as diabetic nephropathy, the pro-fibrotic cytokine transforming growth factor beta1 (TGF-β1) binds to its receptor and recruits SMAD2/3 signalling ahead of changes in gene transcription and up-regulation of Cx43-mediated hemichannels (HC). Uncoupled hemichannels permit the release of adenosine triphosphate (ATP) in to the extracellular space (↑[ATP]e), where ATP binds to the P2X7 purinoreceptor and activates the nucleotide-binding domain and leucine-rich repeat containing (NLR) protein-3 (NLRP3) inflammasome. Inflammation results in epithelial-to-mesenchymal transition (EMT), fibrosis and tubular injury. A major consequence is further loss of ECAD and reduced stickiness between cells, which can be functionally measured as a decrease in the maximum unbinding force needed to uncouple two adherent cells (Fmax). Loss of ECAD feeds forward to further lessen cell-cell coupling exacerbating the switch from GJIC to HC-mediated release of ATP. Reduction in ZO-1 impedes tight junction effectiveness and decreases trans-epithelial resistance (↓TER), resulting in increased paracellular permeability.

中文翻译：

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阻断 Connexin-43 介导的半通道活性可防止实验性慢性肾病的早期肾小管损伤。

肾小管间质纤维化是慢性肾脏病 (CKD) 的关键潜在病理学，但治疗选择仍然有限。在这项研究中，我们研究了连接蛋白43（Cx43）半通道介导的三磷酸腺苷（ATP）释放在早期肾小管损伤中嘌呤能介导的粘附物和紧密连接复合物解体中的作用。人原代近端小管上皮细胞 (hPTEC) 和克隆性肾小管上皮细胞 (HK2) 用转化生长因子 Beta1 (TGF-β1) ± apyrase 或 ATPγS 处理 48 小时。对于抑制剂研究，细胞与 Cx43 模拟肽 5 或嘌呤能受体拮抗剂苏拉明、A438079 或 A804598 共同孵育。免疫印迹、单细胞力谱和跨上皮电阻评估了蛋白质表达、细胞间粘附和细胞旁通透性。羧基荧光素摄取和生物传感测量了半通道活性和实时 ATP 释放，同时单侧输尿管梗阻 (UUO) 杂合 Cx43+/- 小鼠模型评估了 Cx43 在体内的作用。糖尿病肾病患者活检材料的免疫组织化学证实嘌呤能受体 P2X7 表达增加。TGF-β1 增加 hPTEC 和 HK2 细胞中 Cx43 介导的半通道活性和 ATP 释放。细胞因子降低了最大解离力并减少了细胞间粘附，从而导致细胞旁通透性增加。当细胞与肽 5 或 P2-嘌呤受体抑制剂共孵育时，变化发生逆转。在 UUO 纤维化模型中，Cx43+/- 小鼠没有表现出与早期肾小管损伤相关的蛋白质变化。数据表明，Cx43 介导的 ATP 释放通过其对粘附和紧密连接复合物的作用代表了早期肾小管损伤的初始触发因素。由于 Cx43 在肾病中高表达，因此它代表了干预 CKD 肾小管间质纤维化的新靶点。在近端肾小管上皮细胞 (PTEC) 中，紧密连接蛋白（包括 zona ocludens-1 (ZO-1)）有助于上皮完整性，而粘附连接蛋白上皮 (E)-钙粘蛋白 (ECAD) 则维持细胞间偶联，促进连接蛋白 43 (Cx43) 间隙连接介导的细胞间通讯 (GJIC) 以及细胞间小分子和离子的直接转移。在糖尿病肾病等疾病中，促纤维化细胞因子转化生长因子 β1 (TGF-β1) 与其受体结合，并在基因转录变化和 Cx43 介导的半通道 (HC) 上调之前招募 SMAD2/3 信号传导。解偶联的半通道允许将三磷酸腺苷 (ATP) 释放到细胞外空间 (↑[ATP]e)，其中 ATP 与 P2X7 嘌呤受体结合并激活核苷酸结合结构域和富含亮氨酸的重复序列 (NLR) 蛋白 3 (NLRP3) 炎性小体。炎症导致上皮间质转化（EMT）、纤维化和肾小管损伤。一个主要后果是 ECAD 的进一步损失和细胞间粘性的降低，这可以通过功能上测量为解开两个贴壁细胞所需的最大解结合力 (Fmax) 的降低。ECAD 的丧失会进一步减少细胞间的耦合，加剧从 GJIC 到 HC 介导的 ATP 释放的转变。ZO-1 的减少会阻碍紧密连接的有效性并降低跨上皮阻力 (↓TER)，从而导致细胞旁通透性增加。