Gap junctional intercellular communication of renal podocytes

肾小球足细胞损伤或足细胞丢失被认为是局灶节段性肾小球硬化症发生的重要机制。然而，足细胞对损伤的反应机制还知之甚少。在这项研究中，肾小球连接蛋白43（Cx43）间隙连接蛋白的表达进行了检查，在蛋白质和转录水平的足细胞损伤的实验模型，嘌呤霉素氨基糖苷（PAN）肾病。免疫荧光和免疫电镜显示，在PAN肾病的早期到肾炎阶段，足细胞的Cx43信号显著增加。Western blotting和核糖核酸酶保护试验显示PAN肾病时肾小球Cx43蛋白、磷酸化水平和转录水平均明显升高。将荧光染料荧光黄单细胞显微注射到培养的足细胞中，证明足细胞中存在功能性间隙连接细胞间通讯（GJIG）。胎肾免疫荧光显微镜显示，足细胞中Cx43的表达与足细胞分化平行增加。间隙连接的形成需要其他支持连接，如粘附连接。然而，我们没有发现经典的钙粘蛋白或连环蛋白在足细胞在PAN肾病。相反，在足细胞中检测到柯萨奇病毒和腺病毒受体（CAR）。CAR是紧密连接的完整膜组分，并促进同型细胞聚集。足细胞的缝隙连接与紧密连接紧密相连，足细胞间存在Cx43介导的GJIC，提示足细胞可能作为一个完整的肾小球上皮细胞而非单个细胞对损伤作出反应。

Podocyte injury or podocyte loss in the renal glomerulus has been proposed as the crucial mechanism in the development of focal segmental glomerulosclerosis. However, it is poorly understood how podocytes respond to injury. In this study, glomerular expression of connexin43(Cx43) gap junction protein was examined at both protein and transcript levels in an experimental model of podocyte injury, puromycin aminonucleoside(PAN) nephrosis. Inmunofluorescence and immunoelectron microscopy showed a striking increase in signals for Cx43 of podocytes in the early to nephritic stage of PAN nephrosis. Consistently, Western blotting and ribonuclease protection assay revealed a distinct increase of Cx43 protein, phosphorylation and transcript in glomeruli during PAN nephrosis. A single-cell microinjection of fluorescent dye, Lucifer yellow, into cultured podocytes demonstrated the presence of functional gap junctional intercellular communication(GJIG) in podocytes. Immunofluorescence microscopy using fetal kidney showed that expression of Cx43 in podocytes increased in parallel with podocyte differentiation. Gap junction formation needs the other supporting junction such as adherens junction. However, we could not find classic cadherins or catenins in podocytes in PAN nephrosis. Instead, coxsackievirus and adenovirus receptor(CAR) was detected in podocytes. CAR is an integral membrane component of tight junction and promotes homotypic cell aggregation. Gap junction is located closely to tight junction in podocytes.Thus, it is concluded that Cx43-mediated GJIC is present between podocytes, suggesting that podocytes may respond to injury as an integrated epithelium on a glomerulus rather than individually as a separate cell.