Zhao Linning, Yaoita Eishin, Nameta Masaaki, Zhang Ying, Cuellar Lino Munoz, Fujinaka Hidehiko, Xu Bo, Yoshida Yutaka, Hatakeyama Katsuyoshi, Yamamoto Tadashi
Department of Structural Pathology, Institute of Nephrology, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori, Niigata, Japan.
Am J Physiol Regul Integr Comp Physiol. 2008 Jun;294(6):R1856-62. doi: 10.1152/ajpregu.00862.2007. Epub 2008 Mar 26.
Tight junctions rarely exist in podocytes of the normal renal glomerulus, whereas they are the main intercellular junctions of podocytes in nephrosis and in the early stage of development. Claudins have been identified as tight junction-specific integral membrane proteins. Those of podocytes, however, remain to be elucidated. In the present study, we investigated the expression and localization of claudin-6 in the rat kidney, especially in podocytes. Western blot analysis and RT-PCR revealed that the neonatal kidney expressed much higher levels of claudin-6 than the adult kidney. Immunofluorescence microscopy showed intense claudin-6 staining in most of the tubules and glomeruli in neonates. The staining in tubules declined distinctly in adults, whereas staining in glomeruli was well preserved during development. Claudin-6 in glomeruli was distributed along the glomerular capillary wall and colocalized with zonula occludens-1. The staining became conspicuous after kidney perfusion with protamine sulfate (PS) to increase tight junctions in podocytes. Immunoelectron microscopy showed that immunogold particles for claudin-6 were accumulated at close cell-cell contact sites of podocytes in PS-perfused kidneys, whereas a very limited number of immunogold particles were detected, mainly on the basal cell membrane and occasionally at the slit diaphragm and close cell-cell contact sites in normal control kidneys. In puromycin aminonucleoside nephrosis, immunogold particles were also found mainly at cell-contact sites of podocytes. These findings indicate that claudin-6 is a transmembrane protein of tight junctions in podocytes during development and under pathological conditions.
紧密连接在正常肾小球足细胞中很少存在，而在肾病和发育早期，它们是足细胞的主要细胞间连接。Claudins已被确定为紧密连接特异性整合膜蛋白。然而，足细胞中的Claudins仍有待阐明。在本研究中，我们调查了Claudin-6在大鼠肾脏中的表达和定位，特别是在足细胞中的表达和定位。蛋白质印迹分析和逆转录聚合酶链反应显示，新生大鼠肾脏中Claudin-6的表达水平远高于成年大鼠肾脏。免疫荧光显微镜检查显示，新生大鼠的大多数肾小管和肾小球中有强烈的Claudin-6染色。成年大鼠肾小管中的染色明显减少，而肾小球中的染色在发育过程中保存良好。肾小球中的Claudin-6沿肾小球毛细血管壁分布，并与闭合蛋白-1共定位。在用硫酸鱼精蛋白（PS）灌注肾脏以增加足细胞紧密连接后，染色变得明显。免疫电子显微镜显示，在PS灌注的肾脏中，Claudin-6的免疫金颗粒聚集在足细胞紧密的细胞间接触部位，而在正常对照肾脏中，仅在基底细胞膜上检测到非常有限数量的免疫金颗粒，偶尔在裂孔隔膜和紧密的细胞间接触部位检测到。在嘌呤霉素氨基核苷肾病中，免疫金颗粒也主要存在于足细胞的细胞接触部位。这些发现表明，Claudin-6是发育过程中和病理条件下足细胞紧密连接的跨膜蛋白。
