Cadherin-related molecules in slit diaphragms and the other intercellular junctions of podocytes in the kidney

在用简并的RT-PCR方法寻找大鼠肾小球中表达的钙粘蛋白相关分子的过程中，我们分离到了编码脂肪和原钙粘蛋白的cDNA68。FAT是钙粘蛋白超家族的一员，是一种大的跨膜蛋白。为了定位脂肪并推测其在肾脏中的作用，我们用核糖核酸酶保护试验(RPA)、免疫印迹(WB)和免疫电子显微镜(IEM)检测了脂肪在生理状态下的大鼠肾脏和糖尿病肾病大鼠肾脏中的表达。用IEM证实了脂肪是缝隙横隔膜的一种成分。在PAN肾病中，RPA、WB和免疫组织化学染色显示肾小球脂肪表达增加。免疫组织化学显示，脂肪除定位于裂隙隔外，还定位于足细胞的紧密连接型细胞间连接。以上结果表明，脂肪是足细胞细胞间连接的基本成分之一，PCDH68也属于钙粘蛋白超家族。其胞浆结构域序列与已注册的人PCDH68的序列不同。从我们克隆的人PCDH68的序列和大鼠体内Northern杂交的结果来看，不太可能存在PCDH68的同型。RPA在肾、脑、小脑、肺、脾的肾小球中有明显表达。抗Thy-1抗体处理并未降低其在肾小球的表达，表明系膜细胞不表达PCDH68。在PAN肾病中，其在肾小球的表达水平无明显变化。现在，我们假设PCDH68存在于足细胞的紧密连接型细胞间连接中。为了阐明PCDH68的精确定位，我们正在制备针对PCDH68胞浆结构域的抗体。

During a search for cadherin-related molecules expressed in rat glomeruli by PT-PCR with degenerate primers, we have isolated cDNA encoding FAT and protocadherin (PCDH) 68.FAT, a member of the cadherin super family, is a large transmembrane protein. To localize FAT and to speculate its role in the kidney, we examined its expression in-the rat kidney in the physiological condition and PAN nephrosis by using ribonuclease protection assay (RPA), Western blotting (WB) and immunoelectron microscopy (IEM). By IEM, it was demonstrated that FAT is a component of slit diaphragms. In PAN nephrosis, increase in FAT expression in glomeruli was shown by RPA, WB, and immunostaining. IEM showed that FAT is localized in the tight junction-type intercellular junction of podocytes in addition to slit diaphragms. The above findings suggest that FAT is one of basic constituents of intercellularjunctions of podocytes.PCDH68 also belongs to the cadherin superfamily. Its sequence was different in the cytoplasmic domain from that of human PCDH68 that has been registered. From the sequence of our cloned human PCDH68 and the result of Northern blot in the rat, it is unlikely that there are isotypes of PCDH68. RPA revealed its distinct expression in glomeruli in the kidney, cerebrum, cerebellum, lung and spleen. Anti-Thy-1 antibody treatment did not reduce its glomerular expression, indicating that mesangial cells do not express PCDH68. In PAN nephrosis, the level of the expression in glomeruli did not change. Now, we have hypothesized that PCDH68 exists in the tight junction-type intercellular junction of podocytes. To clarify the precise localization of PCDH68, we are preparing the antibody against PCDH68 cytoplasmic domain.