Torres Vicente E, Cai Yiquiang, Chen X I, Wu Guanquing Q, Geng Lin, Cleghorn Kathleen A, Johnson Christopher M, Somlo Stefan
Nephrology Research Unit, Division of Nephrology and Internal Medicine, Mayo Clinic and Mayo Foundation, Rochester, Minnesota.
Renal Biopsy Laboratory, Mayo Clinic and Mayo Foundation, Rochester, Minnesota.
J Am Soc Nephrol. 2001 Jan;12(1):1-9. doi: 10.1681/ASN.V1211.
The expression of polycystin-1 in the vascular smooth muscle cells (VSMC) of elastic and large distributive arteries suggests that some vascular manifestations of autosomal-dominant polycystic kidney disease (ADPKD) result directly from the genetic defect. Intracranial aneurysms have been reported in PKD2, as well as in PKD1 families. To determine whether the vascular expression of polycystin-2 is similar to that of polycystin-1, the expression of PKD2 mRNA and protein in cultured pig aortic VSMC was studied and immunofluorescence and immunohistochemistry were used to study the localization of polycystin-2 in cultured pig aortic VSMC, pig ascending thoracic aorta, and normal elastic and intracranial arteries and intracranial aneurysms obtained at autopsy from patients without or with ADPKD. Tissues derived from Pkd2 wild-type and Pkd2 null mice were used to confirm the specificity of the immunostaining for polycystin-2. Northern blots of VSMC revealed the expected 5.3-kb band. Western blotting detected a 110-kb band in a 100,000 x g fraction of VSMC homogenates. Cultured VSMC as well as VSMC between the elastic lamellae of pig thoracic aorta were positive for polycystin-2 by immunofluorescence. The staining pattern was cytoplasmic. Treatment of the cells before fixation with Taxol, colchicine, or cytochalasin-D altered the pattern of staining in a way suggesting alignment with the cytoskeleton. The immunohistochemical staining for polycystin-2 was abolished by extraction with 0.5% Triton X-100, indicating that polycystin-2 is not associated with the cytoskeleton. Weak immunoreactivity for polycystin-2, which was markedly enhanced by protease digestion, was detected in formaldehyde-fixed normal human elastic and intracranial arteries. Immunostaining of variable intensity for polycystin-2, which was not consistently enhanced by protease digestion, was seen in the spindle-shaped cells of the wall of the intracranial aneurysms. The similar expression of polycystin-1 and polycystin-2 in the vascular smooth muscle is consistent with the proposed interaction of these proteins in a single pathway. These observations suggest a direct pathogenic role for PKD1 and PKD2 mutations in the vascular complications of ADPKD.
多囊蛋白 -1 在弹性和大的分布性动脉的血管平滑肌细胞（VSMC）中的表达表明，常染色体显性多囊肾病（ADPKD）的一些血管表现直接源于基因缺陷。PKD2 以及 PKD1 家族中均有颅内动脉瘤的报道。为了确定多囊蛋白 -2 的血管表达是否与多囊蛋白 -1 相似，研究了培养的猪主动脉 VSMC 中 PKD2 mRNA 和蛋白的表达，并使用免疫荧光和免疫组织化学方法研究了多囊蛋白 -2 在培养的猪主动脉 VSMC、猪胸主动脉升段、正常弹性动脉和颅内动脉以及从患有或未患有 ADPKD 的患者尸检中获得的颅内动脉瘤中的定位。使用来自 Pkd2 野生型和 Pkd2 基因敲除小鼠的组织来确认针对多囊蛋白 -2 的免疫染色的特异性。VSMC 的 Northern 印迹显示出预期大小为 5.3 kb 的条带。Western 印迹在 VSMC 匀浆的 100,000×g 组分中检测到一条 110 kb 的条带。通过免疫荧光检测，培养的 VSMC 以及猪胸主动脉弹性板之间的 VSMC 对多囊蛋白 -2 呈阳性。染色模式为细胞质染色。在用紫杉醇、秋水仙碱或细胞松弛素 -D 固定细胞之前进行处理，以一种表明与细胞骨架排列一致的方式改变了染色模式。用 0.5% Triton X -100 提取后，多囊蛋白 -2 的免疫组织化学染色消失，表明多囊蛋白 -2 不与细胞骨架相关。在甲醛固定的正常人弹性动脉和颅内动脉中检测到对多囊蛋白 -2 的弱免疫反应性，经蛋白酶消化后明显增强。在颅内动脉瘤壁的梭形细胞中观察到对多囊蛋白 -2 的免疫染色强度各异，经蛋白酶消化后并非始终增强。多囊蛋白 -1 和多囊蛋白 -2 在血管平滑肌中的相似表达与这些蛋白在单一途径中所提出的相互作用一致。这些观察结果表明 PKD1 和 PKD2 突变在 ADPKD 的血管并发症中具有直接的致病作用。
