Histochemical study for apolipoprotein abnormalities in lipoprotein glomerulopathy

我们通过免疫电子显微镜、电子显微镜、脂质染色和常规组织学的一系列观察，阐明在脂蛋白肾小球病(LPG)及其实验模型中，特定的载脂蛋白(Apo)E变异体参与了肾小球内脂蛋白血栓的形成。此外，我们还研究了脂蛋白血栓中颗粒大小与血浆脂蛋白谱之间的关系。我们分别使用了apoE-仙台(Arg145Pro)、apoE缺失18个氨基酸和apoE-Tokyo缺失3个氨基酸的LPG患者的活检标本。我们还研究了将apoE-仙台病毒注射到apoE缺陷小鼠体内诱导的LPG模型的组织化学。我们用抗人apoE抗体对活检标本和小鼠肾组织的冰冻切片进行了免疫过氧化物酶的研究，并用倒立包埋法将切片包埋在EPON 812中进行了电子显微镜观察。在人和实验性LPG肾小球内脂蛋白血栓中，apoE阳性染色均见于脂质颗粒周围。为了明确脂蛋白血栓中颗粒的特征，用电子显微镜和苏丹-IV染色观察了EPON包埋块的连续切片。结果表明，脂蛋白血栓主要由甘油三酯组成，甘油三酯由不饱和脂肪酸组成。在EPON包埋过程中，对苯二胺的孵育增强了实验LPG中的研究结果。我们在电子显微镜下检测了肾小球内脂质颗粒的平均直径，并与血浆中的脂蛋白图谱进行了比较。直径与全血和高密度脂蛋白中甘油三酯/胆固醇比值显著相关(r=-0.80，p&lt；0.01)，而在极低密度脂蛋白或低密度脂蛋白中不相关。此外，血浆和高密度脂蛋白中脂质颗粒较小、甘油三酯/胆固醇比值较高的患者在6个月内为肾病或肾病。

We clarified using immunoelectron microscopy and serial observations with electron microscopy and lipid stain as well as routine histology that specific apolipoprotein (apo) E variants are involved in the formation of intraglomerular lipoprotein thrombi in cases of lipoprotein glomerulopathy (LPG) and its experimental model. In addition, we investigated the relationship between sizes of particles in lipoprotein thrombi and lipoprotein profile in plasma.1. We use biopsy specimens from cases of LPG having apoE-Sendai(Arg145Pro), apoE with 18 amino acid deletion and apoE-Tokyo with 3 amino acid deletion, respectively. We also investigated histochemistry of a LPG model induced by the injection of apoE-Sendai virus into apoE-deficient mice.2. We applied immunoperoxidase study with anti-human apoE antibody to snap-frozen sections of biopsy specimens and mouse kidney portions, and embedded the sections in Epon 812 by the handstand-embedding method for electron microscopic observation. Positive stain of apoE were seen around lipid granules in the intraglomerular lipoprotein thrombi of both human and experimental LPG.3. To define the characteristics of particles in lipoprotein thrombi, serial sections of Epon-embedded blocks were observed with electron microscopy and Sudan-IV stain. The results showed that lipoprotein thrombi mainly included triglycerides, which were composed of unsaturated fatty acids. The findings in experimental LPG were enhanced by incubation in p-phenylenediamine during Epon-embedding.4. We examined the mean diameters of intraglomerular lipid particles under electron microscopy and compared with lipoprotein profile in plasma. The diameters were significantly correlated with triglycerides/cholesterol ratios in whole plasma and in HDL fraction (r=-0.80, p<0.01) but not in VLDL or LDL.In addition, the cases showing smaller lipid granules and higher TG/Chol ratios in plasma and in HDL were nephrotic or became nephrotic within 6 months.