白蛋白活化肾小管上皮细胞对肾小管周微血管的影响及机制研究

Influence of albumin-activated renal proximal tubular epithelial cells on capillaries and its potential mechanism

目的探讨人类血清白蛋白（HSA）超负荷损伤肾小管上皮细胞后对肾间质微血管损伤的影响及可能机制。方法激光共聚焦显微镜观察肾小管上皮细胞（HKC）吞饮罗丹明标记白蛋白（TRITC—BSA）以及吞饮受体cubilin siRNA对其的抑制效应。氢化乙锭标记的荧光探针柃测白蛋白刺激HKC产生O2^-以及白蛋白乔饮受体cubilin siRNA和线粒体呼吸链复合物Ⅰ抑制剂鱼藤酮对HKC产生O2^-的抑制作用。培养上清H2O2采用化学比色方法检测。倒置显微镜下观察HSA激活的HKC以及cubilin siRNA或龟藤酮预处理的HKC与脐静脉内皮细胞（HUVEC）共培养后缸管内皮管样结构形成情况。四甲基偶氮哗盐（MTT）比色法测定内皮细胞活力。用流式细胞仪以AnnexinV FITC／PI双染法偷测内皮细胞凋亡率。结果（1）cubilin siRNA可显著抑制HKC译饮白蛋白（P〈0．05）。（2）HSA刺激HKC产生ROS呈剂量及时间依赖性（P〈0．05）；cubilin siRNA及鱼藤酮均可抑制门蛋门超负荷诱导HKC产牛大垃ROS（P〈0．05）。（3）HKC与HUVEC共培养体系中，HSA活化的HKC抑制内皮细胞管样结构的形成，内皮细胞增殖显著减少（P〈0．05），细胞凋亡率显著增高（P〈0．05）；而cubilin siRNA和鱼藤酮干预组内皮细胞管样结构数及内皮细胞活力显著增加（P〈0．05），细胞凋亡率显著降低（P〈0．05）。结论白蛋白可通过乔饮受体cubilin激活肾小管上皮细胞线粒体呼吸链复合物Ⅰ产生大馈ROS，后者可能是慢性肾病时大量蛋白尿活化肾小管上皮细胞进而损伤肾小管周徽血管网的再要介质。

Objective To explore the influence of albumin-activated renal tubular epithelial cells （RTECs）on peritubular capillaries in co-cuhure system and its potential mechanism. Methods Endocytosis of TRITC labeled bovine serum albumin （TRITC-BSA） by HKC was detected by laser scanning confocal fluorescence microscope. HKC or HKC transfeeted with eubilin （endocytic receptor of albumin） siRNA or pre-treated with rotenone was incubated with albumin（20 g/L） for 24 h respectively. Fluorescence probe technique and spectrometry were applied for determination of intracellular superoxide anion O2^- and H2O2 in supernatant. Then, the albuminactivated-HKC, pretreated-HKC with euhilin siRNA or rotenone, was euhured with HUVEC for 24 h in co-cnlture system respectively. HUVEC proliferation was determined by MTF and cellular apoptosis was analyzed by flow cytometry. Tuhnlar morphogenesis of endothelial cells was examined by microscopy. Results TRITC-BSA uptake was obviously lower in HKC transfected with cubilin siRNA. Intracellular generation of O2^- and H2O2 in culture supernatant was increased in dose- and time-dependent manner after stimulating with albumin. The levels of O2^- and H2O2 were suppressed by cubilin siRNA and rotenone. In co-culture system, albumin-activated-HKC induced endothelial ceils apoptosis and inhibited their capillary tubular morphogenesis. Pretreatment of HKC with cubilin siRNA or rotenone could suppress endothelial cells apoptosis and promote capillary tubular morphogenesis. Conclusions There may be a crosstalk between RTECs and peritubular microvascular endothelial cells in renal proteinuric diseases. The generation of ROS by albumin- activated RTECs may play an important role in this process.