Burford James L, Gyarmati Georgina, Shirato Isao, Kriz Wilhelm, Lemley Kevin V, Peti-Peterdi János
Department of Physiology and Biophysics, Zilkha Neurogenetic Institute, University of Southern California, 1501 San Pablo Street, Room ZNI 335, Los Angeles, CA, 90033, USA.
Division of Nephrology, Department of Internal Medicine, School of Medicine, Juntendo University, Tokyo, Japan.
Pflugers Arch. 2017 Aug;469(7-8):965-974. doi: 10.1007/s00424-017-2020-0. Epub 2017 Jun 29.
The development of podocyte injury and albuminuria in various glomerular pathologies is still incompletely understood due to technical limitations in studying the glomerular filtration barrier (GFB) in real-time. We aimed to directly visualize the early morphological and functional changes of the GFB during the development of focal segmental glomerulosclerosis (FSGS) using a combination of transmission electron microscopy (TEM) and in vivo multiphoton microscopy (MPM) in the rat puromycin aminonucleoside (PAN) model. We hypothesized that this combined TEM + MPM experimental approach would provide a major technical improvement that would benefit our mechanistic understanding of podocyte detachment. Male Sprague-Dawley (for TEM) or Munich-Wistar-Frömter (for MPM) rats were given a single dose of 100-150 mg/kg body weight PAN i.p. and were either sacrificed and the kidneys processed for TEM or surgically instrumented for in vivo MPM imaging at various times 2-14 days after PAN administration. Both techniques demonstrated hypertrophy and cystic dilatations of the subpodocyte space that developed as early as 2-3 days after PAN. Adhesions of the visceral epithelium to the parietal Bowman's capsule (synechiae) appeared at days 8-10. TEM provided unmatched resolution of podocyte foot process remodeling, while MPM revealed the rapid dynamics of pseudocyst filling, emptying, and rupture, as well as endothelial and podocyte injury, misdirected filtration, and podocyte shedding. Due to the complementary advantages of TEM and MPM, this combined approach can provide an unusally comprehensive and dynamic portrayal of the alterations in podocyte morphology and function during FSGS development. The results advance our understanding of the role and importance of the various cell types, hemodynamics, and mechanical forces in the development of glomerular pathology.
由于实时研究肾小球滤过屏障（GFB）存在技术限制，目前对各种肾小球疾病中足细胞损伤和蛋白尿的发生发展仍未完全了解。我们旨在利用透射电子显微镜（TEM）和体内多光子显微镜（MPM）相结合的方法，在大鼠嘌呤霉素氨基核苷（PAN）模型中直接观察局灶节段性肾小球硬化（FSGS）发展过程中GFB的早期形态和功能变化。我们假设这种TEM + MPM联合实验方法将带来重大技术改进，有助于我们从机制上理解足细胞脱离。给雄性Sprague-Dawley大鼠（用于TEM）或Munich-Wistar-Frömter大鼠（用于MPM）腹腔注射100 - 150 mg/kg体重的PAN单次剂量，在注射PAN后2 - 14天的不同时间点，要么处死大鼠并将肾脏用于TEM处理，要么进行手术操作以进行体内MPM成像。两种技术均显示早在PAN注射后2 - 3天足细胞下间隙就出现肥大和囊性扩张。8 - 10天时出现脏层上皮与壁层鲍曼囊的粘连（粘连）。TEM提供了足细胞足突重塑的无与伦比的分辨率，而MPM揭示了假囊肿填充、排空和破裂的快速动态过程，以及内皮细胞和足细胞损伤、滤过方向错误和足细胞脱落。由于TEM和MPM的互补优势，这种联合方法能够异常全面且动态地描绘FSGS发展过程中足细胞形态和功能的变化。这些结果推进了我们对各种细胞类型、血流动力学和机械力在肾小球疾病发展中的作用及重要性的理解。
