Supplementary Material for: Podocyte-Released Migrasomes in Urine Serve as an Indicator for Early Podocyte Injury

Background: Levels of urinary microvesicles, which are increased during various kidney injuries, have diagnostic potential for renal diseases. However, the significance of urinary microvesicles as a renal disease indicator is dampened by the difficulty to ascertain their cell source. Objectives: The aim of this study was to demonstrate that podocytes can release migrasomes, a unique class of microvesicle with size ranging between 400 and 2,000 nm, and the urine level of migrasomes may serve as novel non-invasive biomarker for early podocyte injury. Method: In this study, immunofluorescence labeling, electronic microscopy, nanosite, and sequential centrifugation were used to purify and analyze migrasomes. Results: Migrasomes released by podocytes differ from exosomes as they have different content and mechanism of release. Compared to podocytes, renal tubular cells secrete markedly less migrasomes. Moreover, secretion of migrasomes by human or murine podocytes was strongly augmented during podocyte injuries induced by LPS, puromycin amino nucleoside (PAN), or a high concentration of glucose (HG). LPS, PAN, or HG-induced podocyte migrasome release, however, was blocked by Rac-1 inhibitor. Strikingly, a higher level of podocyte migrasomes in urine was detected in mice with PAN-nephropathy than in control mice. In fact, increased urinary migrasome number was detected earlier than elevated proteinuria during PAN-nephropathy, suggesting that urinary migrasomes are a more sensitive podocyte injury indicator than proteinuria. Increased urinary migrasome number was also detected in diabetic nephropathy patients with proteinuria level <5.5 g/day. Conclusions: Our findings reveal that podocytes release the “injury-related” migrasomes during migration and provide urinary podocyte migrasome as a potential diagnostic marker for early podocyte injury.

背景：在多种肾脏损伤过程中，尿液微囊泡（urinary microvesicles）水平会升高，其在肾脏疾病诊断中具有潜在应用价值。然而，由于难以确定其细胞来源，尿液微囊泡作为肾脏疾病标志物的临床应用价值受到显著限制。研究目的：本研究旨在证实足细胞（podocytes）可释放迁移体（migrasomes）——一类尺寸介于400 nm至2000 nm之间的独特微囊泡，且尿液中迁移体水平可作为早期足细胞损伤的新型无创生物标志物。研究方法：本研究采用免疫荧光标记、电子显微镜、纳米位点（nanosite）技术及梯度离心法，对迁移体进行纯化与分析。研究结果：足细胞释放的迁移体与外泌体（exosomes）存在显著差异，二者的内容物组成及释放机制均不相同。相较于足细胞，肾小管上皮细胞分泌的迁移体数量明显更少。此外，在脂多糖（LPS）、嘌呤霉素氨基核苷（PAN）或高浓度葡萄糖（HG）诱导的足细胞损伤模型中，人源或鼠源足细胞的迁移体分泌量均显著上调。而Rac-1抑制剂可有效阻断LPS、PAN或HG诱导的足细胞迁移体释放。尤为值得关注的是，PAN肾病小鼠的尿液中足细胞迁移体水平显著高于对照组小鼠。进一步研究发现，在PAN肾病模型中，尿液迁移体数量的升高早于蛋白尿（proteinuria）水平的升高，这表明尿液迁移体作为足细胞损伤标志物的敏感性优于蛋白尿。在蛋白尿水平<5.5 g/天的糖尿病肾病（diabetic nephropathy）患者中，同样检测到尿液迁移体数量升高。研究结论：本研究结果证实，足细胞在迁移过程中可释放"损伤相关"迁移体，并提出尿液足细胞迁移体可作为早期足细胞损伤的潜在诊断标志物。