Department of Anatomy and Cell Biology, University Medicine Greifswald, Greifswald, Germany; and.
Department of Ophthalmic Research, Cole Eye Institute, Cleveland Clinic, Cleveland, Ohio.
Am J Physiol Renal Physiol. 2014 Dec 15;307(12):F1427-34. doi: 10.1152/ajprenal.00029.2014. Epub 2014 Oct 8.
The zebrafish pronephros is a well-established model to study glomerular development, structure, and function. A few methods have been described to evaluate glomerular barrier function in zebrafish larvae so far. However, there is a need to assess glomerular filtration as well. In the present study, we extended the available methods by simultaneously measuring the intravascular clearances of Alexa fluor 647-conjugated 10-kDa dextran and FITC-conjugated 500-kDa dextran as indicators of glomerular filtration and barrier function, respectively. After intravascular injection of the dextrans, mean fluorescence intensities of both dextrans were measured in the cardinal vein of living zebrafish (4 days postfertilization) by confocal microscopy over time. We demonstrated that injected 10-kDa dextran was rapidly cleared from the circulation, became visible in the lumen of the pronephric tubule, quickly accumulated in tubular cells, and was detectably excreted at the cloaca. In contrast, 500-kDa dextran could not be visualized in the tubule at any time point. To check whether alterations in glomerular function can be quantified by our method, we injected morpholino oligonucleotides (MOs) against zebrafish nonmuscle myosin heavy chain IIA (zMyh9) or apolipoprotein L1 (zApol1). While glomerular filtration was reduced in zebrafish nonmuscle myosin heavy chain IIA MO-injected larvae, glomerular barrier function remained intact. In contrast, in zebrafish apolipoprotein L1 MO-injected larvae, glomerular barrier function was compromised as 500-kDa dextran disappeared from the circulation and became visible in tubular cells. In summary, we present a novel method that allows to simultaneously assess glomerular filtration and barrier function in live zebrafish.
斑马鱼的前肾是研究肾小球发育、结构和功能的成熟模型。迄今为止，已经描述了几种评估斑马鱼幼虫肾小球屏障功能的方法。然而，还需要评估肾小球滤过功能。在本研究中，我们通过同时测量血管内 Alexa fluor 647 标记的 10 kDa 葡聚糖和 FITC 标记的 500 kDa 葡聚糖的血管内清除率，将现有的方法扩展到同时评估肾小球滤过和屏障功能。在血管内注射葡聚糖后，通过共聚焦显微镜在活体斑马鱼（受精后 4 天）的主静脉中随时间测量两种葡聚糖的平均荧光强度。我们证明，注射的 10 kDa 葡聚糖从循环中迅速清除，在肾单位小管的管腔中可见，迅速积聚在管状细胞中，并可在泄殖腔中检测到排泄。相比之下，在任何时间点都无法在管腔中观察到 500 kDa 葡聚糖。为了检查我们的方法是否可以定量评估肾小球功能的变化，我们注射了针对斑马鱼非肌肉肌球蛋白重链 IIA（zMyh9）或载脂蛋白 L1（zApol1）的基因敲低（MO）。虽然注射斑马鱼非肌肉肌球蛋白重链 IIA MO 的幼虫肾小球滤过功能降低，但肾小球屏障功能仍然完整。相比之下，在注射载脂蛋白 L1 MO 的斑马鱼幼虫中，肾小球屏障功能受损，因为 500 kDa 葡聚糖从循环中消失并在管状细胞中可见。总之，我们提出了一种新的方法，可以在活体斑马鱼中同时评估肾小球滤过和屏障功能。
