Lectin-mediated, time-efficient, and high-yield sorting of different morphologically intact nephron segments

Pflugers Arch. 2023 Dec 13. doi: 10.1007/s00424-023-02894-w. Online ahead of print.ABSTRACTThe kidney is a highly complex organ equipped with a multitude of miniscule filter-tubule units called nephrons. Each nephron can be subdivided into multiple segments, each with its own morphology and physiological function. To date, conventional manual approaches to isolate specific nephron segments are very laborious, time-consuming, often limited to only a specific segment, and typically have low yield. Here, we describe a novel, unconventional method that is superior in many aspects to previous protocols by combining low-cost fluorophore-conjugated lectins or agglutinins (Flaggs) with flow sorting. This allows the simultaneous separation of different nephron segments with preserved 3D morphology from mouse or human samples in under 3 h. Using a 200-µm nozzle and 5 psi, glomeruli, proximal, or distal convoluted tubules are sorted with Cy3-labeled Sambucus Nigra agglutinin (SNA-Cy3), Fluorescein-labeled Lotus Tetragonolobus lectin (LTL-FITC), or Pacific Blue-labeled soybean agglutinin (SBA-PB), respectively. Connecting tubules and collecting ducts are sorted by double-positive SBA-PB and SNA-Cy3 signals, while thick ascending limb segments are characterized by the absence of any Flaggs labeling. From two mouse kidneys, this yields 37-521 ng protein/s or 0.71-16.71 ng RNA/s, depending on the specific nephron segment. The purity of sorted segments, as assessed by mRNA expression level ...
Source: Pflugers Archiv : European Journal of Physiology - Category: Physiology Authors: Source Type: research