Quantitative Intravital Imaging of Endo‐Lysosomal System Dynamics in the Kidney Proximal Tubule
Authors: Schuh Claus Dieter , Polesel Marcello , Haenni Dominik , Hall Andrew M. 
Published: June 2021

Low‐molecular weight proteins (LMWPs) are filtered in the kidney and reclaimed by the proximal convoluted tubule (PCT) to prevent their urinary wasting. They are taken up by receptor‐mediated endocytosis before entering the endo‐lysosomal system (ELS). While the uptake pathway in vivo is well understood, the trafficking and fate of LMWPs in the ELS is still a matter of debate. Intravital microscopy offers the potential to study ELS trafficking in the PCT but tracking single vesicles over prolonged periods of time in living animals to generate quantitative data is a major technical challenge, due to issues such as tissue movement and dye bleaching.Physiological ligands for PCT endocytosis like lysozyme and lactoglobulin were conjugated to bright and photo‐stable Atto dyes and injected intravenously into anaesthetized C57Bl/6J‐Rj mice. A custom‐designed multiphoton microscope and stage were used to perform stable imaging of PCT cells over time at high resolution. Structural markers such as SiR Actin were used to identify cell membranes. Trafficking of cargo containing vesicles in the ELS was tracked and quantified using multistep computational analysis.ATTO565‐labeled lysozyme and lactoglobulin showed rapid filtration and uptake into PCT cells in vivo. Figure 1 shows the handling of lysozyme over 5 minutes. Images were acquired every 6 seconds, and dynamic trafficking of labeled vesicles is depicted by temporal color‐coding, where every bright pixel is assigned a color representing the time when it appeared during the image sequence. Actin (in white) delineates the apical brush border and the basement membrane. Single tracks of points clearly show vesicles traveling from the sub‐apical region all the way to the basolateral membrane, in a direction matching the known distribution of microtubules. To investigate in more detail the dynamics of cargo‐loaded vesicle trafficking we recorded zoomed‐in images (47 μm2) every second for 10 to 15 minutes. Afterwards the data was deconvolved and stabilized using Huygens software, before single vesicles were tracked semi‐automatically in the image analysis software Imaris.A subset of labeled vesicles could be identified that traffic rapidly through the cell and reside at the basolateral membrane before returning to the apical side. These vesicles move at maximal speeds up to 800 nm/s, with typical mean speeds of around 150 nm/s. Maximum track distances up to 5.2 μm were observed, meaning that the respective vesicles traversed the cell in approximately 20 seconds. Figure 2 shows an example of trafficking vesicles imaged at high resolution and depicted as both SUM and temporal color‐coded images. Furthermore, quantitative data concerning track distance, speed and duration are provided for a single vesicle. The measured speeds of vesicle movement are consistent with active transport along microtubules.Using LMWPs labeled with new bright dyes, a custom‐designed microscopy setup and advanced computational analysis we can perform quantitative tracking of cargo containing vesicles in the kidneys of living mice. This approach provides the potential to gain important new insights into dynamic ELS function in the PCT.Support or Funding InformationNCCR Kidney. CH; SNSF; CRPP MINZThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.