Fully Automated Evaluation of Total Glomerular Number and... : Journal of the American Society of Nephrology (original) (raw)

Brief Communications

Fully Automated Evaluation of Total Glomerular Number and Capillary Tuft Size in Nephritic Kidneys Using Lightsheet Microscopy

Klingberg, Anika*; Hasenberg, Anja*; Ludwig-Portugall, Isis†; Medyukhina, Anna‡; Männ, Linda*; Brenzel, Alexandra*; Engel, Daniel R.*; Figge, Marc Thilo‡,§; Kurts, Christian†; Gunzer, Matthias*

*Institute for Experimental Immunology and Imaging, University Hospital, University Duisburg-Essen, Essen, Germany;

†Institute for Experimental Immunology, Rheinische-Friedrichs-Wilhelms University of Bonn, Bonn, Germany;

‡Applied Systems Biology, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute, Leibniz-Association, Jena, Germany; and

§Friedrich Schiller University Jena, Jena, Germany

Correspondence: Prof. Matthias Gunzer, University Duisburg-Essen, University Hospital, Institute for Experimental Immunology and Imaging, Hufelandstrasse 55, D-45122 Essen, Germany. Email: [email protected]

Abstract

The total number of glomeruli is a fundamental parameter of kidney function but very difficult to determine using standard methodology. Here, we counted all individual glomeruli in murine kidneys and sized the capillary tufts by combining in vivo fluorescence labeling of endothelial cells, a novel tissue–clearing technique, lightsheet microscopy, and automated registration by image analysis. Total hands–on time per organ was <1 hour, and automated counting/sizing was finished in <3 hours. We also investigated the novel use of ethyl-3-phenylprop-2-enoate (ethyl cinnamate) as a nontoxic solvent–based clearing reagent that can be handled without specific safety measures. Ethyl cinnamate rapidly cleared all tested organs, including calcified bone, but the fluorescence of proteins and immunohistochemical labels was maintained over weeks. Using ethyl cinnamate–cleared kidneys, we also quantified the average creatinine clearance rate per glomerulus. This parameter decreased in the first week of experimental nephrotoxic nephritis, whereas reduction in glomerular numbers occurred much later. Our approach delivers fundamental parameters of renal function, and because of its ease of use and speed, it is suitable for high-throughput analysis and could greatly facilitate studies of the effect of kidney diseases on whole-organ physiology.