Fluorescent Labeling Approach for Visualizing and Quantifying Glomerular Density and Volume in Mouse Kidneys

Glomeruli number and glomerular volume are indicators of kidney health and chronic kidney disease (CKD) progression. Low nephron number is predictive of the development of CKD, and increased glomerular volume is associated with hyperfiltration and hypertension. Accurate estimates of glomeruli number are necessary for relating whole body GFR to single nephron GFR (SNGFR) which is an important parameter for mathematical modeling of transport along the nephron. In addition, previous studies had observed larger volumes and greater SNGFR in juxtamedullary glomeruli compared to cortical glomeruli. To enable routine estimation of cortical glomerular density and the size of cortical and juxtamedullary glomeruli, we optimized a cost- and time-effective method for labeling and imaging glomeruli in mouse models. Mice were injected with Alexa fluor 647 conjugated wheat germ agglutinin (WGA-647; 0.5 mg) intracardially. After 4 minutes circulation time, kidneys were perfusion fixed and harvested. Kidneys were sectioned at the midline at 600 µm up to 999 µm and cleared with ethyl cinnamate. Cross sections were imaged on Leica Sp8 with either 10x or 25x objective and 15 µm z-step size. This method enabled visualization of perfused glomerular capillaries and kidney vasculature. The cortical region was identified based on autofluorescence (488 nm laser). Individual glomeruli were identified and counted by automated 3D segmentation in Python. The diameters of individual glomeruli were measured in higher magnification images using Leica LAS X software. Juxtamedullary glomeruli were identified as those within the inner third of the cortex, nearest the medulla. On average, juxtamedullary glomeruli had larger diameters compared to cortical glomeruli. However, the extent of WGA-647 perfusion within individual glomeruli varied along the interlobular vessels. Including heparin (500 U/L) in the WGA-647 injection resulted in better filling or perfusion of glomeruli, especially in the superficial cortex, and as a result the estimated average glomerular diameter increased. In future, this methodology will be applied to compare cortical glomerular density and average glomerular volume in male and female mice and in genetic kidney disease models. National Institutes of Health T32 DK007052, R01 DK125049, U54 DK137329 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.