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Abstract: FR-OR098

Assessment of Renal Function by Advanced Light Sheet Microscopy and 3D Image Analyses: Quantification of Albumin Filtration/Reabsorption at the Single-Nephron Level in Rodents

Session Information

Category: Pathology and Lab Medicine

  • 1601 Pathology and Lab Medicine: Basic

Authors

  • Østergaard, Mette Viberg, Gubra ApS, Hørsholm, Denmark
  • Roostalu, Urmas, Gubra ApS, Hørsholm, Denmark
  • Pedersen, Annemarie Aarup, Gubra ApS, Hørsholm, Denmark
  • Pedersen, Tanja X., Gubra ApS, Hørsholm, Denmark
  • Fink, Lisbeth N., Gubra ApS, Hørsholm, Denmark
  • Skytte, Jacob Lercke, Gubra ApS, Hørsholm, Denmark
  • Vrang, Niels, Gubra ApS, Hørsholm, Denmark
  • Hecksher-Sørensen, Jacob, Gubra ApS, Hørsholm, Denmark
Background

Drug development in diabetic kidney disease (DKD) and chronic kidney disease (CKD) is halted by poor translatability of preclinical animal models. Using 3D imaging techniques, we aimed to develop a new method for quantification of renal function, glomerular filtration and proximal tubular albumin absorption to assess the morphology and functionality of populations of single nephrons in preclinical rodent models of kidney disease.

Methods

Healthy mice and rats as well as 5/6 nephrectomised (Nx) rats were perfused with fluorescently labelled lectin, albumin, and 10 kDa dextran under terminal anesthesia, and intact kidneys were cleared for scanning by light sheet microscopy (LSM). Using 3D image analysis, distribution of glomerular size and nephron morphometry were determined, while proximal tubular albumin absorption was quantified based on albumin-intensity. To characterize kidney function using standard methodologies, 2D histology, plasma, and urine analyses were applied.

Results

In healthy mice and rats, LSM and 3D nephron reconstruction revealed intact nephrons with glomeruli connected to proximal then distal tubules extending from the cortex into the medulla. Juxtaglomerular proximal tubules were clearly delineated by tubular epithelial cells that were fluorescently labelled by absorbed albumin and followed by tubular epithelial cells labelled by dextran. In sharp contrast, imaging analyses suggested functional defects in the kidney remnants of 5/6 Nx rats. Nephrons in 5/6 Nx rats appeared fragmented and with limited tubular absorption of albumin and dextran, which obstructed tubular tracking from the cortex to the medulla. These findings were corroborated by albuminuria, plasma urea and creatinine, and 2D renal histopathology in 5/6 Nx vs sham-operated rats.

Conclusion

Development of advanced microscopy and 3D imaging technologies allows for assessment of renal function at a single-nephron level and by characterization of populations of single nephrons. Thereby, this 3D imaging technique can support 2D end-point histological analyses and functional endpoints in rodent models to refine the use of rodent models to study disease mechanisms and test novel therapies for DKD and CKD.