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Abstract: SA-PO576

3D Organellar Morphological Dynamics in Kidney Tissue Using SIM Super-Resolution Microscopy

Session Information

Category: Acute Kidney Injury

  • 103 AKI: Mechanisms

Authors

  • Taguchi, Kensei, Vanderbilt University Medical Center/Division of Nephrology & Hpertension, Nashville, Tennessee, United States
  • Brooks, Craig R., Vanderbilt University Medical Center/Division of Nephrology & Hpertension, Nashville, Tennessee, United States
Background

Organellar dynamics, mitochondrial morphology, endoplasmic reticulum (ER) morphology and autophagosome formation, are key components in the pathology of kidney disease. Imaging organellar dynamics in isolated cells has been a cornerstone to our understanding of cellular biology. Visualizing changes in these organelles in kidney tissue, however, remains difficult and largely limited to electron microscopy (EM), which provides a very narrow two dimensional image of the cellular/organellar structure. Thus, our understanding of organelle structure in vivo, and how this structure changes with disease, remains limited. Here, we investigate whether structured illumination microscopy (SIM) could be used to visualize three dimensional organellar dynamics in kidney tissue.

Methods

Human biopsies or kidneys from sham, aristolochic acid- or ischemic reperfusion-injured C57BL/6 mice were fixed and paraffin embedded following standard protocols. Sections of the kidneys were mounted on silane-treated coverslips and stained for intracellular organelles markers, and then imaged by Nikon N-SIM microscope. Kidney tissue was co-stained for markers of kidney injury, such as kidney injury molecule 1 (KIM-1) and markers of the cell cycle. Changes in organelle structure were quantified with Imaris software.

Results

SIM reveals a dramatic increase in the number and size of autophagosomes in injured tubular cells marked with KIM-1. Likewise, the ER morphology is significantly altered in injured kidneys. Mitochondrial morphology was found to be far more complex than suggested by EM. 3D rendering of the mitochondria demonstrates that within one cell in uninjured kidneys the mitochondrial network is nearly continuous. After injury, this network breaks down into many smaller mitochondria. Importantly, SIM analysis of human kidney tissue exhibits dramatic changes in mitochondrial and other organelle structures.

Conclusion

SIM imaging provides a novel approach to analyze organellar dynamics and interfaces of organelles in kidney tissue and enables a 3D morphological analysis. KIM-1 staining is associated with increased autophagy and mitochondrial fragmentation. When combined with Injury or other markers SIM imaging can help delineate the pathways involved in organelle dynamics in vivo in human disease or animal models.