Kensei Taguchi1, Bertha C Elias1, Evan Krystofiak2, Subo Qian1, Snehal Sant1, Haichun Yang3, Agnes B Fogo3, Craig R Brooks1,4. 1. Division of Nephrology and Hypertension, School of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, 37232, USA. 2. Cell Imaging Shared Resource, Vanderbilt University, Nashville, Tennessee, 37232, USA. 3. Department of Pathology, Microbiology and immunology, Vanderbilt University School of Medicine, Nashville, Tennessee. 4. Department of Cell and Developmental Biology, Vanderbilt University, Nashville, Tennessee, 37232, USA.
Abstract
Background: The root of many kidney diseases in humans can be traced to alterations or damage to subcellular organelles. Mitochondrial fragmentation, endoplasmic reticulum (ER) stress, and lysosomal inhibition, among others, ultimately contribute to kidney injury and are the target of therapeutics in development. Although recent technological advancements allow for the understanding of disease states at the cellular level, investigating changes in subcellular organelles from kidney tissue remains challenging. Methods: Using structured illumination microscopy, we imaged mitochondria and other organelles from paraffin sections of mouse tissue and human kidney biopsy specimens. The resulting images were 3D rendered to quantify mitochondrial size, content, and morphology. Results were compared with those from transmission electron microscopy and segmentation. Results: Super-resolution imaging reveals kidney tubular epithelial cell mitochondria in rodent and human kidney tissue form large, interconnected networks under basal conditions, which are fragmented with injury. This approach can be expanded to other organelles and cellular structures including autophagosomes, ER, brush border, and cell morphology. We find that, during unilateral ischemia, mitochondrial fragmentation occurs in most tubule cells, and they remain fragmented for >96 hours. Promoting mitochondrial fusion with the fusion promotor M1 preserves mitochondrial morphology and interconnectivity and protects against cisplatin-induced kidney injury. Conclusions: We provide, for the first time, a nonbiased, semiautomated approach for quantification of the 3D morphology of mitochondria in kidney tissue. Maintaining mitochondrial interconnectivity and morphology protects against kidney injury. Super-resolution imaging has the potential to both drive discovery of novel pathobiologic mechanisms in kidney tissue and broaden the diagnoses that can be made on human biopsy specimens.
Background: The root of many kidney diseases in humans can be traced to alterations or damage to subcellular organelles. Mitochondrial fragmentation, endoplasmic reticulum (ER) stress, and lysosomal inhibition, among others, ultimately contribute to kidney injury and are the target of therapeutics in development. Although recent technological advancements allow for the understanding of disease states at the cellular level, investigating changes in subcellular organelles from kidney tissue remains challenging. Methods: Using structured illumination microscopy, we imaged mitochondria and other organelles from paraffin sections of mouse tissue and human kidney biopsy specimens. The resulting images were 3D rendered to quantify mitochondrial size, content, and morphology. Results were compared with those from transmission electron microscopy and segmentation. Results: Super-resolution imaging reveals kidney tubular epithelial cell mitochondria in rodent and human kidney tissue form large, interconnected networks under basal conditions, which are fragmented with injury. This approach can be expanded to other organelles and cellular structures including autophagosomes, ER, brush border, and cell morphology. We find that, during unilateral ischemia, mitochondrial fragmentation occurs in most tubule cells, and they remain fragmented for >96 hours. Promoting mitochondrial fusion with the fusion promotor M1 preserves mitochondrial morphology and interconnectivity and protects against cisplatin-induced kidney injury. Conclusions: We provide, for the first time, a nonbiased, semiautomated approach for quantification of the 3D morphology of mitochondria in kidney tissue. Maintaining mitochondrial interconnectivity and morphology protects against kidney injury. Super-resolution imaging has the potential to both drive discovery of novel pathobiologic mechanisms in kidney tissue and broaden the diagnoses that can be made on human biopsy specimens.
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