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Quantitative Analyses of Foot Processes, Mitochondria, and Basement Membranes by Structured Illumination Microscopy
Ayumi Matsumoto1, Isao Matsui1, Yusuke Katsuma1
1Department of Nephrology, Osaka University Graduate School of Medicine, Osaka, Japan.
Introduction:
Foot process effacement and mitochondrial fission associate with kidney disease pathogenesis. Electron microscopy is the gold-standard method for their visualization, but the observable area of electron microscopy is smaller than light microscopy. It is important to develop alternative ways to quantitatively evaluate these microstructural changes because the lesion site of renal diseases can be focal.
Methods:
We analyzed elastica-Masson trichrome (EMT) and periodic acid-Schiff (PAS) stained kidney sections using structured illumination microscopy (SIM).
Results:
EMT staining revealed three-dimensional (3D) structures of foot process, whereas ponceau xylidine acid fuchsin azophloxine solution induced fluorescence. Conversion of foot process images into their constituent frequencies by Fourier transform showed that the concentric square of (1/4)2-(1/16)2 in the power spectra (PS) included information for normal periodic structures of foot processes. Foot process integrity, assessed by PS, negatively correlated with proteinuria. EMT-stained sections revealed fragmented mitochondria in mice with mitochondrial injuries and patients with tubulointerstitial nephritis; Fourier transform quantified associated mitochondrial injury. Quantified mitochondrial damage in patients with immunoglobulin A (IgA) nephropathy predicted a decline in estimated glomerular filtration rate (eGFR) after kidney biopsy but did not correlate with eGFR at biopsy. PAS-stained sections, excited by a 640 nm laser, combined with the coefficient of variation values, quantified subtle changes in the basement membranes of patients with membranous nephropathy stage I.
Conclusions:
Kidney microstructures are quantified from sections prepared in clinical practice using SIM.
Insights
Structured illumination microscopy (SIM) quantifies kidney microstructures like foot processes and mitochondria from standard clinical sections. This method correlates foot process integrity with proteinuria and predicts kidney function decline in IgA nephropathy patients.
Area of Science:
- Nephrology
- Biomedical Imaging
- Pathology
Background:
- Foot process effacement and mitochondrial fission are key in kidney disease pathogenesis.
- Electron microscopy, while gold-standard, has limited field of view for focal lesions.
- Alternative quantitative methods are needed for microstructural changes in kidney diseases.
Purpose of the Study:
- To develop and validate quantitative methods for evaluating kidney microstructural changes using structured illumination microscopy (SIM).
- To assess foot process integrity and mitochondrial damage in kidney disease.
- To correlate microstructural findings with clinical parameters like proteinuria and estimated glomerular filtration rate (eGFR).
Main Methods:
- Analyzed elastica-Masson trichrome (EMT) and periodic acid-Schiff (PAS) stained kidney sections with SIM.
- Utilized Fourier transform analysis of EMT-stained sections to assess foot process structure and mitochondrial integrity.
- Employed coefficient of variation analysis on PAS-stained sections to quantify basement membrane changes.
Main Results:
- EMT staining with SIM revealed 3D foot process structures and quantified mitochondrial fragmentation.
- Power spectra analysis of foot process images showed integrity negatively correlated with proteinuria.
- Quantified mitochondrial damage in IgA nephropathy patients predicted future eGFR decline.
- PAS staining quantified subtle basement membrane changes in membranous nephropathy.
Conclusions:
- Kidney microstructures can be quantitatively assessed from clinically prepared sections using SIM.
- SIM offers a viable alternative for evaluating microstructural damage in kidney diseases.
- This approach aids in understanding disease pathogenesis and predicting clinical outcomes.
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