Abstract
IntroductionFoot 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.MethodsWe analyzed elastica-Masson trichrome (EMT) and periodic acid-Schiff (PAS) stained kidney sections using structured illumination microscopy (SIM).ResultsEMT 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.ConclusionsKidney microstructures are quantified from sections prepared in clinical practice using SIM.
Highlights
Foot process effacement and mitochondrial fission associate with kidney disease pathogenesis
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
Quantified mitochondrial damage in patients with immunoglobulin A (IgA) nephropathy predicted a decline in estimated glomerular filtration rate after kidney biopsy but did not correlate with eGFR at biopsy
Summary
We analyzed elastica-Masson trichrome (EMT) and periodic acid-Schiff (PAS) stained kidney sections using structured illumination microscopy (SIM). Kidney biopsy samples were obtained from inpatients of the Nephrology Unit at Osaka University Medical. Characteristics of the patients at the time of renal biopsy were obtained from their medical records and patients who were younger than 16 years of age were excluded from the study. The eGFR slope of each patient (ml/min/1.73 m2 per year) was estimated using a mixed-effects linear regression model including all available data on eGFR after kidney biopsy, with individual patients regarded as a random effect. Images for the foot process analyses (Figure 1c), coefficient of variation (CV) values (Figure 2b, c, and d), and damage indices (Figure 3c and d) were obtained by a physician who was blinded to patients’ information. The other images were not obtained in a blinded manner
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