Electron Microscopy

Transmission electron microscopy and podocyte definition were performed by standard protocol described previously.¹¹ Briefly, the arithmetic mean of foot process width (FPW) was estimated as follow: 

∑GBM length is the measurement of total GBM length in electron microscope photographs of each AAV patient, where ∑foot process is the calculation of total number of foot processes. The constant π is served for the correction of presumed random variation, which exists inevitably in the angle of section relative to the long axis of the podocyte.^19,20 The intraobserver variability of the FPW was assessed by the coefficient of variation (the ratio of the SD to the mean) between 2 measurements of the same object by 1 observer.¹⁵ The interobserver variability in this study was assessed by the intraclass correlation coefficient.²¹

Podocyte density per glomerulus [Nv(epi/glom)] was used to estimate podocyte detachment (detailed in the Supplementary Material). In brief, the numerical density of podocytes in each glomerulus was calculated as Nv(epi/glom)=(1/1.55) × [Na(epi-nuclei/glom)³/Vv (nuclei/glom)]^1/2, where 1.55 is the shape factor for the podocyte nuclei. The calculation of both Na(epi-nuclei/glom) and Vv(epi-nuclei/glom) was performed according to the standard protocol described previously.²²

The GBM width was estimated by the orthogonal intercept method in 10,000 × electron microscope micrographs.²³