Transmission Electron Microscopy Analysis of Glomerular Ultramorphologic Lesions.

We processed fresh kidney samples for transmission electron microscopy (TEM) as described (Szeto et al., 2017). Freshly isolated renal tissue was fixed in 4% paraformaldehyde, followed by postfixation in 1% osmium tetroxide, dehydration in graded alcohols, and embedding in Epon. Ultrathin sections (200–400 Å) were cut on nickel grids, stained with uranyl acetate and lead citrate, and examined in the Weill Cornell Microscopy Core using a digital transmission electron microscope (JEM-1400; JEOL, Ltd., Akishima, Japan).

We evaluated renal TEM sections for the following ultramorphologic changes: 1) diffuse glomerular basement membrane (GBM) thickening, 2) mesangial expansion, 3) podocytopenia, 4) diffuse foot process effacement, 5) electron‐dense areas of hyalinosis in sclerotic nodules, 6) cellular lipid droplets (LDs), and 7) diabetic fibrillosis. We performed quantitative analysis of GBM thickening using the orthogonal intercept method of Jensen (Jensen et al., 1979). Briefly, 10 digital images were captured per animal (five capillary loops per glomerulus, five glomeruli per animal). A grid mask with intercepts was applied to each image, and GBM thickness was measured in pixels at each intercept from the basal endothelial to the basal podocyte plasma membranes and converted to nanometers using the image processing software iTEM (Olympus-SiS, Münster, Germany). Quantitative analysis of mesangial expansion (volume density of mesangium and mesangial matrix) was conducted as described in Guo et al. (2005), podocyte number as in Weibel and Gomez (1962), and diffuse foot process effacement according to Deegens et al. (2008). We evaluated and quantitated ultramorphologic changes to renal TEM sections in a blinded manner as described (Weibel and Gomez, 1962; Jensen et al., 1979; Guo et al., 2005; Deegens et al., 2008).