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Samples were prepared using standard procedures [4 (link)] and observed using a transmission electron microscope, JEOL JEM-1400 TEM (JEOL, Tokyo, Japan). Images were recorded on an Olympus Quemesa digital camera, using iTEM software (Olympus Soft Imaging Solutions GmbH, Munster, Germany). SP diameter was measured using an image processing program designed for scientific multidimensional images, ImageJ (National Institutes of Health, Bethesda, Maryland, USA). SP sizes are presented as the mean ± SD.

Thin carbon support films were prepared by sublimation of a carbon thread onto a freshly cleaved mica surface. Lysate containing the sacculi was adsorbed onto a carbon film for 1 min and negatively stained with 1% (w/v) aqueous uranyl acetate, pH 5.0 (Valentine et al., 1968 (link)). After air-drying, samples were examined in a TEM 910 transmission electron microscope (Carl Zeiss, Oberkochen, Germany) at an acceleration voltage of 80 kV and calibrated magnifications using a line replica. Images were recorded digitally with a Slow-Scan CCD-Camera (ProScan, 1024x1024, Scheuring, Germany) with ITEM-Software (Olympus Soft Imaging Solutions, Münster, Germany).

Kidney biopsy samples were routinely analyzed by light microscopy (hematoxylin-eosin, Periodic Acid-Schiff, Masson trichrome, and Jones methenamine silver stain), immunofluorescent analysis (IgG, IgA, IgM, C3, C1q, kappa, and lambda light chains), and EM. The stages of interstitial fibrosis and tubular atrophy (IFTA) were defined as mild (stage 1), affecting less than 25% of the specimen; moderate (stage 2), affecting 25%–50%; and severe (stage 3), affecting more than 50% of the specimen. In all participants (except one patient without glomeruli in tissue prepared for EM), the GBM thickness was measured on EM images using iTEM software (Olympus Soft Imaging Solutions GmbH). Altogether three measurements of the GBM thickness per capillary loop on ten random capillary loops per specimen were made (i.e., 30 measurements per patient) using a modified direct method of measuring GBM thickness as previously described by Haas [1 (link)]. Furthermore, the distance between the podocyte and endothelium cell membrane was measured with a predefined threshold for the thin GBM according to the standardized method of GBM thickness measurements established at the Department of Nephropatology and Electron Microscopy, Dubrava University Hospital [46 ].

A 10 μL drop of EV in PBS was applied to nitrocellulose carbon-coated PELCO® Cu grids (Ted Pella Inc., Redding, CA, USA) and incubated for 1 min. Liquid was removed by touching the top of the grid with filter paper. This grid was exposed to a 10 μL drop of 2% uranyl acetate, followed by 10 s incubation with further removal of the moisture by touching the grid with the filter paper. Samples were examined in a transmission electron microscope, JEOL JEM-1400 TEM (JEOL, Tokyo, Japan) attached to an Olympus Quemesa digital camera and analyzed by iTEM software (Olympus Soft Imaging Solutions GmbH, Munster, Germany).

The morphological characteristics of the CNCs were evaluated via transmission electron microscopy (TEM), using a LEO 912AB TEM with an Omega energy filter (Zeiss, Oberkochen, Germany) at an accelerating voltage of 120 kV. For the microscopic observations, drops of dilute aqueous suspension of CNCs (≈4 wt %), previously sonicated for 1 min, were deposited on carbon-coated electron microscope grids, negatively stained with 2% uranyl acetate, and allowed to dry. Representative micrographs were selected for measuring the diameter (D), length (L), and aspect ratio (L/D) of the nanocrystals by digital image analysis (iTEM, software, Olympus Soft Imaging Solutions GmbH, Münster, Germany). In addition, the dimensions of CNCs from all heating programs, in dilute suspensions (at pH 8), were also investigated by dynamic light scattering (DLS) measurements (mod. Litesizer500, Anton Paar, Graz, Austria), performed at 25.0 ± 0.1 °C with a 35 mW laser diode light (λ = 658 nm), and collecting the scattered light at 15° and 90°. By applying correlation analysis and the Stokes–Einstein relation, the equivalent hydrodynamic diameters (D_Hy), the polydispersity index (PDI), and size distributions of the scatters were calculated. Four runs were performed, withdrawing three different aliquots for each set of experimental conditions.

Tissues were fixed in 2.5% electron microscopy grade glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.4 (Science Services, Munich, Germany), postfixed in 2% aqueous osmium tetraoxide³⁷ , dehydrated in gradual ethanol (30–100%) and propylene oxide, embedded in Epon (Merck, Darmstadt, Germany) and cured for 48 hours at 60 °C. Semithin sections were cut and stained with toluidine blue. Ultrathin sections of 50 nm were collected onto 200 mesh copper grids, stained with uranyl acetate and lead citrate before examination by transmission electron microscopy (Zeiss Libra 120 Plus, Carl Zeiss NTS GmbH, Oberkochen, Germany). Pictures were acquired using a Slow Scan CCD-camera and iTEM software (Olympus Soft Imaging Solutions, Münster, Germany).