Renal Biopsy Evaluation and Scoring

Independent evaluation of all renal biopsies was performed by two renal pathologists, who were blinded to data assessment and data analysis. Within each renal biopsy specimen, each glomerulus was separately evaluated for present crescents, global sclerosis, and necrosis. The percentage of glomeruli with any of these pathologies was calculated as a fraction of the total number of glomeruli in each renal biopsy. Renal biopsies were scored in accordance with the current version of the Banff score for allograft pathology, as recently described [47 (link),48 (link)]. Kidney sections (formalin-fixed, paraffin-embedded) were deparaffinized in xylene and then rehydrated in ethanol containing distilled water. Utilizing antibodies against C3c (1:10,000, A0062, Agilent Dako, Santa Clara, CA, USA) and C4d (1:50, 503-17344, Zytomed, Berlin, Germany), tissue sections were stained. According to the manufacturer’s protocol, labeling was conducted using the Novolink^TM Polymer Detection System (Leica Biosystems, Wetzlar, Germany). Nuclear counterstaining was performed using Mayer’s Hematoxylin Solution (Sigma, St. Louis, MO, USA). C3c and C4d deposits in the glomerular tuft, interlobular arteries, peritubular capillaries, and venules were evaluated for presence, as recently described [12 (link),41 (link)].