Immunohistochemical Analysis of Kidney Injury Markers

Immunohistochemical and immunofluorescent studies were performed as described previously (Jaimes et al. 2007 (link); Lan et al. 2018 (link)). Primary antibodies were rabbit anti-Grem1 (Novus Biologicals, NBP1-31150, 1:100), rabbit anti-Nephrin (Abcam, Cambridge, MA, ab58968, 1:100), rabbit anti-P-Smad2/3 (Abcam, ab272332, 1:100), rabbit anti-P-Smad1/5/8 (Sigma-Aldrich, Burlington, MA, AB3848-I, 1:100). Periodic acid-Schiff (PAS) staining was conducted by specialists at the Pathology Department, Long Island-Jewish Hospital (Queens, NY), following standard staining protocols. Masson trichrome staining was performed by specialists at the Pathology Department, Shanghai Gefan Biotechnology Co., Ltd (Shanghai, China), following standard staining protocols.