Nephrin

Sections (3 μm) from kidneys, lungs and Matrigel plugs were cut and stained with hematoxylin and eosin or with Masson’s trichrome stain. For immunohistochemical analysis, sections were deparaffinated in xylene and rehydrated in a graded series of ethanol. The antigen-retrieval process was carried out by a 3-min microwave incubation of sections with citrate solution (BioGenex). Endogenous peroxidase was blocked by incubation in 3 % hydrogen peroxide and sections were incubated with anti-αSMA (Leica Biosystems), anti-podocin (Abcam), anti-Willms Tumor (WT1) (sc-192; Santa Cruz Biotechnology), anti-CD31 (Leica Biosystems), mouse monoclonal anti-CD105 (Dako) or anti-CD68 (Dako). Then, sections were washed in PBS and incubated with the Novolink Polymer Detection System (Novocastra), followed by reaction with 3,3′diaminobenzidine as chromogen. Negative controls were performed in the absence of the primary antibody. For ThinPrep cytology, urine cytological slides were prepared by using a liquid-based method technique following the manufacturer’s guidelines (ThinPrep 2000; Cytyc) and stained using either the standard hematoxylin technique or immunohistochemistry against αSMA as above. Urine samples were used to determine the concentration of SolEng (R&D Systems), nephrin (Exocell) and podocalyxin (Wuhan EIAAB Science), using ELISA kits.