Figure 3

Podocytes isolated out of the urine of patients with nephrotic syndrome express ADAM10. Cells isolated out of the urine of a patient with nephrotic syndrome were analyzed by flow cytometry (A+E), Western blot (B+C), RT-PCR (C lower panel), and immunofluorescence (D+F). (A) Cells isolated from the urine were stained with ADAM10 or L1 adhesion molecule and analyzed with Cellquest software from Becton Dickinson (Heidelberg, Germany). (B) Urinary cells were lysed and western blots (WB) with ADAM10 and L1 (L1 11A) specific antibodies were performed. (C) Lower panel: RT-PCR with α3, β1, and podocin specific primers on cDNA of cells isolated from the urine. Upper panel: Western blot analysis with α3, β1 and podocin specific antibodies in lysats of cells isolated from the urine. (D) Immunofluorescence double staining of cells isolated from the urine with podocyte specific marker proteins α3, nephrin, podocyin antibodies. Images were documented with a Zeiss camera. (E) Urinary cells were investigated by intracellular FACS staining using WT1 (podocyte specific marker protein) and ADAM10 antibodies. Stained cells were analyzed with Cellquest software from Becton Dickinson (Heidelberg, Germany). (F) Immunofluorescence staining of untreated (control) and IFN-γ treated urinary podocytes with L1 specific primary antibodies followed by Alexa 488 coupled secondary antibodies. Nuclei of urinary podocytes were stained and visualized with DAPI. Images were documented with a Zeiss camera. (G) Glomeruli from human kidney were isolated and glomerular lysats were prepared, proteins were loaded on a SDS gel and western blot analysis were performed using a polyclonal antibody against the cytoplasmic tail of L1.