Receptor Activator of NF-kappaB and Podocytes: Towards a Function of a Novel Receptor-Ligand Pair in the Survival Response of Podocyte Injury
Figure 4
RANK Expression was Upregulated in a Model of Podocyte Injury in Vitro.
(A) RANK expression co-localizes with synaptopodin. RANK expression was localized to the cell plasma membrane and cytoplasm. Synaptopodin (green) positive cells were positive for RANK (red) and showed morphology consistent with podocytes. Nuclei stained with DAPI (blue). (B) RANK positive controls are SJRH30 cells. RANK immunofluorescence staining of acetone-fixed SJRH30 cells showed membrane localization. Nuclei stained with DAPI (blue). (C) For determination of whether RANK was increased after PA (25 µg/ml) exposure, RANK mRNA was measured by real-time RT-PCR at 24 h and 48 h after PA exposure. Densitometric analyses indicate a 1.8- and 1.5-fold induction of RANK at 24 h and 48 h, respectively. Results were obtained from three independent experiments. *Compared with control, P<0.05. (D) Whole-cell lysates were harvested 24 or 48 h after PA injury or control conditions, and RANK protein expression was determined by using Western blot analysis with RANK antibody. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) immunoblotting served as a loading control and confirmed equal protein loading between samples. RANK protein was in low abundance in the absence of PA injury. In contrast, RANK expression was increased after PA injury at 24 h and 48 h. Densitometric analyses revealed that RANK was upregulated a 3.8- and 2.4-fold at 24 h and 48 h, respectively. RANK immunoblotting revealed that RANK is activated in an autocrine manner upon PA injury in podocytes, reaching a maximum within 24 h of PA treatment. (E) SJRH30 cells are RANK positive controls. RANK protein was confirmed by Western blot in SJRH30 cells.