Receptor Activator of NF-kappaB and Podocytes: Towards a Function of a Novel Receptor-Ligand Pair in the Survival Response of Podocyte Injury
Figure 5
RANKL and RANK Protects Mouse Podocytes from Apoptosis.
(A) Mouse podocytes were transfected with RANK siRNA at a concentration of 100 nM. (B) Flow cytometry fluorescence of podocytes not transfected with siRNA-Cy3 was measured as negative control; the value in podocytes was 4.1%. (C) Transfection efficiency was measured by counting fluorescence-positive cells by flow cytometry; the value in podocytes was 71.6%. (D) RANK immunoblotting of podocytes revealed that RANK was downregulated with RANK siRNA. RANK protein was low abundance in the podocytes 3 d after transfection (lane 1 and lane 2). (E) The percentage of apoptotic cells was measured by flow cytometry. In cells transfected with RANK siRNA, RANK knockdown was associated with mild increase in apoptosis compared with control siRNA (24.0±1.8% versus 22.8±1.1%; p>0.05) after podocytes were exposed to PA. However, RANKL (40 ng/ml) decreased PA-induced apoptosis of podocytes with RANK siRNA (RANKL 16.5±1.5% versus control 22.8±1.1%). (F) Quantification of apoptosis of podocytes with PA and RANKL. Apoptosis was measured by flow cytometry in control podocytes without PA induction (G) (8.7±0.97%) and podocytes that were exposed to RANKL (40 ng/ml) without PA after 48 h (H) (5.7±0.81%). (I) PA increased the apoptosis of podocytes. Apoptosis was measured by flow cytometry of podocytes exposed to PA (25 µg/ml) for 48 h (26.3±3.6%). (J) Exogenous RANKL protected podocytes from PA induction apoptosis. RANKL (40 ng/ml) decreased PA-induced apoptosis (15.5±2.2%). All the experiments were conducted in three times. **Compared with control, p<0.01; *Compared with control, p<0.05.