Figure 2: Formoterol induced MB in podocytes.

(A & B) FCCP uncoupled maximum OCR measurements were made in cultured human podocytes treated with 30 nM formoterol for 24 h and showed significantly enhanced mitochondrial oxygen consumption. *P≤0.05, 2-tailed t-test. (C) qPCR analysis of podocytes treated with either control vehicle or 30nM formoterol showed relative fold change in the expression of PGC-1a, ATPase 6, CYTB, FN1 and CO1 genes that are involved in MB. All experiments were performed at least in triplicates. Data are presented in mean±SEM and p-values were calculated using a 2-tailed t-test. *P≤0.05, **P≤0.01, ***p≤001 vs. control. (D) Rat glomeruli were stained with β₂-AR (Green) and Synaptopodin (Red) antibodies (arrows mark the presence of β₂-AR in podocytes). Scale bar 10μm (E) Cultured human podocytes immuno-stained with phalloidin (Green) and β₂-AR (Red) showed membrane and nuclear staining of β₂-AR. Scale bar 20μm (F) Western blot analysis of β₂-AR expression in podocyte cell lysate. (G) β₂-AR knockdown (β₂-AR-KD) podocytes were generated using five different sets of shRNA and the qPCR analysis showed maximal (~80%) knockdown using shRNA # 83, which was subsequently used for all experiments. (H) mtDNA copy number analysis showed induction of mtDNA by formoterol, whereas, β₂-AR-KD blunted formoterol-induced increase in mtDNA copy number. Data are presented in mean±SEM. *P<0.05, control-KD+vehicle vs control-KD+formoterol; #p<0.05, control-KD+vehicle vs β₂-AR-KD+ vehicle.