Figure 7. CaMKIV regulates mTOR and autophagy in the kidney.
A, Immunoblot analysis of renal cortex lysate detecting mTOR, p-Ser9-GSK3β, FBXW7, GSK3β, and tubulin from WT and CaMKIV−/− mice (representative blot of n=4 independent experiments where 2 mice per group is shown; N=8 mice total per group for all 4 experiments combined). Corresponding densitometry blots compare mTOR and p-Ser9-GSK3β from WT and CaMKIV−/− mice. B, The effect of endotoxemia (LPS 5mg/kg) on autophagy in the renal cortices of WT and CaMKIV−/− mice was assessed by immunoblot for LC3B (16KD). Representative blot is shown at 18h timepoint from n=4 independent experiments where 3 mice per group is shown (N=12 mice total per group for all 4 experiments combined). Immunofluorescent microscopy (20X) of renal cortex in WT and CaMKIV−/− mice harvested at 18h after LPS (5mg/kg). LC3 (Cy3, red), actin (Alex488, green), nucleus (Hoechst, blue). Scale bar=20μm. Representative images of n=3 independent experiments (N=9 mice total per group for all 3 experiments combined). Transmission electron microscopy (5×104 and 5×105 X) of renal cortex of WT and CaMKIV−/− mice 18h after endotoxemia. Middle row scale bar=2μm; Bottom row scale bar=500nm. Representative images of n=3 independent experiments (N=9 mice total per group for all 3 experiments combined). C, Human proximal renal tubular epithelial (HKC-8) cells were subjected to RNAi using Non-target (NT) or mTOR siRNA. After 72 h, cells were exposed to LPS (10ng/mL) for 30 minutes. Total cellular lysate (10μg per lane) was then isolated and analyzed for the expression of mTOR, LC3B, and tubulin by immunblot (representative blot of 3 independent experiments).