Induction of Grp78 in response to tunicamycin administration and activation of autophagy in the kidney and during renal ischemia-reperfusion injury.

<p><b>A.</b> Kidneys from mice treated with tunicamycin (TN) or vehicle (30% DMSO + 70% saline) for 48 hours were processed for immunofluorescence staining as described in Methods. Deparaffinized 5 μm sections were immunostained with a polyclonal rabbit Grp78 antibody followed with an anti-rabbit Alexafluor-488-labeled secondary antibody and images were recorded using an Olympus BX51 fluorescence microscope (40x magnification). The results represent three independent experiments. <b>B.</b> Kidney tissue from mice treated with tunicamycin (TN) or vehicle (30% DMSO + 70% saline) for 48 hours was homogenized with lysis buffer containing protease inhibitors. Tissue lysate (50 μg protein) was subjected to western blot analysis using antibodies to Grp78 and β-actin. β-actin was used as a loading control. The results represent three independent experiments. <b>C.</b> Mice were treated with tunicamycin or vehicle (30% DMSO +70% saline) for 48 hours and then were subjected to IR. Chloroquine was administered i.p. one hour before IR surgery. Kidneys were processed for immunofluorescent staining as described in Methods. Deparaffinized 5 μm sections were immunostained with polyclonal rabbit anti-LC3 followed with anti-rabbit Alexafluor-488-labeled secondary antibody and pictures recorded on an Olympus BX51 fluorescence microscope at 40x magnification. The results represent three independent experiments. <b>D.</b> Mice were treated with tunicamycin or vehicle (30% DMSO +70% saline) for 48 hours and then were subjected to IR. Chloroquine was administered i.p. one hour before IR surgery. Kidney tissue lysates were subjected to western blot analysis using antibody to p62. GAPDH was used as a loading control. The results represent three independent experiments.