AMPK

Renee Rao, Kazuhiro Omi, Roshan Rajani, Hui Li, Nuria M. Pastor-Soler

The ex vivo kidney slice technique has been used extensively in the fields of kidney physiology and cell biology. Our group and others have used this method to study epithelial traffic of transport proteins in situ in kidney tissue. In this methodology chapter, we summarize our adaptation of this classic protocol for the study of the effect of AMPK in the modulation of transport protein regulation, especially in kidney epithelial cells. Briefly, slices were obtained by sectioning freshly harvested rodent (rat or mouse) kidneys using a Stadie-Riggs tissue slicer. The harvested kidney and the kidney slices are kept in a physiological buffer equilibrated with 5% CO2at body temperature (37 °C) in the presence of different AMPK activating agents vs. vehicle control followed by rapid freezing or fixation of the slices to prevent non-specific AMPK activation. Thus, homogenates of these frozen slices can be used to study AMPK activation status in the tissue as well as the downstream effects of AMPK on kidney proteins via biochemical techniques, such as immunoblotting and immunoprecipitation. Alternatively, the fixed slices can be used to evaluate AMPK-mediated subcellular traffic changes of epithelial transport proteins via immunolabeling followed by confocal microscopy. The resulting micrographs can then be used for systematic quantification of AMPK-induced changes in subcellular localization of transport proteins.