Chapter 28
Using Ex Vivo Kidney Slices to Study AMPK Effects
on Kidney Proteins
Renee Rao, Kazuhiro Omi, Roshan Rajani, Hui Li,
and Nuria M. Pastor-Soler
Abstract
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% CO 2 at body temperature (37C) 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.
Key wordsHanks buffer, Ringer buffer, Kidney lysate, Homogenization1 Introduction
The kidney is one of the most metabolically active tissues in the
body. For example, via gluconeogenesis, the kidney generates more
glucose per gram of tissue than any other organ [1]. The kidney is
less than 1% of the total body mass, yet it receives 20% of the cardiac
output and utilizes approximately 7% of the oxygen consumed by
the body. Proper kidney function requires that a large portion of
the energy available for cellular function be used toward epithelial
membrane transport [2]. Therefore, the subject of the regulation of
membrane transport processes in the kidney has had a special focusDietbert Neumann and Benoit Viollet (eds.),AMPK:MethodsandProtocols, Methods in Molecular Biology, vol. 1732,
https://doi.org/10.1007/978-1-4939-7598-3_28,©Springer Science+Business Media, LLC 2018
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