trying to uncover links between metabolism and transport, espe-
cially in kidney epithelial cells.
One of the important results in this search is that of the discov-
ery that the metabolic sensing kinase AMP-activated protein kinase
(AMPK) is a key regulator of membrane transport in kidney epi-
thelial cells [3, 4]. The cellular heterogeneity of the nephron and
the structural intricacy of kidney tissue have traditionally presented
many technical difficulties to uncovering kidney regulatory
mechanisms. Researchers have used segment-specific immortalized
cell culture models for studies of membrane transport regulation.
However, these cell culture models only reliably replicate proximal
tubule and collecting duct principal cell epithelia [5]. There are
other cell culture models that replicate intercalated cells and inner
medullary collecting duct epithelial cells [6–10]. On the other
hand, reliable models of the distal convoluted tubule, thick ascend-
ing limb, and thin limb are either not available or still being estab-
lished. Therefore, research that involves evaluation of tubular
function by micropuncture and isolated perfused tubules are still
being used to confirm cell culture findings.
In our laboratory, we have been using the kidney slice system to
evaluate the links between transport and metabolism, especially in
kidney intercalated cells. Using this method with rat kidney tissue,
we have studied how AMPK regulates the vacuolar H+-ATPase
(V-ATPase) in proximal tubule, the potassium channel KCNQ1
in collecting duct, the creatine transporter in proximal tubule,
and aquaporin-2 in collecting duct principal cells [11–13]. This
method is useful in evaluating acute changes in transporter subcel-
lular localization by immunolabeling, as well as in determining
protein abundance and measuring the levels of posttranslational
modifications (e.g., levels of phosphorylated protein) by
performing immunoblots of treated slice lysates. We are currently
trying to establish the use of kidney slices in using mouse tissue, and
although preliminary results show that the tissue is equally respon-
sive to rat tissue, the size of the mouse kidney significantly limits the
number of conditions that can be used to treat kidney slices in one
experiment. On the other hand, the availability of more mouse
models that are genetically engineered to knock out or to over-
express certain proteins offers advantages to study regulatory cas-
cades in situ in kidney tissue without having to use pharmacologic
inhibitors or activators that may have non-specific effects.
AMPK is a Ser/Thr kinase ubiquitously expressed in the body
and throughout the kidney, which senses cellular metabolic stress.
Under hypoxic or energy stress conditions, this kinase is exquisitely
sensitive to cellular increases in the [AMP]/[ATP] ratio leading to
AMPK activation [3, 14]. When AMPK is activated, threonine-172
in the alpha subunit (catalytic subunit) becomes phosphorylated
(phospho-Thr172) [15]. This phosphorylation event can occur via
upstream kinases or autophosphorylation. As AMPK becomes450 Renee Rao et al.