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Protocol no. 63
a-METHYL GLUCOSE UPTAKE IN ISOLATED
PROXIMAL TUBULAR CELLS
The inhibition by a test compound of the uptake
of the glucose analogue, a-methyl glucose by freshly isolated proximal
tubular cells from rat kidney is used as a measure of acute early-stage
nephrotoxicity.
CONTACT
Dr J.F. Nagelkerke
The Division of Toxicology Center for Bio-Pharmaceutical
Sciences
Sylvius Laboratories P.O. Box 9503, 2300 RA Leiden
The Netherlands
Tel: 31-(0)71-2766226 Fax: 31-(0)71-2776292
RATIONALE
Owing to its structure and function, the kidney
is a particularly susceptible target organ to a range of chemicals to which
humans may be exposed in the environment, the work place or most commonly
through medical treatment. The kidney is a complex structure, and nephrotoxins
exert their effects in well-defined regions of the organ depending on the
specific characteristics of cells in each region i.e. susceptibility of
specific targets in certain cells, capacity to transport the toxin and
the type of metabolite activating enzymes present. The cells of the proximal
convoluted tubule are a primary target of nephrotoxins since they are the
first cells exposed to the glomerular filtrate, they actively transport
a range of organic and inorganic compounds, and are able to perform a wide
range of drug metabolising reactions. Previous investigations into nephrotoxicity
have measured endpoints such as vital dye exclusion and enzyme leakage.
These parameters limit assessment to the final stages of toxicity that
lead to cell death. The procedure described in this protocol was developed
as a means of assessing earlier signs of cell dysfunction. Since the presence
of glucose in the urine is one of the earliest signs of proximal tubular
cell (PTC) damage, glucose transport was selected as an in vitro indicator
of early PTC damage. However, glucose itself cannot be used to assess this
transport system in single cells in vitro since it is taken up from the
medium at one side of the cell and excreted back into the medium from the
other side. The system described in this protocol involves measuring the
uptake of an analogue of glucose, a-methylglucose (a-MG), as an endpoint
for cell viability. a-MG cannot be metabolised, however it is a substrate
for the active sodium-glucose co-transport carrier at the apical membrane,
but not for the carrier that facilitates transport out of the cell at the
basolateral membrane, therefore a-MG becomes trapped within the cell and
its rate of uptake from the surrounding medium can thus be determined.
BASIC PROCEDURE
Rat renal proximal tubular cells (PTC) are isolated
by successive EGTA and collagenase perfusions and are purified by filtration
and isopycnic centrifugation on a discontinuous Nycodenz gradient. The
cells are pre-incubated for 15 minutes, exposed to test chemicals, and
a-MG uptake is determined after 0, 60, 120 and 180 minutes. To determine
a-MG uptake, an aliquot of the cell suspension/test compound mixture is
removed and incubated with radiolabelled a-MG. After labelling, an aliquot
is removed and placed into a buffer prior to separation of cells from medium
by centrifugation. Following washing, the cells are lysed and deproteinated
with trichloroacetic acid. The mixture is centrifuged and the radioactivity
of the supernatant is determined by liquid scintillation counting. a-MG
uptake is calculated from the total amount of acid soluble 14C. The total
procedure takes about 75 minutes from the operation to the cell purification.
Cannulation takes approximately 5 minutes, the perfusion 40 minutes, and
cell purification 30 minutes.
CRITICAL ASSESSMENT
A variety of in vitro nephrotoxicity testing
systems have been developed, including the use of isolated perfused kidney
(IPK), renal slices, perfused isolated nephrons, and isolated tubules in
suspension. All of these systems have their advantages and drawbacks. The
IPK is the in vitro system that most closely models the in vivo system,
however, the interactions between the different cell types make it difficult
to evaluate the functioning of a single type. Another limitation is due
to the fact that it is only possible to monitor the constituents of the
medium that enter and leave the organ, therefore it is not possible to
derive specific information on the processes that go on in between. Results
from the IPK system are sensitive to the perfusate composition and perfusion
pressure, therefore careful control of these factors is fundamental to
the reproducibility of the results. Renal slices have been employed for
over 40 years in nephrotoxicology as a rapid and simple technique, however
there are two major disadvantages associated with the system. The first
is that the slices comprise a heterogenous cell population; and the second
is that many tubules on the exposed surface are damaged during preparation
and lack cellular integrity. Although the tubules below the surface are
intact, they are not in direct contact with the incubation medium and therefore
lack a controlled supply of nutrients, oxygen and test compound. These
problems have largely been overcome by the procedure of ultra-thin sectioning,
followed by quantitative examination under the light microscope, however,
this adaptation of the renal slice system is more time consuming and involves
more complicated preparative techniques, and is therefore less well suited
to the large scale screening of potential nephrotoxins. Perfused isolated
nephrons are rarely used, despite the fact that they permit highly specialised
studies of transport phenomena and electrophysiology, because it is only
possible to study very small amounts of tissue and the results are not
very reliable since the toxicity of some compounds alters the integrity
of the tubules. The use of isolated tubules in suspension offers the advantage
that fairly large amounts of tissue can be obtained for multiple testing.
The respiratory and metabolic integrity of these preparations is better
than that of renal slice preparations, and they provide a more sensitive
assay system. The major drawback of this system is that the tubule lumens
are liable to close, therefore restricting contact with the incubation
medium and treatments. The use of single cells in suspension is the most
simple system that retains cellular integrity, and ensures that exposure
of the cells to experimental conditions is carefully controlled, since
each cell is bathed in incubation medium. It is possible to isolate and
culture sufficiently large quantities of tissue for studying toxicity under
different conditions in cells from the same preparation. Many nephrotoxins
have been found to exhibit target cell-specific toxicity, therefore, the
results obtained from the procedure described here are suitable for assessing
toxic effects on PTC, but may not necessarily be applicable to toxicity
in the kidney as a whole, or to other specific regions. Isolated cells
have an advantage over established cell lines, since the latter do not
yield reliable results owing to changes in cellular characteristics and
metabolising capacity. Previously described methods for the isolation of
PTC have involved mechanical and enzymatic digestion. The method of Ormstad
et al, (1980) gives the best yield and viability of PTC, however the purity
is not good with 30% of the yield comprising other cell types. This procedure
has been modified in order to isolate PTC with both a high yield and high
purity. The procedure described in this protocol improves the yield by
extending the recirculating perfusion collagenase digestion from 10-13
minutes as in the original method, to a total of 18 minutes. Longer periods
than this were found to result in medullary contamination and more dead
cortical cells (Boogaard, 1990). The cortical cell suspension is heterogenous
when viewed by light microscopy; it contains glomeruli, small blood vessels
and connective tissue, distal cells, collecting duct cells and small tubular
fragments. The cellular debris is removed by a washing procedure. Most
of the glomeruli, and all of the blood vessels and connective tissue, are
removed by sieving through two layers of 80mm nylon gauze. Isopycnic centrifugation,
using a discontinuous gradient, is used to separate the PTC from other
cell types, which are of different densities and, therefore, collect at
the respective interfaces. Nycodenz was selected as being the most appropriate
density medium, since it is a non-ionic renal contrast medium that is not
toxic to renal tissue. Of the various combinations of density-cushions
of Nycodenz that were tested, a layer of 11.3% with one of 6.8% w/v Nycodenz
produced the greatest enrichment of PTC. A viable, single cell fraction
can be collected at this interface (although cell clumps may be present
on some occasions). Dead cells (which may make up to 15% of the crude cell
suspension) have a higher cell density, and therefore will always form
a pellet at the bottom of the tubes. The single cell fraction at the 11.3
and 6.8% w/v Nycodenz gradient is not contaminated by intact tubules, filtered
glomeruli, or small fragments of undigested tissue, since these have a
higher density. This isolation procedure should routinely yield approximately
30 x 106 cells/g kidney, comprising 90% PTC, with a viability of 97_3 %.
Measurements of initial ATP content revealed that it is low on isolation,
but following a 30 minute post-isolation recovery period, the ATP content
rises to 16_2 nmol/mg protein, which is comparable to that found in the
intact kidney. The ATP content remains constant for up to 3 hours of incubation
but then declines. This needs to be taken into consideration when defining
lengths of exposure to test compounds, since low intracellular ATP levels
can seriously affect transport and metabolic processes. The decline in
functional integrity after 2-3 hours is the major disadvantage of using
isolated PTC as not all nephrotoxins will show their effects in the time
available. Another disadvantage of using isolated tubules and cell suspensions
is the loss of the overall polarisation that is present within the tissue
in vivo, where the apical membrane is in contact with primary urine, and
the basolateral membrane with the blood. Both these problems can be overcome
by establishing primary cultures from PTC, as reported by Boogaard (1990),
who used them for chronic exposure studies. a-methylglucose uptake The
principal role of the proximal tubular cells is the active transport of
compounds from the primary urine to the blood and vice versa. To facilitate
this, the proximal tubular cells have carriers for the uptake of compounds
at one side of the cell and carriers for excretion at the other side. Studies
with intact renal tissue assess the functional integrity of the tissue
by monitoring the transport of organic ions such as p-aminohippurate (p-AH),
tetraethylammonium and N-methylnicotinamide. However it is not possible
to use these as endpoints in in vitro studies, since these compounds would
be taken up from, and excreted back into the medium, therefore, monitoring
the transport processes would not be feasible. Glucose is not suitable
for the same reasons. However, the analogue of glucose, a-MG, (which has
been successfully used in various studies of glucose transport) is taken
up by PTC but is not excreted, and therefore a-MG uptake measurements can
be used to assess the early effects of nephrotoxins on PTC. a-methylglucose
is actively taken up by the proximal tubular cell via the apical Na+/glucose-cotransporter,
which is driven by the Na+ gradient over the cellular membrane. This gradient
is maintained by the Na+/K+-ATPase in the basolateral membrane. The activity
of the Na+/K+-ATPase is dependent on the intracellular ATP concentration,
which in turn is dependent on mitochondrial activity. If any of these processes
is disturbed, the uptake of a-MG will be affected. It is therefore possible
that a toxic compound can affect a-MG uptake independently of the ATP content
of the cell via inhibition of the Na+/glucose or Na+/K+-ATPase transporters.
This feature makes a-MG uptake a more specific indicator for early nephrotoxicity
than ATP measurements. a-MG uptake may also be directly affected by shortage
of ATP due to reduced mitochondrial activity, which may be caused by limited
substrate availability or loss of mitochondrial function. To further elucidate
the mode of action of a nephrotoxin, additional endpoints can be measured.
For example, measurements of intracellular K+ concentrations will indicate
whether the Na+/K+-ATPase activity has been affected, alternatively measurements
of O2 consumption will provide information on the mitochondrial capacity
for ATP synthesis. The optimum incubation period for cells with [14C] a-MG
was determined by preliminary investigations in which cells were pre-incubated
for 15 minutes, treated with a-MG and then incubated for periods ranging
from 30 seconds to 20 minutes. a-MG uptake was found to be time-dependent
and linear for at least 2 minutes. Therefore an incubation period of 2
minutes is used in the protocol. The normal rate of [14C] a-MG uptake is
15.2_1.3nmol/g protein/minute. Boogaard, (1990) investigated the capacity
of a-MG uptake measurements to predict nephrotoxicity in the known, acute
nephrotoxins Cd2+, Hg2+, UO22+, p-aminophenol, and bis-2,3-dibromopropyl
phosphate. The a-MG uptake measurements were compared directly with ATP
measurements (see figure 1). Low concentrations of each of these compounds
were found to cause inhibition of a-MG uptake. Studies using drugs that
have been implicated in nephrotoxicity following chronic administration,
such as gentamicin, cephaloridine, phenacetin and acetaminophen, caused
inhibition of a-MG uptake only at high concentrations in this system (see
figure 2). The sensitivity of the endpoint is evident in a comparison of
two studies, in which rat PTC were exposed to 100mm cysteine-S-conjugate
of chlorotri-fluoroethylene for 3 hours. Dohn et al (1985) reported a 39%
decrease in viability assessed by Trypan Blue exclusion, and a 47% increase
in LDH leakage, while Boogaard et al (1989a) found an 82% inhibition of
a-MG uptake. Boogaard et al, (1989b) in a study of various nephrotoxins
found that an inhibition of a-MG uptake was evident at lower concentrations
than required to induce LDH leakage. Nevertheless, Boogaard (1990) considers
that an alternative, independent parameter of cell viability be used in
combination with a-MG uptake, in order to minimise the likelihood of artefacts.
Conclusion This PTC isolation procedure should routinely provide high yields
of 30 x 106 cells/g kidney, of >90% purity. The cells that are isolated
maintain a viability similar to that of intact kidney cells for up to 3
hours and retain the metabolising capacity that established cell cultures
tend to lose. a-MG uptake is a suitable endpoint as it is simple to measure,
and can act as a sensitive indicator of dysfunction of the glucose transport
mechanism in early stages of nephrotoxicity. In comparison to in vivo testing,
very little of the test substance is required in vitro. The uptake of a-MG
by isolated PTC is an appropriate system for the study of acute nephrotoxicity,
especially at its early stages.
TEST STATUS
In-house development
CHEMICALS TESTED
Aminooxyacetic acid Anthglutin Bis-(2,3-dibromo-propyl)-phosphate
Bismuth nitrate pentahydrate Cadmium chloride (CdCl2) Cephaloridine Chlorotrifluoroethylene-cysteine
conjugate Chlorotrifluoroethylene-mercapturate conjugate Cis-diamminedichloro-platinum(II)
Dibromodifluoroethylene-cysteine conjugate Dibromodifluoroethylene-mercapturate
conjugate Dichlorovinyl-cysteine Gentamicin Mercuric chloride (HgCl2) 4-methylthiobenzoic
acid p-amino phenol S-(1,1-difluoro-2,2-di-chloroethyl)-L-cysteine N-acetyl-1-S-(1,1-difluoro-2,2-dichloroethyl)-L-cysteine
Tetrafluoroethylene-cysteine conjugate Tetrafluoroethylene-mercapturate
conjugate Uranyl nitrate (UO2(NO3)2)
REFERENCES
- Boogaard, P.J. (1990) Development of proximal
tubular cell systems to study nephrotoxicity in vitro. Phd. Thesis, Leiden
University. Boogaard, P.J., Commandeur, J.N.M., Mulder, J.G., Vermeulen,
N.P.E., & Nagelkerke, J.F. (1989a) Toxicity of the cysteine-S-conjugates
and mercapturic acids of four structurally related difluorethylenes in
isolated proximal tubular cells from rat kidney. Uptake of the conjugates
and activation to toxic metabolites. Biochem. Pharmacol., 38, 3731-3741.
- Boogaard, P.J., Mulder, G.J., & Nagelkerke,
J.F. (1989b) Isolated proximal tubular cells from rat kidney as an in vitro
model for studies on nephrotoxicity. II. a-Methylglucose uptake as a sensitive
parameter for mechanistic studies of acute toxicity by xenobiotics. Toxicol.
Appl. Pharmacol., 101, 144-157.
- Dohn, D.R., Leninger, J.R., Lash, L.H., Quebbemann,
A.J. & Anders, M.W. (1985) Nephrotoxicity of S-(2-chloro-1,1,2,-trifluoroethyl)-glutathione
and S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine, the glutathione and cysteine
conjugates of chlorotrifluoroethylene. J. Pharmacol. Exp. Ther., 235, 851-857.
- Ormstad, K., Jones, D.P. & Orrenius, S. (1980)
Characteristics of glutathione biosynthesis by freshly isolated rat kidney
cells. J. Biol. Chem. 255, 175-181.
IP-63 © November 1992
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