Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences Little Rock, Arkansas 72205-7199
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This study examined the repair of renal proximal tubule cellular (RPTC) functions following sublethal injury induced by the nephrotoxicant S-(1,2-dichlorovinyl)-L-cysteine (DCVC). DCVC exposure resulted in 31% cell death and loss 24 h following the treatment. Monolayer confluence recovered through migration/spreading but not proliferation after 6 days. Basal, uncoupled, and ouabain-sensitive oxygen consumption (QO2) decreased 47, 76, and 62%, respectively, 24 h after DCVC exposure. Na+-K+-ATPase activity and Na+-dependent glucose uptake were inhibited 80 and 68%, respectively, 24 h after DCVC exposure. None of these functions recovered over time. Addition of epidermal growth factor (EGF) following DCVC exposure did not prevent decreases in basal, uncoupled, and ouabain-sensitive QO2 values and Na+-K+-ATPase activity but promoted their recovery over 4-6 days. In contrast, no recovery of Na+-dependent glucose uptake occurred in the presence of EGF. These data show that: 1) DCVC exposure decreases mitochondrial function, Na+-K+-ATPase activity, active Na+ transport, and Na+-dependent glucose uptake in sublethally injured RPTC; 2) DCVC-treated RPTC do not proliferate nor regain their physiological functions in this model; and 3) EGF promotes recovery of mitochondrial function and active Na+ transport but not Na+-dependent glucose uptake. These results suggest that cysteine conjugates may cause renal dysfunction, in part, by decreasing RPTC functions and inhibiting their repair.
renal proximal tubular cells; cysteine conjugate; S-(1,2-dichlorovinyl)-L-cysteine; sublethal cell injury; regeneration; cell repair; mitochondrial function; oxygen consumption; sodium-potassium adenosinetriphosphatase; active sodium transport; sodium-coupled glucose uptake; ascorbic acid; epidermal growth factor
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE KIDNEY has the potential for complete recovery from acute renal failure (ARF) following toxicant- and ischemia/reperfusion-induced injury (5, 8). ARF is often associated with tubular damage and cell death, particularly of renal proximal tubular cells (RPTC), due to their active transport functions and selective accumulation of xenobiotics. However, renal dysfunction may be due also to sublethal injury of the tubular epithelium that is associated with the loss of RPTC physiological functions without producing necrosis (35). The most common RPTC alterations observed following toxicant exposure or ischemia/reperfusion are 1) loss and/or internalization of the brush-border membrane microvilli, 2) loss of cell polarity, 3) mitochondrial dysfunction and ATP depletion, 4) inhibition of the Na+-K+-ATPase, and 5) alterations in ion homeostasis and transport functions (1, 2, 21, 35, 39).
Pathological changes in RPTC can be repaired over time, and the return of RPTC physiological functions is critical for the restoration of normal renal function (35, 36). Numerous studies suggest that tubular repair is composed of several steps and begins with the exfoliation of dead cells from the basement membrane. This is followed by the migration/spreading of noninjured cells into the denuded area, dedifferentiation, proliferation, migration, and differentiation of RPTC, ultimately resulting in relining of the damaged tubule.
Epidermal growth factor (EGF) is a potent mitogen for RPTC and is synthesized within the kidney (12, 23, 25, 29). In addition to its well-documented mitogenic activity, EGF exerts effects on a number of important cellular responses including oxygen consumption, glycolysis, gluconeogenesis, arachidonic acid metabolism, intracellular calcium levels, glycosaminoglycans and collagen synthesis, ion exchange, and tubular transport (4, 10, 18, 25, 34). Endogenous EGF levels increase in the kidney following ARF, enhance renal tubule cell regeneration and repair, and promote the recovery of renal function in postischemic ARF (12, 24, 31, 32).
Halogenated hydrocarbons represent a large group of
compounds that are used as chemical intermediates,
solvents, and pesticides and produce toxicity after their enzymatic
conversion to reactive intermediates. Haloalkanes and haloalkenes
(e.g., trichloroethylene) are biotransformed in a series of steps that
ultimately result in the formation of nephrotoxic cysteine
S-conjugates [e.g.,
S-(1,2-dichlorovinyl)-L-cysteine (DCVC)]. In the RPTC, these conjugates are biotransformed by
cysteine conjugate -lyase to thiol-containing reactive metabolites
that produce nephrotoxicity by their covalent binding to target
molecules within the cell (6). DCVC is a model halocarbon
nephrotoxicant that is selective for RPTC and produces RPTC necrosis
and ARF (7, 33).
Acute exposure of RPTC to DCVC results in thiol depletion, loss of calcium homeostasis, mitochondrial dysfunction and ATP depletion, lipid peroxidation, DNA damage, loss of brush border enzymes, and inhibition of transport functions (16, 17). These changes are associated with the reorganization of RPTC cytoskeleton including the loss and depolymerization of F-actin, loss of actinin, redistribution of talin, and disturbance of focal adhesions and are followed by cell detachment and death (38). Long-term exposure of LLC-PK1 cells to low concentrations of DCVC results in cellular dedifferentiation characterized by alterations in cellular morphology, composition of nuclear matrix and intermediate filament proteins, loss of membrane polarity, and impairment of apical glucose uptake and pH-dependent ammonia production (37).
Recently, we have demonstrated that primary cultures of RPTC grown in improved culture conditions that promote oxidative metabolism and transport functions of RPTC (26) undergo complete morphological regeneration of the monolayer following sublethal injury induced by an oxidant [tert-butyl hydroperoxide (TBHP)] and that this process is due to both proliferation and migration/spreading (27, 28). Furthermore, the decrease in oxidative metabolism, ATP content, Na+-K+-ATPase activity, active Na+ transport, and Na+-coupled glucose uptake observed after RPTC exposure to TBHP is followed by repair and complete recovery of RPTC functions with cellular proliferation and monolayer regeneration preceding the recovery of mitochondrial and transport functions (28). Thus RPTC cultured in these conditions are able to undergo a complete morphological and functional repair following sublethal oxidant injury.
Numerous studies have demonstrated inhibitory effect of DCVC exposure on RPTC physiological functions. However, the ability of RPTC to restore these functions following DCVC-induced injury has not been examined. It is also unknown whether EGF can promote the repair of RPTC functions following DCVC-induced injury. Therefore, the aim of this study was 1) to determine the effect of DCVC on mitochondrial and brush-border and basolateral membrane functions in RPTC; 2) to determine whether RPTC recover these functions following sublethal injury induced by DCVC; and 3) to examine whether EGF treatment following DCVC exposure protects against the loss of mitochondrial and transport functions of RPTC and/or promotes recovery of these functions.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials. Female New Zealand White
rabbits (1.5-2.0 kg) were purchased from Myrtle's Rabbitry
(Thompson Station, TN). DCVC was a generous gift from Dr. T. W. Petry
(Pharmacia Upjohn, Kalamazoo, MI) and was synthesized according to the
method of Moore and Green (22).
L-Ascorbic acid-2-phosphate
magnesium salt and cell culture media were obtained from Wako
BioProducts (Richmond, VA) and Sigma Chemical (St. Louis, MO),
respectively. EGF (recombinant, human) was supplied by R&D Systems
(Minneapolis, MN). Methyl
-D-glucopyranoside (MGP),
[glucose-14C(U)] (sp
act 282 mCi/mmol), was purchased from DuPont-New England Nuclear
(Boston, MA). The sources of the other reagents have been described
previously (25, 26).
Isolation of proximal tubules and culture conditions. Rabbit renal proximal tubules were isolated by iron oxide perfusion method and grown in 35-mm culture dishes in improved conditions as described previously (25, 26). The purity of the renal proximal tubular S1 and S2 segments isolated by this method is ~96%. The culture medium was a 50:50 mixture of DMEM and Ham's F-12 nutrient mix (without phenol red, pyruvate, and glucose) supplemented with 15 mM NaHCO3, 15 mM HEPES, and 6 mM lactate (pH 7.4, 295 mosmol/kg). Human transferrin (5 µg/ml), selenium (5 ng/ml), hydrocortisone (50 nM), bovine insulin (10 nM), and L-ascorbic acid-2-phosphate (0.05 mM) were added to the medium immediately before daily media change (2 ml/dish).
Toxicant treatment of RPTC monolayer. RPTC cultures reached confluence within 4-5 days and were treated with DCVC on the sixth day. RPTC were treated with 0.2 mM DCVC (dissolved in water; 10 µl/dish) for 1.5 h to obtain ~30% cell death and loss. Following toxicant exposure, the remaining cellular monolayer was washed with fresh culture medium. In some experiments, RPTC were treated daily with EGF (10 ng/ml) starting with the media change following DCVC exposure. In other experiments, RPTC were cultured in the presence of 5 mM glucose with or without EGF (10 ng/ml) following DCVC treatment. Samples of RPTC were taken at various time points after exposure for measurements of cellular functions. Prior to measurement of any functions, RPTC were washed several times with warm culture media to remove nonviable cells.
Oxygen consumption. RPTC monolayers were washed with 37°C culture medium and gently detached from the dishes with a rubber policeman, suspended in 37°C culture medium, and transferred to the oxygen consumption (QO2) measurement chamber. QO2 was measured polarographically in RPTC suspended in the culture medium using Clark-type electrode as described previously (25, 26). For measurements of ouabain-sensitive and uncoupled QO2, 0.1 mM ouabain and 1 µM FCCP (final concentrations) were used, respectively.
Na+-coupled glucose uptake. Na+-coupled glucose uptake was assessed using the nonmetabolizable glucose analog MGP as described previously (26). MGP uptake was measured in glucose-free medium used for RPTC culture and corrected for Na+-independent (phloridzin-insensitive) and time 0 uptakes.
Enzyme assays. Na+-K+-ATPase activity was determined in cellular lysates by measuring the difference between total ATPase activity and ouabain-insensitive ATPase activity as described previously (26). ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Monolayer regeneration. Exposure of
confluent RPTC monolayers to 0.2 mM DCVC for 1.5 h resulted in 17%
cell loss, as indicated by DNA content, at 4 h and 31% at 24 h
following the treatment (Fig. 1). Monolayer
DNA content in DCVC-injured RPTC did not return to control values
during the recovery period. The protein-to-DNA ratio decreased at 24 h
and returned to control values by day 4 following DCVC treatment (Table
1). Visual inspection demonstrated that
monolayer confluence recovered by day
6 as reported previously (14). These results show that
monolayer recovery was due to migration/spreading of sublethally
injured RPTC and not proliferation or hypertrophy.
|
|
Mitochondrial function. Basal and
uncoupled
QO2 were
used as markers of RPTC mitochondrial function. Specifically, uncoupled QO2 served
as a marker of electron transport chain integrity. DCVC exposure
decreased basal
QO2 in
sublethally injured RPTC by 33% and 47% at 4 and 24 h, respectively
(Fig.
2A).
Uncoupled QO2 was
decreased by 64% and 76% at 4 and 24 h, respectively, after DCVC
exposure (Fig. 2B). No significant
changes in basal and uncoupled
QO2
occurred in sublethally injured RPTC during the 6-day recovery period
following DCVC exposure. Ouabain-insensitive QO2
decreased by 37% at 24 h (13.3 ± 1.3 vs. 8.7 ± 1.8 nmol
O2 · min1 · mg
protein
1 in control and
DCVC-treated RPTC, respectively) and remained decreased by 20% until
day 6 following the exposure. These
data show that DCVC treatment decreases mitochondrial function in RPTC and that mitochondrial function in sublethally injured RPTC does not
recover over time.
|
Basolateral membrane function. Active
Na+ transport was used as a marker
of basolateral membrane function. The assessment of active
Na+ transport in RPTC was done by
measurements of ouabain-sensitive QO2 and
Na+-K+-ATPase
activity. DCVC exposure in RPTC resulted in 50% and 62% decreases in
ouabain-sensitive
QO2 at 4 and 24 h, respectively (Fig.
3A).
Na+-K+-ATPase
activity was inhibited by 62% and 80% at 4 and 24 h, respectively (Fig. 3B). Neither ouabain-sensitive
QO2 nor
Na+-K+-ATPase
activity recovered over time (Fig. 3). These data show that DCVC
decreases active Na+ transport in
RPTC and that the repair of this function does not occur in sublethally
DCVC-injured RPTC.
|
Brush-border membrane function.
Na+-dependent glucose uptake and
GGT activity were used as markers of brush-border membrane function in
RPTC. Na+-dependent glucose uptake
was reduced by 25% and 68% at 4 and 24 h, respectively, following
DCVC exposure (Fig. 4). No recovery of this
function occurred over the 6-day period after DCVC treatment. In
contrast, DCVC had no effect on GGT activity (452 ± 45 vs. 437 ± 46 mU/mg protein in DCVC-treated and control RPTC, respectively, at 4 h following the exposure, and 408 ± 33 vs. 457 ± 31 mU/mg protein in DCVC-treated and control RPTC, respectively, at 24 h
following the exposure). These data show that DCVC-induced injury is
associated with a marked decrease in
Na+-dependent glucose uptake and
lack of recovery of this function. The results also show that not all
brush-border membrane proteins are altered by DCVC.
|
Another aim of this study was to investigate whether EGF can promote recovery of RPTC functions following DCVC-induced injury. EGF (10 ng/ml) was added to control and DCVC-injured monolayers daily with fresh media starting with the media change following DCVC removal.
Effect of EGF on recovery of mitochondrial
function. The addition of EGF to RPTC monolayers during
the first 24 h following DCVC exposure had no effect on DCVC-induced
decreases in basal and uncoupled
QO2 (Fig.
5). However, in the presence of EGF, basal and uncoupled
QO2 in
DCVC-injured RPTC returned to control values on day
6 (Fig. 5). EGF had no
effect on the decrease in ouabain-insensitive QO2 in
DCVC-treated RPTC at 24 h (37% vs. 36% in DCVC-treated RPTC in the
absence and presence of EGF, respectively). However, in the presence of
EGF, ouabain-insensitive
QO2 in
DCVC-injured RPTC returned to control values on day
4 (14.8 ± 0.9 vs. 16.9 ± 1.6 nmol
O2 in the absence and presence of
EGF, respectively). These data show that EGF does not protect against
DCVC-induced decreases in RPTC respiration but promotes the repair of
mitochondrial function in RPTC following DCVC injury.
|
Effect of EGF on recovery of basolateral membrane
function. Figure
6A shows
that addition of EGF following DCVC exposure had no effect on the
DCVC-induced decrease in ouabain-sensitive
QO2. However, ouabain-sensitive
QO2 in
DCVC-injured RPTC cultured in the presence of EGF returned to control
levels on day 6 (Fig. 6A). Addition of EGF to DCVC-injured
RPTC diminished the decrease in
Na+-K+-ATPase
activity on day 1 following the
exposure (83 vs. 59% decrease in presence and absence of EGF,
respectively).
Na+-K+-ATPase
activity in DCVC-injured RPTC cultured in the presence of EGF returned
to control level on day 4 (Fig.
6B). These data show that:
1) EGF does not prevent the
DCVC-induced decrease in active
Na+ transport in RPTC but promotes
the repair of this function following DCVC exposure;
2) EGF reduces the DCVC-induced
decrease in
Na+-K+-ATPase
activity in RPTC and promotes recovery of
Na+-K+-ATPase
activity following DCVC exposure; and
3) EGF-mediated recovery of
Na+-K+-ATPase
activity following DCVC exposure precedes the recovery of active
Na+ transport.
|
Effect of EGF on recovery of brush-border membrane
function. The data presented in Fig.
7 show that culturing control RPTC in the
presence of EGF decreased
Na+-dependent glucose uptake by 38 and 36% on days 4 and
6, respectively. Addition of EGF to
DCVC-injured RPTC had no effect on the decrease in
Na+-dependent glucose uptake
during the first 24 h following the exposure. However, continuous
presence of EGF in DCVC-injured RPTC further decreased
Na+-dependent glucose uptake by 47 and 50% on days 4 and
6, respectively (Fig. 7). GGT activity
was not affected by EGF treatment either in control or DCVC-injured
RPTC (data not shown). These results show that EGF causes further
decrease in Na+-dependent glucose
uptake in DCVC-injured RPTC.
|
Effect of EGF on RPTC proliferation following DCVC
injury. Figure
8A shows
that administration of EGF following DCVC exposure did not induce RPTC
proliferation. This suggested that the lack of RPTC proliferation
following DCVC-induced injury may be due to the inability of RPTC to
respond to the mitogen or to synthesize DNA from substrates present in
the culture medium. Supplementation of the culture medium with glucose
(5 mM), a common source of precursors for DNA synthesis in cultured
cells (30), following DCVC exposure did not change DNA
content in injured monolayers (Fig.
8B). However, EGF treatment in the
presence of glucose resulted in RPTC proliferation and complete
recovery of monolayer DNA content on day
4 after DCVC exposure (Fig.
8B). These results show that: 1) DCVC-injured RPTC lack a
mitogenic signal necessary to induce proliferation following injury,
and 2) DCVC-treated RPTC are capable of a mitogenic response and of synthesizing new DNA with glucose supplementation.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Renal failure caused by toxicant exposure is often due to RPTC injury, death, and detachment. However, in many cases, toxicant-induced renal failure is associated with RPTC dysfunction without apparent necrosis and cell loss (35). In these conditions, sublethally injured RPTC have decreased physiological functions and the recovery of normal renal function occurs if RPTC repair their functions. There is a possibility, however, that some toxicants may contribute to renal dysfunction not only by inducing RPTC injury but also by inhibiting the repair process. Our previous study showed that RPTC grown in improved cell culture conditions recover their functions following sublethal injury induced by an oxidant, TBHP (28). This recovery is not dependent on addition of exogenous mitogens or factors stimulating cellular repair, which suggests that RPTC grown in these conditions have autocrine mechanisms inducing cellular repair. Present studies were undertaken to determine whether recovery of RPTC functions is a common process that follows sublethal injury induced by a nephrotoxicant and whether EGF stimulates the return of RPTC functions after toxicant exposure. The toxicant used in this study was DCVC, a cysteine conjugate that is the key metabolite of the nephrotoxic and nephrocarcinogenic chemicals, trichloroethylene and dichloroacetylene.
Our data show that a DCVC exposure that results in the death and loss of 30% of RPTC from the monolayer and sublethal injury to remaining cells is not followed by cellular proliferation and recovery of the monolayer DNA content. Interestingly, the confluence of DCVC-injured monolayers recovered 6 days after the treatment. Sublethally injured RPTC enlarged but did not become hypertrophic, as protein-to-DNA ratios did not increase within 6 days after DCVC exposure. The decrease in the protein-to-DNA ratio at 24 h following DCVC exposure may reflect leakage or degradation of intracellular proteins in injured RPTC. The results suggested that the recovery of the monolayer confluence following DCVC exposure was due to migration and spreading and remain in contrast to our previous observation that RPTC proliferation occurs following an oxidant (TBHP) exposure that produces a similar amount of RPTC death and loss (28).
The absence of RPTC proliferation following DCVC-induced injury could be due to numerous reasons including the lack of a mitogenic signal, the inability to respond to this signal, damage to the DNA synthesis apparatus and/or inability to generate sufficient DNA precursors from substrates present in the culture medium. To test the hypothesis that the lack of proliferation is due to the absence of a mitogenic signal, we supplemented the culture medium with EGF, a potent mitogen for RPTC in vivo and in vitro (12, 23, 25). EGF did not induce RPTC proliferation during 6 days after DCVC exposure. The absence of proliferation could be due also to the lack of appropriate DNA precursors. To test this hypothesis, we supplemented the culture medium with glucose, a common substrate for DNA precursors in cultured cells (30), and demonstrated that glucose alone did not promote proliferation in DCVC-injured RPTC. However, EGF in conjunction with glucose induced DNA synthesis and proliferation in DCVC-injured monolayers, which suggests that the inability of RPTC to proliferate after DCVC exposure is due to the lack of a mitogenic signal and the appropriate source of DNA precursors. These results also suggest that in this model RPTC maintain their ability to respond to mitogenic signals following DCVC injury and that EGF-stimulated DNA synthesis requires an alternate or additional source of DNA precursors. In contrast, TBHP-induced injury in RPTC is followed by proliferation that does not require the presence of EGF and additional sources of DNA precursors (28).
Mitochondrial function, active Na+ transport, Na+-K+-ATPase activity, and Na+-dependent glucose uptake are major targets of DCVC in RPTC. A decrease in these functions was observed immediately after DCVC removal (time 0) from the monolayers, when RPTC did not exhibit any evidence of cell death. Further decreases of mitochondrial function, active Na+ transport and Na+-K+-ATPase activity, and Na+-dependent glucose uptake were observed 24 h later when most lethally injured RPTC were dead and lost. A decrease in mitochondrial function in DCVC-injured RPTC is consistent with previous reports that demonstrated a decrease in state 3 respiration, dissipation of the mitochondrial membrane potential, and mitochondrial damage in RPTC subjected to DCVC exposure (9, 38). This study shows that the mitochondrial electron transport chain is a target of DCVC in sublethally injured RPTC and that both electron transport chain integrity and mitochondrial respiration are not repaired over the 6-day recovery period in this model. The lack of repair of mitochondrial function following DCVC-induced injury remains in contrast to the complete recovery of this function after oxidant-induced injury in RPTC (28).
These results also demonstrate that DCVC decreases active Na+ transport, the basolateral membrane function that is critical for Na+ reabsorption by renal proximal tubules in vivo. This decrease is due, in part, to the inhibition of Na+-K+-ATPase activity in sublethally injured RPTC. To our knowledge, this is the first report showing the inhibitory effect of DCVC exposure on active Na+ transport and Na+-K+-ATPase activity in renal cells. The mechanism of this inhibition remains to be determined. The decrease in the Na+-K+-ATPase activity may be due to DCVC binding to Na+-K+-ATPase protein, as DCVC binds covalently to RPTC proteins (3, 9). It is also likely that DCVC exposure results in the loss of Na+-K+-ATPase protein from sublethally injured RPTC. It has been shown that DCVC causes depolymerization of F-actin and disorganization of RPTC cytoskeleton (38). As Na+-K+-ATPase is associated with the cytoskeleton through F-actin, depolymerization of actin my cause a partial loss of Na+-K+-ATPase protein from injured cells. Independently, the loss of mitochondrial function and the resulting ATP depletion would decrease Na+-K+-ATPase activity and active Na+ transport. The lack of repair of active Na+ transport and Na+-K+-ATPase activity in RPTC following DCVC-induced injury remains in contrast to complete recovery of these functions in RPTC after oxidant injury (28).
DCVC-induced injury in RPTC is associated also with a decrease in Na+-dependent glucose uptake, the brush-border membrane function that is critical in vivo for reabsorption of glucose from renal filtrate. Our results obtained using cultured RPTC are in agreement with a previous report that demonstrated impaired reabsorption of glucose by the isolated perfused rat kidney following administration of DCVC in vivo (13). The decrease in Na+-dependent glucose uptake in DCVC-injured RPTC may be due to the loss of mitochondrial function, decreased Na+ gradient, reduced number of Na+-dependent glucose transporters on brush-border membrane, or/and direct impairment of the function or binding properties of Na+-dependent glucose transporters by DCVC. Regardless of the mechanism, DCVC-induced decreases in Na+-dependent glucose uptake by RPTC persisted, and no repair of this function occurred during the 6-day recovery period following the exposure, whereas Na+-dependent glucose uptake completely recovered following TBHP-induced injury (28).
Not all brush-border membrane proteins are targets of DCVC. GGT activity was not altered by DCVC exposure or during the recovery period. Ilinskaja and Vamvakas (13) showed increased activity of brush border enzymes in urine after DCVC exposure in vivo in rats, which suggested that DCVC induced brush-border membrane damage or RPTC loss and lysis into urine.
The effect of growth factors on cellular repair following injury is not limited to stimulation of cellular proliferation. Growth factors may limit cell injury by decreasing the effect of agents that induce the damage and/or promote cell repair by supporting the reestablishment of cell-extracellular matrix interactions and/or cell-cell integrity (11). It has been demonstrated that administration of EGF in vivo accelerates both structural and functional recovery of the kidney following acute renal injury (12, 24, 32). Although the stimulation of proliferation in injured RPTC by EGF is well documented (12, 32), it is not clear whether EGF promotes recovery of other RPTC functions following toxicant-induced injury. Our data demonstrate that addition of EGF after DCVC exposure induced repair of the electron transport chain and resulted in the return of basal QO2 and ouabain-insensitive QO2, whereas no repair of these functions occurred in DCVC-injured RPTC cultured without EGF. These results show that EGF does not protect against DCVC-induced damage to the mitochondria but induces the repair of mitochondrial function in RPTC following the injury.
This study also demonstrates that EGF does not affect the decrease in active Na+ transport in RPTC following DCVC-induced injury but stimulates return of this function. Ouabain-sensitive QO2 returned to control levels in DCVC-injured RPTC cultured for 6 days in the presence of EGF but not in cells cultured without this growth factor. The data suggest that the stimulation of repair of ouabain-sensitive QO2 was due, in part, to the protective effect of EGF on Na+-K+-ATPase activity during first 24 h following DCVC-induced injury. Subsequently, the return of the Na+-K+-ATPase activity in RPTC cultured in the presence of EGF was observed on day 4 following DCVC exposure in conjunction with the return of mitochondrial function, and was followed by complete recovery of ouabain-sensitive QO2 on day 6.
Our results also show that EGF did not stimulate the return of Na+-dependent glucose uptake and suggest that EGF does not mediate the repair of this function in sublethally injured RPTC following DCVC exposure. These data indicate that EGF does not promote the repair of all physiological functions in sublethally injured RPTC.
In conclusion, our results show that: 1) DCVC decreases proliferation, mitochondrial function, active Na+ transport, Na+-K+-ATPase activity, and Na+-dependent glucose uptake in sublethally injured RPTC; 2) none of these functions return over time, which suggests that DCVC inhibits the repair of RPTC functions in this model; 3) EGF stimulates proliferation of sublethally injured RPTC following DCVC exposure but only in the presence of glucose as the source of DNA precursors; 4) EGF promotes the repair of mitochondrial function and active Na+ transport in DCVC-injured RPTC but not Na+-dependent glucose uptake; and 5) stimulation of recovery of active Na+ transport following DCVC exposure is due, in part, to the protective effect of EGF on Na+-K+-ATPase activity and stimulation of mitochondrial function in sublethally injured RPTC. Our results suggest that nephrotoxic cysteine conjugates may cause renal dysfunction not only by decreasing RPTC functions but also by inhibiting their repair and that the beneficial effect of EGF in sublethally injured RPTC is not limited to the stimulation of proliferation.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Thomas W. Petry (Upjohn Pharmacia, Kalamazoo, MI) for the generous gift of S-(1,2-dichlorovinyl)-L-cysteine.
![]() |
FOOTNOTES |
---|
This work was supported by National Institute of Environmental Health Sciences Grant ES-04110. Parts of this work were presented at the 29th Annual Meeting of the American Society of Nephrology, November 3-6, 1996, New Orleans, LA (abstract no. 2985).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: R. G. Schnellmann, Univ. of Arkansas for Medical Sciences, Dept. of Pharmacology and Toxicology, 4301 W. Markham St., Slot 638, Little Rock, AR 72205-7199.
Received 6 July 1998; accepted in final form 8 October 1998.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Alejandro, V. S.,
W. J. Nelson,
P. Huie,
R. K. Sibley,
D. Dafoe,
P. Kuo,
J. D. Scandling,
and
B. D. Myers.
Postischemic injury, delayed function and Na+/K+-ATPase distribution in the transplanted kidney.
Kidney Int.
48:
1308-1315,
1995[Medline].
2.
Anderson, R. J.,
and
R. W. Schrier.
Acute renal failure.
In: Diseases of the Kidney, edited by R. W. Schrier,
and C. W. Gottschalk. Boston, MA: Little Brown, 1997, p. 1069-1113.
3.
Chen, Q.,
T. W. Jones,
and
J. L. Stevens.
Early cellular events couple covalent binding of reactive metabolites to cell killing by nephrotoxic cysteine conjugates.
J. Cell. Physiol.
161:
293-302,
1994[Medline].
4.
Creely, J. J.,
S. J. DiMari,
A. M. Howe,
C. P. Hyde,
and
M. A. Haralson.
Effects of epidermal growth factor on collagen synthesis by an epithelioid cell line derived from normal rat kidney.
Am. J. Pathol.
136:
1247-1257,
1990[Abstract].
5.
Cuppage, F. E.,
and
A. Tate.
Repair of the nephron following injury with mercuric chloride.
Am. J. Pathol.
51:
405-429,
1967[Medline].
6.
Dekant, W.,
S. Vamvakas,
and
M. W. Anders.
Formation and fate of nephrotoxic and cytotoxic glutathione S-conjugates: cysteine conjugate -lyase pathway.
Adv. Pharmacol.
27:
115-162,
1994[Medline].
7.
Elfarra, A. A.,
I. Jackson,
and
M. W. Anders.
Mechanisms of S-(1,2-dichlorovinyl) glutathione-induced nephrotoxicity.
Biochem. Pharmacol.
35:
283-288,
1986[Medline].
8.
Glaumann, B.,
H. Glaumann,
I. K. Berezesky,
and
B. F. Trump.
Studies on cellular recovery from injury. II. Ultrastructural studies on the recovery of the pars convoluta of the proximal tubule of the rate kidney from temporary ischemia.
Virchows Arch.
24:
1-18,
1977.
9.
Groves, C. E.,
P. J. Hayden,
E. A. Lock,
and
R. G. Schnellmann.
Differential cellular effects in the toxicity of haloalkene and haloalkane cysteine conjugates to rabbit renal proximal tubules.
J. Biochem. Toxicol.
8:
49-56,
1993[Medline].
10.
Harris, R. C.
Potential physiological roles for epidermal growth factor in the kidney.
Am. J. Kidney Dis.
17:
627-630,
1991[Medline].
11.
Harris, R. C.
Growth factors and cytokines in acute renal failure.
Adv. Ren. Replace. Ther.
4:
43-53,
1997[Medline].
12.
Humes, H. D.,
D. A. Cieslinski,
T. M. Coimbra,
J. M. Messana,
and
C. Galvao.
Epidermal growth factor enhances renal tubule cell regeneration and repair and accelerates the recovery of renal function in postischemic acute renal failure.
J. Clin. Invest.
84:
1757-1761,
1989[Medline].
13.
Ilinskaja, O.,
and
S. Vamvakas.
Alterations of the renal function in the isolated perfused rat kidney system after in vivo and in vitro application of S-(1,2-dichlorovinyl)-L-cysteine and S-(2,2-dichlorovinyl)-L-cysteine.
Arch. Toxicol.
70:
224-229,
1996[Medline].
14.
Kays, S. E.,
and
R. G. Schnellmann.
Regeneration of renal proximal tubule cells in primary culture following toxicant injury: response to growth factors.
Toxicol. Appl. Pharmacol.
132:
273-280,
1995[Medline].
15.
Labarca, C.,
and
K. Paigen.
A simple, rapid, and sensitive DNA assay procedure.
Anal. Biochem.
102:
344-352,
1980[Medline].
16.
Lash, L. H.
Role of metabolism in chemically induced nephrotoxicity.
In: Mechanisms of Injury and Renal Disease and Toxicity, edited by R. S. Goldstein. Boca Raton, FL: CRC, 1994, p. 207-237.
17.
Lash, L. H.,
and
M. W. Anders.
Mechanism of S-(1,2-dichlorovinyl)-L-cysteine- and S-(1,2-dichlorovinyl)-L-homocysteine-induced renal mitochondrial toxicity.
Mol. Pharmacol.
32:
549-556,
1987[Abstract].
18.
Lin, F.,
A. Rios,
J. R. Falck,
Y. Belosludtsev,
and
M. L. Schwartzman.
20-Hydroxyeicosatetraenoic acid is formed in response to EGF and is a mitogen in rat proximal tubule.
Am. J. Physiol.
269 (Renal Fluid Electrolyte Physiol. 38):
F806-F816,
1995
19.
Lowry, O. H.,
N. J. Rosebrough,
A. L. Farr,
and
R. J. Randall.
Protein measurement with the Folin phenol reagent.
J. Biol. Chem.
193:
265-275,
1951
20.
Meister, A.,
S. S. Tate,
and
O. W. Griffith.
Reduced proximal tubule Na+ transport following ischemic injury.
J. Membr. Biol.
107:
119-127,
1989[Medline].
21.
Molitoris, B. A.,
L. K. Chan,
J. I. Shapiro,
J. D. Conger,
and
S. A. Falk.
Loss of epithelial polarity: a novel hypothesis for reduced proximal tubule Na+ transport following ischemic injury.
J. Membr. Biol.
107:
119-127,
1989[Medline].
22.
Moore, R. B.,
and
T. Green.
The synthesis of nephrotoxin conjugates of glutathione and cysteine.
Toxicol. Environ. Chem.
17:
153-162,
1988.
23.
Norman, J.,
B. Badie-Dezfooly,
E. P. Nord,
I. Kurtz,
J. Schlosser,
A. Chaudhari,
and
L. G. Fine.
EGF-induced mitogenesis in proximal tubular cells: potentiation by angiotensin II.
Am. J. Physiol.
253 (Renal Fluid Electrolyte Physiol. 22):
F299-F309,
1987
24.
Norman, J.,
K. Tsau,
A. Backay,
and
L. G. Fine.
Epidermal growth factor accelerates functional recovery from ischaemic acute tubular necrosis in the rat: role of the epidermal growth factor receptor.
Clin. Sci. (Colch.)
78:
445-450,
1990[Medline].
25.
Nowak, G.,
and
R. G. Schnellmann.
Integrative effects of EGF on metabolism and proliferation in renal proximal tubular cells.
Am. J. Physiol.
269 (Cell Physiol. 38):
C1317-C1325,
1995
26.
Nowak, G.,
and
R. G. Schnellmann.
L-Ascorbic acid regulates growth and metabolism of renal cells: improvements in cell culture.
Am. J. Physiol.
271 (Cell Physiol. 40):
C2072-C2080,
1996
27.
Nowak, G.,
and
R. G. Schnellmann.
Renal cell regeneration following oxidant exposure: inhibition by TGF-1 and stimulation by ascorbic acid.
Toxicol. Appl. Pharmacol.
145:
175-183,
1997[Medline].
28.
Nowak, G.,
M. D. Aleo,
J. A. Morgan,
and
R. G. Schnellmann.
Recovery of cellular functions following oxidant injury.
Am. J. Physiol.
274 (Renal Physiol. 43):
F509-F515,
1998
29.
Rall, L. B.,
J. Scott,
G. I. Bell,
R. J. Crawford,
J. D. Penschow,
H. D. Niall,
and
J. P. Coghlan.
Mouse prepro-epidermal growth factor synthesis by the kidney and other tissues.
Nature
313:
228-231,
1985[Medline].
30.
Reitzer, L. J.,
B. M. Wice,
and
D. Kennell.
The pentose cycle. Control and essential function in HeLa cell nucleic acid synthesis.
J. Biol. Chem.
255:
5616-5626,
1980
31.
Schaudies, R. P.,
and
J. P. Johnson.
Increased soluble EGF after ischemia is accompanied by a decrease in membrane-associated precursors.
Am. J. Physiol.
264 (Renal Fluid Electrolyte Physiol. 33):
F523-F531,
1993
32.
Schaudies, R. P.,
D. Nonclercq,
L. Nelson,
G. Toubeau,
J. Zanen,
J. A. Heuson-Stiennon,
and
G. Laurent.
Endogenous EGF as a potential renotrophic factor in ischemia-induced acute renal failure.
Am. J. Physiol.
265 (Renal Fluid Electrolyte Physiol. 34):
F425-F434,
1993
33.
Stevens, J. L.,
P. Hayden,
and
G. Taylor.
The role of glutathione conjugate metabolism and cysteine conjugate beta-lyase in the mechanism of S-cysteine conjugate toxicity in LLC-PK1 cells.
J. Biol. Chem.
261:
3325-3332,
1986
34.
Tashjian, A. H., Jr.,
E. F. Voelkel,
W. Lloyd,
R. Derynck,
M. E. Winkler,
and
L. Levine.
Actions of growth factors on plasma calcium: epidermal growth factor and human transforming growth factor-alpha cause elevation of plasma calcium in mice.
J. Clin. Invest.
78:
1405-1409,
1986[Medline].
35.
Toback, F. G.
Regeneration after acute tubular necrosis.
Kidney Int.
41:
226-246,
1992[Medline].
36.
Toback, F. G.,
S Kartha,
and
M. M. Walsh-Reitz.
Regeneration of kidney tubular epithelial cells.
Clin. Investig.
71:
861-866,
1993[Medline].
37.
Vamvakas, S.,
H. Richter,
and
D. Bittner.
Induction of dedifferentiated clones of LLC-PK1 cells upon long-term exposure to dichlorovinylcysteine.
Toxicology
106:
65-74,
1996[Medline].
38.
Van de Water, B.,
J. J. Jaspers,
D. H. Maasdam,
G. J. Mulder,
and
J. F. Nagelkerke.
In vivo and in vitro detachment of proximal tubular cells and F-actin damage: consequences for renal function.
Am. J. Physiol.
267 (Renal Fluid Electrolyte Physiol. 36):
F888-F899,
1994
39.
Venkatachalam, M. A.,
D. B. Jones,
H. G. Rennke,
D. Sandstrom,
and
Y. Patel.
Mechanism of proximal tubule brush border loss and regeneration following mild renal ischemia.
Lab. Invest.
45:
355-365,
1981[Medline].