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* The William Harvey Research Institute, St. Bartholomew’s and the Royal London School of Medicine and Dentistry, London, EC1M 6BQ, U.K.;
Institute of Pharmacology, University of Messina, Messina 98123, Italy; and
Institute of Pathology, University of Mainz, D-55101 Mainz, Germany
1Correspondence: The William Harvey Research Institute, St. Bartholomew’s and the Royal London School of Medicine and Dentistry, Charterhouse Square, London, EC1M 6BQ, U.K. E-mail: p.k.chatterjee{at}mds.qmw.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: kidney • proximal tubule • reactive oxygen species • reperfusion injury • poly (ADP-ribose) synthetase inhibitors
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Reperfusion of previously
ischemic renal tissue initiates a complex and interrelated sequence of
events that results in injury and the eventual death of renal cells due
to a combination of apoptosis and necrosis (2)
. Although
reperfusion is essential for the survival of ischemic tissue, there is
good evidence that reperfusion itself causes additional cell injury
(reperfusion injury) (3)
, which has been attributed to
adenosine triphosphate (ATP) depletion, intracellular calcium
accumulation, phospholipase activation and membrane lipid alterations,
cytoskeletal dysfunction, neutrophil infiltration, and the generation
of reactive oxygen species (ROS) (3
, 4)
. ROS-mediated cell
injury and death has been implicated in the pathogenesis of renal
ischemia-reperfusion injury and associated renal failure
(5)
. The situation is particularly complex in the kidney,
as ischemia itself can cause early irreversible damage, which also
appears to be mediated by ROS (3
, 5)
.
ROS produce cellular injury and necrosis via several mechanisms
including peroxidation of membrane lipids, protein denaturation, and
DNA damage (4)
. Evidence from studies using cultured cells
have demonstrated that ROS produce strand breaks in DNA that trigger
activation of the nuclear enzyme poly (ADP-ribose) synthetase [PARS,
EC 2.4.2.30, also referred to as poly (ADP-ribose) polymerase (PARP) or
poly (ADP-ribose) transferase (pADPRT) (6)
]. PARS is an
abundant, chromatin-bound enzyme constitutively expressed in most cell
types (7)
. It is an energy-consuming enzyme thought to be
involved in DNA repair (6)
, although its true
physiological role is still unclear (8)
. Once activated,
PARS catalyzes the transfer of ADP-ribose moieties from NAD to nuclear
proteins, including histones, and onto PARS itself (automodification)
with the concomitant formation of nicotinamide (NIC) (9)
.
However, there is now good evidence that exposure of cells to oxidant
stress results in strand breaks in DNA leading to an excessive
activation of PARS, resulting in the depletion of its substrate NAD
in vitro and a reduction in the rate of glycolysis
(10
, 11)
. Since NAD functions as a cofactor in glycolysis
and the tricarboxylic acid cycle, NAD depletion leads to a rapid fall
in intracellular ATP levels (12
13
14
15)
. Furthermore,
nicotinamide formed by PARS activation can be recycled back to NAD via
a mechanism that also consumes ATP (13)
. Thus, activation
of PARS leads to a fall in ATP via two different mechanisms, leading to
cellular dysfunction and ultimately cell death. Overall, this process
has been termed the ‘PARS suicide hypothesis’ (15)
.
PARS inhibitors include benzamide analogs such as 3-aminobenzamide
(3-AB), NIC, and recently discovered and more potent isoquinoline
derivatives such as 1,5-dihydroxyisoquinoline (ISO) (16
, 17)
. Such compounds have previously been used in both in
vivo and in vitro studies to investigate their
ameliorative role in various pathophysiological conditions ranging from
ischemia-reperfusion injury to diabetes mellitus (17
, 18)
.
PARS inhibitors have been shown to attenuate the fall in ATP (and NAD)
and improve the survival of several cultured cell types exposed to
reactive oxygen or nitrogen species such as
H2O2 or peroxynitrite
(18
19
20
21
22
23)
. Three years ago we reported that the PARS
inhibitors 3-AB, ISO, NIC reduce ischemia-reperfusion injury in the
rabbit heart and skeletal muscle in vivo (24)
.
To date, there has been little research into the role of PARS within
the kidney in vivo under normal or pathophysiological
conditions. We have recently discovered that 3-AB, ISO, and NIC protect
primary cultures of rat proximal tubular (PT) cells against
H2O2-mediated oxidant
stress (25)
. The possibility of pharmacological inhibition
of PARS activation in the kidney could lead to the development of novel
therapies for the treatment of renal ischemia-reperfusion injury and
associated acute renal failure, either alone or in combination with
other recognized therapies, since 1) reperfusion injury
plays a major role in clinical renal failure, 2) PARS
activation has been implicated in ischemia-reperfusion injury in
several other organs, and 3) the beneficial effects of PARS
inhibitors have been recognized in nonrenal models of
ischemia-reperfusion injury. Therefore, the aim of this study was to
investigate the effect of PARS inhibitors in an in vivo
model of renal ischemia-reperfusion injury in the anesthetized rat.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animal preparation
This study was carried out using 113 male Wistar rats (Tuck,
Rayleigh, Essex, U.K.) weighing 250–350 g, receiving a standard diet
and water ad libitum, and cared for in accordance with the
Home Office Guidance in the Operation of the Animals (Scientific
Procedures) Act 1986, published by HMSO, London, U.K. Of these
animals, four died during the course of the experiment, giving an
overall mortality of 3.9%. All results obtained from rats that died
during the course of the experiment were excluded from the data
analysis and numbers (n) provided in the text refer to the
‘survivors’ of the entire experimental period only. All animals were
anesthetized with sodium thiopentone (Intraval sodium, 120 mg/kg i.p;
Rhone Merieux Ltd., Essex, U.K.), and anesthesia was maintained by
supplementary infusions of sodium thiopentone. The animals were placed
onto a thermostatically controlled heating mat (Harvard Apparatus Ltd.,
Kent, U.K.) and body temperature was maintained at 38 ± 1°C by
means of a rectal probe attached to a homeothermic blanket. A
tracheotomy was performed to maintain airway patency and facilitate
spontaneous respiration. The right carotid artery was cannulated (PP50,
I.D. 0.58 mm, Portex, Kent, U.K.) and connected to a pressure
transducer (Senso-Nor 840, Horten, Norway) for the measurement of mean
arterial blood pressure (MAP) and derivation of the heart rate (HR)
from the pulse waveform, which were displayed on a data acquisition
system (MacLab 8e, AD Instruments, Hastings, U.K.) installed on an
Apple MacIntosh computer. MAP and HR were monitored for the duration of
each experiment. The jugular vein was cannulated (PP25, I.D. 0.40 mm,
Portex, Kent, U.K.) for the administration of drugs. A midline
laparotomy was performed and the kidneys located, after which the renal
pedicles, containing the renal artery, vein, and nerve supplying each
kidney were carefully isolated.
Renal ischemia-reperfusion
Rats were allowed to stabilize for 30 min before they were
subjected to bilateral renal occlusion for 45 min using artery clips to
clamp the renal pedicles. Reperfusion began once the artery clips were
removed (‘control’ animals). Occlusion was verified visually by
change in the color of the kidneys to a paler shade and reperfusion by
a blush. Other rats were subjected to sham operation (‘sham-operated’
animals; identical surgical procedures but without bilateral renal
clamping) and maintained under anesthesia for the duration of the
experiment. At the end of all experiments, animals were killed by an
overdose of sodium thiopentone. Kidneys were then excised and bisected
for histological assessment and measurement of PARS activity as
described below. Another group of anesthetized rat were
tracheostomized, and the carotid artery and bladder were cannulated as
described above for the collection of plasma and urine samples,
respectively, after the 30 min stabilization period. These animals are
described as ‘normal animals’ in this report.
Experimental protocol
On completion of surgical procedures, the animals were grouped
randomly as described in Table 1
. At 1 min before reperfusion began, animals received a bolus injection
of either vehicle (saline, 4 ml/kg, i.v.) or one of the following
inhibitors of PARS activity: 3-AB (3, 10, or 30 mg/kg in saline, i.v.),
ISO (1 mg/kg in 10% v/v DMSO in saline, i.v.), or NIC (10 mg/kg in
saline, i.v.). Additional groups of rats received the following
inactive analogs of the PARS inhibitors (negative controls):
3-aminobenzoic acid (3-ABA; 10 mg/kg in 10% v/v DMSO in saline, i.v.),
or nicotinic acid (NICA; 10 mg/kg in saline, i.v.). Another group of
rats received desferrioxamine mesylate (DEF; 40 mg/kg in saline, i.v.)
as a positive control (see Discussion). As the vehicle for both ISO and
3-ABA was 10% v/v DMSO, another group of animals were administered a
bolus of 10% v/v DMSO in saline, 4 ml/kg, i.v. The corresponding
groups then received a continuous infusion of one of the following
throughout the reperfusion period: vehicle (saline, 4 ml/kg/h, i.v. or
10% v/v DMSO in saline, 4 ml/kg/h, i.v.), 3-AB (1.5, 5, or 15 mg/kg/h
in saline, i.v.), ISO (0.5 mg/kg/h in 10% v/v DMSO in saline, i.v.),
NIC (5 mg/kg/h in saline, i.v.), 3-ABA (5 mg/kg/h in 10% v/v DMSO in
saline), NICA (5 mg/kg/h in saline, i.v.), or DEF (40 mg/kg/h in
saline, i.v.).
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To elucidate the effects of the PARS inhibitors on hemodynamics and
organ function in sham-operated rats, separate groups of animals
received the treatments described (outlined in Table 1
). In all the
groups of animals described above, hemodynamic parameters were recorded
for the duration of the experiment.
Quantification of renal function and injury
At the end of the reperfusion period, blood (1 ml) samples were
collected via the carotid artery into S1/3 tubes containing serum gel.
The samples were centrifuged (6000 r.p.m. for 3 min) to separate
plasma, which was analyzed for biochemical parameters within 24 h
after collection (Vetlab Services, Sussex, U.K.). Plasma concentrations
of urea were measured as an indicator of impaired renal function and/or
increased catabolism (26)
and creatinine as an indicator
of reduced glomerular filtration rate (26)
. Urine samples
were also collected during the reperfusion period and the volume of
urine produced was recorded. Urine concentrations of creatinine and
Na+ were also measured (Vetlab Services) and used
in conjunction with plasma creatinine and Na+
concentrations to calculate glomerular filtration rate (GFR) and
fractional excretion of Na+
(FENa) respectively, using standard formulas.
Plasma and urine samples were also collected from normal,
sham-operated, and control animals and those treated with vehicle, PARS
inhibitors, negative controls, or DEF.
Renal histopathology
Kidneys were fixed in 10% w/v neutral buffered
paraformaldehyde, embedded in paraffin, cut into 4 µm sections,
dewaxed, and stained with hematoxylin-eosin, Fuchsin, and Luxol-fast
blue. Histopathological examination for specific lesions characteristic
of renal injury (27)
was performed by a pathologist
blinded to the animal treatment using light microscopy.
Histochemical measurement of PARS activation
PARS activation was assessed in kidneys fixed in 10% w/v
neutral buffered paraformaldehyde and 8 µm sections were prepared
from paraffin embedded tissues. After deparaffination, endogenous
peroxidase was quenched using 0.3% v/v
H2O2 in 60% methanol for
30 min. The sections were permeablized with 0.1% w/v Triton X-100 in
phosphate-buffered saline (PBS, 0.01 M, pH 7.4) for 20 min. Nonspecific
adsorption was minimized by incubating the section in 2% v/v normal
goat serum in PBS for 20 min. Endogenous avidin and biotin binding
sites were blocked by sequential incubation for 15 min using avidin
(DBA, Milan, Italy) and biotin (DBA), respectively. Sections were then
incubated overnight (18 h) in a 1:500 dilution of primary anti-poly
(ADP-ribose) antibody (DBA) or with control solutions consisting of PBS
alone or nonspecific purified rabbit IgG (1:500 dilution, DBA).
Specific labeling was detected using a biotin-conjugated goat
anti-rabbit IgG (DBA) and avidin-biotin peroxidase (DBA). Samples were
then viewed under a light microscope.
Statistical analysis
Data are expressed as mean ± SE obtained from
hemodynamic or biochemical measurements obtained from n
animals. Statistical analysis was carried out using GraphPad
Prism/Instat (GraphPad Software, Calif.). Data were analyzed using
one-way ANOVA, followed by Dunnett’s post hoc test, and a
P value of less than 0.05 was considered to be significant.
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RESULTS |
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, Fig. 2A
). Plasma urea and creatinine concentrations demonstrated a
steady increase over 2, 4, and 6 h of reperfusion. After 6 h
of reperfusion, plasma levels of urea had increased by 3.5-fold, from
8.2 ± 1.1 mmol/l (sham-operated) to 28.3 ± 0.9 mmol/l (6 h
reperfusion controls), n = 12, P < 0.05.
Plasma creatinine concentrations increased by 5.7-fold, from 35.3 ± 2.3 µmol/l (sham operated) to 201.1 ± 7.4 µmol/l (6 h
reperfusion controls), n = 12, P < 0.05.
Plasma levels of urea and creatinine beyond 45 min ischemia and 6 h reperfusion were not determined in this study.
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In conjunction with plasma creatinine and Na+
concentrations, measurement of urine production (urine flow, ml/min)
and urinary concentrations of creatinine and Na+
allowed the calculation of GFR and FENa,
respectively. GFR, used as an indicator of glomerular function,
remained within physiological levels in normal and sham-operated rats
(1.27±0.13 ml/min, n=11 and 1.01±0.12 ml/min,
n=6, respectively, Fig. 3A
). However, ischemia followed by reperfusion produced a
significant reduction in GFR, which was maintained throughout the
6 h reperfusion period (Fig. 3A
). After 6 h
reperfusion, GFR was reduced by 97.0% from 1.01 ± 0.12 ml/min
(sham-operated), n = 6, to 0.03 ± 0.01 ml/min (6
h reperfusion controls), n = 12, P < 0.05.
Calculations of FENa were used as an indicator of
PT function. FENa calculated from normal and
sham-operated rats remained within normal parameters (0.31±0.04%,
n=11 and 0.44±0.09%, n=6, respectively;
Fig. 4A
). However, ischemia for 45 min, followed by 2, 4, or 6 h reperfusion, produced a significant increase in
FENa that was maintained throughout the
reperfusion period. After 6 h reperfusion,
FENa increased by 98.6% from 0.44 ± 0.09%
(sham-operated), n = 6, to 32.1 ± 3.6% (6 h
reperfusion controls), n = 12, P < 0.05
(Fig. 4A
).
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Effect of PARS inhibitors on plasma and urinary biochemical
parameters
Administration of 3-AB produced a dose-dependent reduction in
plasma concentrations of urea and creatinine compared to values
obtained from control animals (i.e., 45 min ischemia+6 h reperfusion
only) (Fig. 1B
, Fig. 2B
). 3-AB produced a
significant reduction in plasma urea and creatinine levels at the two
higher concentrations administered (10 mg/kg bolus followed by 5
mg/kg/h infusion and 30 mg/kg bolus followed by 15
mg·kg-1·h-1 infusion)
(Fig. 1B
, Fig. 2B
). 3-AB also produced a
significant improvement in GFR at these concentrations (Fig. 3B
). 3-AB reduced FENa in a
dose-dependent manner in comparison with FENa
values obtained from control animals; a significant reduction was
obtained using the two highest concentrations (10 mg/kg bolus followed
by 5 mg/kg/h infusion and 30 mg/kg bolus followed by 15 mg/kg/h
infusion) (Fig. 4B
).
Administration of the PARS inhibitors 3-AB and ISO produced small, but
significant, reductions in plasma urea concentrations compared to
control values (Fig. 1C
). 3-AB and ISO reduced plasma urea
concentrations by ~15 (P<0.05) and 41%
(P<0.05), respectively. However, NIC did not reduce urea
concentrations compared to control urea concentrations. All three PARS
inhibitors produced a significant reduction in plasma creatinine
concentrations compared to control values (Fig. 2C
).
Compared with control levels, 3-AB, ISO, and NIC reduced plasma
creatinine concentrations by ~32 (P<0.05), 44
(P<0.05), and 32% (P<0.05), respectively (Fig. 2C
).
All three PARS inhibitors, given before and during 6 h
reperfusion, significantly increased GFR (Fig. 3C
) and
decreased FENa (Fig. 4C
) compared to
control values. 3-AB, ISO, and NIC produced an 85%
(P<0.05), 79 (P<0.05) and 80%
(P<0.05) increase in GFR, respectively, compared to control
GFR values (Fig. 3C
). The PARS inhibitors also produced
significant decreases in FENa of 61%
(P<0.05), 57% (P<0.05), and 48%
(P<0.05), respectively, vs. FENa
values obtained from controls (Fig. 4C
).
Administration of 3-ABA and NICA [structural analogs of 3-AB and NICA
that do not inhibit PARS activity (16)
and were used as
negative controls in this study] had no effect on plasma urea or
creatinine concentrations when compared with values obtained from
control animals (Fig. 1D
, Fig. 2D
). However,
administration of the iron chelator DEF (positive control) before and
during reperfusion produced significant reductions in plasma urea and
creatinine concentrations of ~36 (P<0.05) and 30%
(P<0.05), respectively (Fig. 1D
, Fig. 2D
).
Neither 3-ABA nor NICA had any effect on GFR or
FENa compared with control values (Fig. 3D
, Fig. 4D
). However, administration of DEF
before and during reperfusion produced a significant increase in GFR
(70%; P<0.05) and a significant decrease in
FENa (55%; P<0.05), respectively,
compared to control values (Fig. 3D
, Fig. 4D
).
Administration of any of the drugs used in this study to sham-operated animals for the duration of the experiment had no effect on plasma urea and creatinine levels, GFR, or FENa and were similar to those obtained from normal and sham-operated animals (data not shown).
Renal histopathology and effects of PARS inhibitors
Compared with sham-operated rats (Fig. 5A
), rats that underwent ischemia for 45 min, followed by
reperfusion, demonstrated the recognized features of renal injury
including characteristic histological signs of glomerular and tubular
damage (27)
. After 6 h reperfusion, degeneration of
the glomeruli, tubular dilatation, tubular swelling and necrosis,
luminal congestion, and the presence of eosinophilia were observed
(Fig. 5B
). The histological signs of renal injury were also
apparent after 2 and 4 h of reperfusion (data not shown), but
increased noticeably after 6 h reperfusion (Fig. 5B
).
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Kidneys obtained from rats treated with PARS inhibitors 1
min prior to and during reperfusion demonstrated reduced histological
features of renal injury when compared with kidneys obtained from
control animals (Fig. 5C
). The example illustrated (Fig. 5C
) shows an example of rat kidney obtained after
administration of 3-AB. Although substantial glomerular degradation is
still observed, there appears to be reduced tubular swelling and
dilatation, luminal congestion, and eosinophilia compared with that
observed in kidneys obtained from control animals.
Immunohistochemical localization of PARS and actions of PARS
inhibitors
Kidneys obtained from rats subjected to ischemia-reperfusion
demonstrated noticeable staining for PARS when compared with kidneys
obtained from sham-operated rats (Fig. 6A, B
), suggesting the activation of PARS after 45 min
ischemia, followed by 6 h reperfusion. In these control rats,
staining for PARS (P) was most marked in the PT cells, with surrounding
structures largely unaffected (Fig. 6B
). The same level and
distribution of staining for PARS were obtained from kidneys removed
after 2 and 4 h of reperfusion (data not shown).
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Kidneys obtained from rats subjected to ischemia-reperfusion in the
presence of the PARS inhibitors demonstrated markedly reduced staining
for PARS protein when compared with kidneys obtained from control
animals (Fig. 6C
, suggesting a reduction in the activation
of PARS after 6 h reperfusion. Figure 5C
shows a
section from a representative rat kidney obtained after administration
of 3-AB.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2) ROS produce
strand breaks in the DNA of renal tissues (28)
, and
3) strand breaks in DNA activate PARS (10)
.
Therefore, our hypothesis is that the activation of PARS may
contribute, in part, to the injury associated with ischemia-reperfusion
of the kidney and contributes to the associated acute renal failure and
eventual end-stage renal failure. We have recently discovered that
three recognized PARS inhibitors (3-AB, ISO, and NIC) reduce the
cellular injury and death caused by
H2O2 in PT cell cultures
isolated from rat kidneys (25)
. We now demonstrate here,
for the first time, that the same PARS inhibitors reduce
ischemia-reperfusion injury in the rat kidney in vivo.
Ischemia followed by reperfusion of rat kidneys produced significant
increases in plasma concentrations of urea, suggesting impaired renal
function and/or increased catabolism, as well as increased plasma
concentrations of creatinine, indicating reduced GFR and hence renal
failure (26)
. In this study, a significantly reduced GFR
and increased FENa were recorded from control
animals, indicating marked reduction in glomerular and PT function,
respectively. These biochemical indicators of renal dysfunction
appeared to be dependent on the time period of reperfusion, with
maximal changes in urea, creatinine, GFR, and
FENa observed after 6 h reperfusion. These
indicators of renal function were not measured beyond 6 h.
However, it has recently been demonstrated that there are no
significant increases in plasma urea and creatinine levels between 6
and 24 h of reperfusion in a similar model of renal
ischemia-reperfusion in the rat (29)
. Histological
evaluation confirmed renal injury after 6 h of reperfusion, with
the characteristic morphological markers of tissue damage present in
kidneys obtained from control animals.
The ability of ROS, including hydroxyl radicals, to induce renal
injury has been demonstrated (3
, 5
, 28)
. The renal
dysfunction produced during reperfusion of previously ischemic rat
kidneys in this study appears to be secondary to the generation of
hydroxyl radicals, as it was attenuated by desferrioxamine.
Desferrioxamine, an iron chelator that binds and subsequently reduces
the availability of Fe2+, which in combination
with H2O2 promotes the
generation of hydroxyl radical, has previously been shown to protect
rabbit kidneys against both cold and warm ischemic injury (30
, 31)
. ROS-mediated mechanisms of renal tissue injury and necrosis
such as DNA damage are likely to be a major player in renal
ischemia-reperfusion injury, as it is still unclear whether lipid
peroxidation is a major contributor to the renal dysfunction associated
with ischemia-reperfusion (32
, 33)
.
The results obtained in this study suggest that the activation of PARS
contributes, in part, to the renal dysfunction and injury observed
after reperfusion of previously ischemic tissue. This hypothesis is
supported by the following key findings: 1) three chemically
distinct PARS inhibitors significantly reduced plasma urea and
creatinine levels, increased GFR, and reduced
FENa when administered before and during
reperfusion; 2) PARS activation in the PT was demonstrated
histochemically; 3) 3-ABA and NICA, analogs of 3-AB and NIC,
which do not inhibit PARS activity (16)
, did not reduce
the renal dysfunction/injury caused by ischemia-reperfusion. Thus, we
propose that ischemia-reperfusion causes an increase in PARS activity
in the PT, which in turn contributes to the cellular injury and death
observed under these conditions. This hypothesis is reinforced by our
earlier findings that the PARS inhibitors can protect primary cultures
of rat PT cells against oxidant stress-mediated cell injury and death
(25)
.
Ischemia-reperfusion of the kidney causes glomerular and tubular
dysfunction and injury (34)
; in this study,
ischemia-reperfusion produced a marked reduction in GFR (an indicator
of glomerular dysfunction) and also an increase in
FENa (an indicator of tubular dysfunction), both
of which were maintained throughout the reperfusion period.
Administration of PARS inhibitors during this period produced a modest
but significant increase in GFR, suggesting some degree of recovery of
glomerular function. This was reflected in the reduced plasma urea and
creatinine concentrations in rats administered PARS inhibitors during
reperfusion. However, it could be argued that although plasma urea
concentrations were reduced in the presence of 3-AB and ISO, NIC did
not have an effect. This is not surprising, since it is known that urea
clearance as a measure of renal function is imprecise as it can be
influenced both by extrarenal factors and urine flow (26)
.
Therefore, assessment of creatinine clearance is a much better marker
of renal function (26)
.
Although the PARS inhibitors produced a small, but significant,
recovery in GFR, the effects of the PARS inhibitors on the increase in
FENa caused by ischemia-reperfusion were more
pronounced. This suggests that the protection afforded by the PARS
inhibitors is greater against the PT effects of reperfusion injury than
those in the glomerulus. This is confirmed by the immunohistochemical
staining for PARS, which is present only in the PT in kidneys obtained
from control rats but absent in kidneys from rats administered the PARS
inhibitors. Further evidence has been reported in our earlier findings
that PARS inhibitors protect cultured rat PT cells against oxidant
stress-mediated cellular injury and death (25)
.
In this study, 3-AB, which we have previously shown to produce a
dose-dependent inhibition of PARS activity in vitro in
primary cultures of rat PT cells (25)
, produced a
significant and dose-dependent improvement in renal function. The
concentrations of the three PARS inhibitors used in this study
correspond to those used in previous in vivo studies
(24
, 35)
. Furthermore, in the case of 3-AB, the
concentration used here in vivo corresponds to a plasma
concentration that is 1) within the concentration range that
selectively inhibits PARS activity in vitro
(36)
and 2) corresponds to the concentrations
required to inhibit PARS activation by
H2O2 in primary cultures of
rat PT cells (25)
. The continuous infusion regimen
selected for the administration of the PARS inhibitors during the
reperfusion period was based on the observation that the calculated
half-life of 3-AB in the rat in vivo is relatively short at
90 min (37)
.
To date, there has been relatively little research into the role of
PARS within the kidney or in specific renal structures such as the PT
under normal and pathophysiological conditions. Previous reports have
described how renal carcinogens produce DNA strand breaks and
subsequently increase in poly (ADP-ribosyl)ation of nuclear proteins
both in the rat after in vivo administration and in
LLC-PK1 cell cultures, a renal cell line with
several PT cell characteristics (38
, 39)
. Increased PARS
activity in murine kidneys has been reported after exposure to cold
ischemia in vitro produced by storage at 0°C for up to
72 h (40)
. This paper demonstrates for the first time
how inhibitors of PARS activation can protect the rat kidney from
ischemia-reperfusion injury in vivo. We propose that potent
and specific PARS inhibitors may be useful in conditions of oxidative
stress that prevail during reperfusion of previously ischemic renal
tissues (5)
. As ischemic renal injury is becoming one of
the main causes of end-stage renal failure (41)
, the
findings described in this study may stimulate interest in the
development of more potent and specific PARS inhibitors that could be
used either alone or in combination for the treatment of the renal
dysfunction associated with reperfusion injury. This is reinforced by
the fact that the endogenous PARS inhibitor nicotinamide is currently
undergoing clinical trials to investigate its beneficial actions in
diabetes mellitus (42)
. We therefore propose that the
inhibition of PARS activity represents a novel approach for the therapy
of renal disorders associated with oxidant stress.
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ACKNOWLEDGMENTS |
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r Kardiologie. C.T. is the recipient of a British Heart Foundation
Senior Research Fellowship (FS/96018). |
FOOTNOTES |
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REFERENCES |
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