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Key words: glomerular filtration rate, renal plasma flow, acute renal
failure, peroxynitrite, superoxide, nitric oxide, l-arginine, kidney transplantation.
Received for publication May 25, 2001
and in revised form September 24, 2001
Accepted for publication November 13, 2001
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ability of the substrate l-Arg, as suggested in hypercholesterolemia and diabetes mellitus [18, 19]. Alternatively,
the reduction in biological activity of NO could be due to
increased inactivation of NO. In certain disease states, the
bioactivity of NO is markedly reduced by its reaction with
superoxide anions (O2) yielding peroxynitrite (ONOO)
[20]. ONOO, unlike NO, is highly reactive and, as an
in vivo footprint [20], nitrates protein tyrosine moieties.
This nitrosative stress response [21, 22] has been suggested
to be the cause of cytotoxicity in a number of toxic, ischemic and inflammatory disease models [20]. However, it
is unclear whether ONOO is the cause or a consequence
of these diseases. Endothelial cells are capable of producing both NO and O2. Important cellular sources of O2
are xanthine oxidase, nicotinamide adenine dinucleotide
phosphate (NADPH) oxidase [23], and NOS, which at
sub-saturating levels of l-Arg also can generate O2 [24].
Notwithstanding, cells have evolved antioxidant defense
mechanisms against O2, among them superoxide dismutases (SODs) [25, 26].
Although the use of exogenous l-Arg as a substrate
for NOS in experimental ARF seems promising, little is
known about its adverse potential since excess production of NO has been suggested to be associated with cytotoxicity [17, 27, 28] involving the formation of ONOO
[20]. However, with respect to the suitability of NT-IR
as an in vivo marker for ONOO, it was demonstrated
recently that tyrosine nitration may not necessarily require ONOO and de novo NO formation. Indeed, myeloperoxidase (MPO) can catalyze tyrosine nitration
from nitrite independently of ONOO [29, 30]. To our
knowledge, none of the previously mentioned parameters of oxidative and nitrosative stress have been investigated in ARF.
Beyond experimental ARF, in the early phase of kidney transplantation, that is, during organ harvesting, preservation and implantation, ischemia can result in impaired
renal function, too. The use of the immunosuppressant,
cyclosporine A (CsA), enhances graft survival but this effect is compromised by hypertension and nephrotoxicity,
suggesting endothelial dysfunction [31]. Since the previously mentioned effects of l-Arg in experimental ARF
are likely to be associated with endothelial dysfunction,
we hypothesized that the early administration of l-Arg
prior to and immediately after human kidney transplantation could exert equally beneficial effects. This hypothesis is supported by the observation that addition of
l-Arg to preservation solutions enhances graft survival
[32]. Moreover, a recent animal study showed beneficial
effects of l-Arg supplementation on renal function after
kidney transplantation [33]. Data from clinical studies
with l-Arg in renal transplantation also showed that
l-Arg improved renal hemodynamics but only in patients
not receiving cyclosporine. These studies, however, are
(Eq. 1)
1425
Nitrite/nitrate measurement
Aliquots of each plasma or urine sample were diluted
as necessary. Nitrate was then converted to nitrite by nitrite reductase and assayed by a modification of the Griess
method [37].
Protein immunoblotting
(Eq. 2)
1426
using an enhanced chemiluminescence kit from Amersham (Braunschweig, Germany). All samples were analyzed in duplicate.
Protein nitrotyrosine dot blot analysis
Proteins were denatured by heating to 95C for five
minutes in 1% sodium dodecyl sulfate (SDS) in the absence of mercaptoethanol. Approximately 25 g protein
was loaded into each well of a Dot Blot template (BioDot; Biorad, Melville, NY, USA) according to the manufacturers instructions and allowed to be absorbed onto
the membrane at 4C overnight. Primary anti-NT (monoclonal anti-NT-BSA) were added to each well (dilution
1:4000) and allowed to equilibrate for one hour at 4C.
After repeated washing, peroxidase-conjugated anti-mouse
IgG in a dilution of 1:2000 (Amersham) was added to
the wells and allowed to equilibrate for one hour at room
temperature. All samples were analyzed in triplicate.
Determination of NOS activity
Catalytic activities of NOS in kidney homogenate and
supernatant fractions were assayed by the standard conversion of tritiated l-Arg to l-citrulline [37]. Tissue homogenates (50 L) were incubated under Vmax conditions,
that is, for 15 minutes at 37C in a total volume of 0.1 mL
of 50 mmol/L Tris buffer (pH 7.0) containing 50 nmol/L
calmodulin, 250 mol/L cyclophosphamide, hexamethylmelamine, (3-[(3-cholamidoproyl)dimethylammonio]-lpropane-sulfonate) (CHAPS), 5 mol/L flavin adenine
dinucleotide (FAD), 5 mol/L flavin mononucleotide
(FMN), 1 mmol/L NADPH, 0.7 mol/L CaCl2, 7 mmol/L
GSH, 30 mol/L l-Arg and 10 mol/L H4biopterin. For
detection of NOS-II activity, CaCl2 was omitted from
the assay buffer and replaced by 0.5 mmol/L EDTA. All
samples were analyzed in triplicate.
Superoxide production
NADPH oxidase and xanthine oxidase activities in
kidney homogenate were measured at 37C in a final volume of 1 mL by lucigenin-induced, SOD inhibitable luminescence in 50 mmol/L phosphate buffer (pH 7.0) containing 1 mmol/L EDTA, 150 mmol/L sucrose, 500 mol/L
lucigenin, in the absence or presence of either 1 mmol/L
NADPH or xanthine. SOD inhibitors were not included.
The reaction was started by the addition of 100 g of
protein (per 100 L homogenate) to 900 L of the abovementioned freshly prepared reagent mixture. Luminescence was monitored every 15 seconds for 15 minutes in
a luminometer (Lumat LB 9501; Berthold, Munchen,
Germany). No activity could be measured in the absence
of NADPH, or was beyond detection limit with lucigenin
concentrations 500 mol/L.
Determination of L-Arg levels by HPLC
l-Arginine measurements in S10 fractions of rat kidney were performed by reversed-phase high-pressure liq-
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Control
ARF
ARF l-Arg
Tissue l-Arg
N
lmol/L
Changes in % of control
5
4
6
24.2 0.4
18.6 0.8a
37.2 5.7ab
23 3.3
54 24
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Clinical data
In a randomized double-blind study, 54 kidney transplant recipients were divided into two groups: one received l-Arg and the other received saline (Methods
section). The patients characteristics are described in
Table 2. l-Arg was administered two hours prior to surgery and continuously infused for an additional three
days. GFR and RPF, monitored by scintigraphy for up
to 10 days after surgery, gradually improved during that
time in both groups (Table 3). Moreover, GFR and RPF
were better improved in the l-Arg treated group as compared to control groups (from day 1 to day 5; however,
this did not reach statistical significance). These trends
were reversed at day 10 post-surgery (Table 3), that is,
seven days after l-Arg treatment was stopped, suggesting
that l-Arg indeed improved suppressed renal function
in the early phase of kidney transplantation. To analyze
further whether a specific subset of patients particularly
benefitted from l-Arg treatment, GFR and RPF at day 3
(after day 3 l-Arg infusion was stopped) were correlated
against several parameters [NOx, cGMP, donor sex, disease stage, HLA-mismatches, organ donor age and cold
ischemia time (CIT)]. However, only organ donor age
and CIT were found to affect the outcome after l-Arg
administration (Fig. 5 AD). Indeed, in patients receiving kidneys with CITs below 20 hours or from donors
younger than 45 years of age l-Arg increased RPF when
compared to the saline-treated control group (Fig. 5
E, F). Also, GFR on day 1 was increased for patients
with CIT less than 20 hours (20 3 vs. 32 5 mL/min,
P 0.05), for donors younger than 45 years (20 3 vs.
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Fig. 5. Effect of L-Arg in renal transplant patients. (A, B) Linear regression analysis of the relationship between donor age and the effect of l-Arg
on renal plasma flow (RPF). (C, D) Linear regression analysis of the relationship between the duration of cold ischemia time (CIT) and the effect
of l-Arg on RPF. There was no significant correlation in the control group (A, C), but there was a significant negative correlation in the l-Arg
treatment group (B, D). (E ) Mean data SEM for RPF when including recipients with donor age 45 years in control (N 16) and l-Arg
treated groups (N 15). (F ) Mean data SEM for RPF when including recipients with duration of cold ischemia time 20 hours in the control
(N 19) and l-Arg treated groups (N 16). Symbols are: () control group; () l-Arg treated group. l-Arg was infused from day 1 to day 3.
Differences between control and l-Arg treatment were analyzed by the unpaired Student t test; NS indicates not significant; *P 0.05 compared
to control, which was accepted as level of significance.
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Control
l-Arg
26
0.65 0.09
49.4 2.33
39.5 2.98
72.8 2.64
41.2 1.82
19.4 0.99
3.3 0.35
28
0.65 0.09
45.6 1.94
40.3 2.83
71 2.58
40.6 1.99
19.4 0.66
4.3 0.52
None of the differences between control group and l-Arg group was significant.
GFR mL/min
RPF mL/min
Treatment
Day 1
Day 3
Day 5
Day 10
Control
l-Arg
Control
l-Arg
23 2
26 3
142 14
158 21
27 3
31 4
191 28
222 31
31 3
33 4
188 22
219 29
40 4
38 4
209 24
177 22
Mean data showing glomerular filtration rate (GFR) and renal plasma flow
(RPF) in kidney transplant recipients from control (N 26) and l-Arg treatment
group (N 28). l-Arg was infused from day 1 to day 3. Results were expressed
as mean SEM. There were no significant differences between the control and
l-Arg groups.
APPENDIX
Abbreviations used in this study are: ADMA, asymmetric N,dimethyl-l-arginine; ARF, acute renal failure; l-Arg, l-arginine; CAT,
cationic amino acid transporter; CIT, cold ischemia time; CsA, cyclosporine A; GFR, glomerular filtration rate; MPO, myeloperoxidase;
l-NNA, N-nitro-l-arginine; NO, nitric oxide (nitrogen monoxide);
NOS, nitric oxide synthase; NT-IR, 3-nitrotyrosine immunoreactivity;
O2, superoxide; ONOO, peroxynitrite; RPF, renal plasma flow; SOD,
superoxide dismutase.
22.
23.
24.
25.
26.
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