Antiviral Therapy 8:295-299
Vasodilator agents protect against indinavir
nephrotoxicity
Magali de Araujo1,2 and Antonio Carlos Seguro1*
1
Laboratório de Pesquisa Básica LIM/12, Disciplina de Nefrologia, Faculdade de Medicina USP, São Paulo, Brazil
Universidade Federal de São Paulo (UNIFESP), São Paulo, Brazil
2
*Corresponding author: Tel/Fax: +55 3088 2267; E-mail: [email protected]
Indinavir (IDV) is a protease inhibitor widely used in AIDS
treatment. A sustained elevation of creatinine was identified in IDV-treated patients. We have previously
demonstrated that IDV causes renal vasoconstriction in
rats. The objective of this study was to investigate the
mechanism of IDV-induced vasoconstriction and the
effect that the vasodilator agents L-arginine (LA),
nifedipine (NF), as well as magnesium supplementation
(Mg), have on IDV-induced nephrotoxicity. Male Wistar
rats were kept on fast overnight and given free access to
water. IDV (80 mg/kg BW) and NF (3 mg/kg BW) were
given by gavage for 15 days. LA (1.5%) and MgCl2.6H2O
(1%) were added to drinking water. Six groups were
studied: Control (n=6): normal rats treated with vehicle,
a 0.05 M citric acid solution; IDV (n=7): IDV-treated rats;
IDV+LA (n=6): IDV- and LA-treated rats; IDV+NF (n=7):
IDV- and NF-treated rats; IDV+Mg (n=7): IDV- and
MgCl2-treated rats; IDV+Mg+L-NAME (n=9): IDV- and
MgCl2-treated rats, supplemented with L-NAME (2.5
mg/l in drinking water). Clearance studies and evaluations of urinary nitrite (NO2) excretion were performed
on day 16. No changes in blood pressure were observed.
NO2 excretion decreased in IDV-treated rats. LA and NF
protected against IDV effects, improving GFR (IDV+LA,
1.95 ±0.10; IDV+NF, 1.94 ±0.07 vs IDV, 1.15 ±0.07 ml/min,
P<0.001) and RBF (IDV+LA, 7.83 ±0.09; IDV+NF, 7.63
±0.14 vs IDV, 6.17 ±0.25 ml/min, P<0.001). These results
suggest that IDV-induced vasoconstriction is mediated by
NO and Ca2+ channels. Magnesium also ameliorated GFR
and RBF in IDV-treated rats (GFR IDV+Mg, 1.77 ±0.08
ml/min, P<0.001; RBF IDV+Mg, 7.35 ±0.158 ml/min,
P<0.001). Magnesium protection is not NO-mediated
since it was not blocked by L-NAME. In conclusion, LA, NF
and Mg protect against IDV-induced nephrotoxicity in
rats. This study may have potential clinical implications
for prevention of IDV-induced nephrotoxicity.
Introduction
Indinavir (IDV), a protease inhibitor used in AIDS
treatment, is a well known cause of crystal-induced
acute renal failure, nephrolithiasis, crystalluria and
tubulointerstitial nephritis [1–5].
Boubaker et al. demonstrated that prolonged use of
IDV is associated with increased serum creatinine in
AIDS patients [6]. Van Rossum et al. also showed that
children treated with IDV have a high cumulative incidence of persistent sterile leukocyturia. In the absence
of clinical symptoms of nephrolithiasis, these children
frequently had serum creatinine levels >50% above
normal [7].
More recently, we have demonstrated that IDV
causes renal vasoconstriction and decreases glomerular
filtration rate, suggesting that this haemodynamic
effect may be at least partly responsible for the renal
failure associated with IDV [8].
Several vasodilator agents have been shown to be
protective in both human and animal pathologies in
which renal vasoconstriction causes nephrotoxicity,
such as that associated with the use of cyclosporine or
radiocontrast [9,10].
©2003 International Medical Press 1359-6535/02/$17.00
L-arginine (LA) is a nitric oxide precursor that
improves endothelium-dependent vasodilation [11,12].
Nifedipine (NF), a calcium channel blocker, has been
shown to enhance vasodilation in coronary arteries
with functional and structural alterations, and to
improve endothelial function in cases of essential
hypertension [13,14].
Magnesium (Mg) is a potent vasodilator agent and
smooth muscle relaxant. Magnesium infusions, even
when given at physiological concentrations, produce
vasodilation of systemic vasculature and coronary
arteries [15,16].
The present study was designed to investigate the
effects that the vasodilator agents L-arginine,
nifedipine and magnesium have on IDV-induced
nephrotoxicity.
Materials and methods
For this study, male Wistar rats (200–254 g) provided
by the University of São Paulo Medical School were
kept on standard rat chow (Nuvilab) and tap water
295
M de Araujo & AC Seguro
until the day of the experiment, and maintained on fast
overnight with free access to water prior to IDV
administration. Six groups were studied: 1) Control
(n=6): normal rats that received a 0.5 ml/100 g BW of a
0.05 M citric acid solution, administered by gavage for
15 days; 2) IDV (n=7): rats treated with IDV (80 mg/kg
BW) dissolved in the citric acid solution, administered
by gavage for 15 days; 3) IDV+LA: (n=6): rats treated
with IDV (80 mg/kg BW), administered by gavage
and supplemented with L-arginine (1.5% in the
drinking water) for 15 days; 4) IDV+NF (n=6): rats
treated with IDV (80 mg/kg BW) and with nifedipine
(3 mg/kg BW), both administered by gavage, for 15 days;
5) IDV+Mg (n=7): rats treated with IDV (80 mg/kg BW),
administered by gavage and supplemented with
MgCl2.6H2O (1% in the drinking water) for 15 days;
6) IDV+Mg+L-NAME (n=9): rats treated with IDV
(80 mg/kg BW) by gavage and supplemented with a
solution of N-nitro-L-arginine methylester (L-NAME,
2.5 mg/l) and MgCl2.6H2O (1%) for 15 days.
On day 16, the animals were anaesthetized with
sodium pentobarbital (50 mg/kg BW). The trachea was
cannulated with a PE-240 catheter and the animals
were maintained under spontaneous breathing conditions. The jugular veins were cannulated with a PE-60
catheter for infusion of inulin and fluids. The right
femoral artery was catheterized with a PE-50 catheter
to control mean arterial pressure and to allow blood
sampling. The urinary bladder was cannulated with a
PE-240 catheter by a suprapubic incision in order to
collect urine samples. Measurements of renal blood flow
(RBF) were obtained by means of a median incision. The
left renal pedicle was carefully dissected and the renal
artery was isolated with care to avoid disturbing the
renal nerves. An electromagnetic flow probe (Transonic
Systems, Bethesda, Md., USA) was placed around the
exposed renal artery and RBF was measured with an
eletromagnetic flowmeter (Transonic Systems, T 106
XM). After completion of the surgical procedure, a
loading dose of inulin (100 mg/kg BW diluted in 1 ml of
0.9% saline) was administered through the jugular vein,
followed by a constant infusion of inulin (10 mg/kg BW
in 0.9% saline) at 0.04 ml/min throughout the experiment. Three urine samples were collected at 30 min
intervals. Blood samples were obtained at the beginning
and at the end of the experiment.
Inulin clearance values represent the mean of the
three periods. Blood and urine inulin were determined
by the anthrone method, and sodium and potassium
concentrations were measured by flame photometry
(model 143; Instrumentation laboratory, Lexington,
Mass., USA). Plasma magnesium in groups 1, 2, 5 and
6 was determined using a Labtest diagnostic kit.
Glomerular filtration rate and fractional excretion of
sodium and water were calculated by standard
296
formulas. Renal vascular resistance was calculated by
dividing the value of blood pressure by that of RBF and
was expressed in mmHg/ml/min.
On day 15, rats from both Control and IDV groups
were kept in metabolic cages and urine was collected
over the 24 h period for measurement of nitrite (NO2),
performed by NOA (Nitric Oxide Analyzer) model
280, Sievers Instruments.
Parametric data were evaluated by analysis of variance
(ANOVA) followed by a Student-Newman-Keuls
multiple comparison post-test and the results are reported
as mean ±SEM. Non-parametric data (fractional excretion of water and sodium) were analysed by a
Kruskal-Wallis test followed by a Dunn post-test, and the
results are reported as mean ±SD. NO2 excretion of control
and IDV groups was analysed by non-paired T test.
Statistically significance was established at P<0.05.
Results
Water ingestion was similar among the groups given
adulterated drinking water: IDV+LA, 35.8 ±3.0 ml/day;
IDV+Mg, 32.5 ±3.0 ml/day; IDV+Mg+L-NAME, 39.2
±2.5 ml/day.
The results of clearance studies are shown in Table
1. There were no differences in the body weights and
in the blood pressures of all animals studied. Urine
volume was significantly higher in IDV+NF rats
compared to those in other groups (P<0.001).
As we have shown in our previous study, IDV induced
vasoconstriction in rats, decreasing inulin clearance (GFR),
RBF and increasing renal vascular resistance (RVR) (Table
1). Figure 1 illustrates that urinary excretion of nitrite
(NO2) was significantly reduced in IDV-treated rats
compared to normal rats (0.15 ±0.02 µg/24 h vs 0.94 ±0.31
µg/24 h, P<0.001), suggesting that IDV vasoconstriction
may be mediated by nitric oxide (NO).
L-arginine supplementation protected completely
against nephrotoxic effects of IDV. GFR was significantly higher in the IDV+LA group, compared to IDV
group (1.95 ±0.10 ml/min vs 1.15 ±0.07 ml/min,
P<0.001), with values similar to the control group
(Table 1). The same pattern of protection was observed
in RBF and RVR, with values significantly higher than
the IDV group and similar to the control group.
Rats treated with IDV and NF also had an improvement in renal function, with values of GFR, RBF and
RVR similar to those of normal rats and significantly
higher than IDV-treated rats (Table 1).
Magnesium supplementation also protected rats
against IDV-induced nephrotoxicity. GFR was significantly higher in the IDV+Mg group compared to IDV
group (1.77 ±0.08 ml/min vs 1.15 ±0.07 ml/min,
P<0.001). RBF and RVR also returned to values
similar to those of the control group (Table 1).
©2003 International Medical Press
Vasodilators protect against indinavir nephrotoxicity
Table 1. Renal function and haemodynamic measurements in normal rats (control) and in rats treated 15 days with indinavir
(IDV), indinavir plus L-arginine (IDV+LA), indinavir plus nifedipine (IDV+NF), indinavir plus magnesium supplementation
(IDV+Mg) and indinavir plus magnesium supplementation and L-NAME (IDV+Mg+L-NAME)
Control (n=6)
IDV (n=7)
IDV+LA (n=6)
IDV+NF (n=7)
IDV+Mg (n=7)
IDV+Mg+ L-NAME (n=9)
BW
V
GFR
RBF
BP
RVR
FEH2O
FENa
228 ±13
254 ±11
254 ±7.0
227 ±6.0
250 ±5.0
251 ±7.0
12.9 ±1.8
8.0 ±0.1
9.6 ±0.1
29.2 ±7.0*
11.0 ±1.8
8.7 ±1.4
1.98 ±0.09
1.15 ±0.07*
1.95 ±0.10
1.94 ±0.07
1.77 ±0.08
1.75 ±0.07
7.88 ±0.51
6.17 ±0.25*
7.83 ±0.09
7.63 ±0.14
7.35 ±0.15
7.35 ±0.09
117 ±5.2
119 ±3.9
123 ±2.8
114 ±1.3
122 ±4.7
126 ±2.8
15.1 ±1.13
19.6 ±1.0 †
15.9 ±0.56
14.9 ±0.43
17.3 ±0.57
17.2 ±0.35
0.66 ±0.22
0.71 ±0.11
0.49 ±0.16
1.50 ±0.88
0.60 ±0.19
0.48 ±0.17
0.66 ±0.25
0.61 ±0.22
0.44 ±0.16
1.47 ±0.78
0.52 ±0.19
0.44 ±0.27
Data expressed as mean ±SEM and mean ±SD (FEH2O and FENa). BW, body weight (g); V, urine volume (µl/min); GFR, inulin clearance (ml/min); RBF, renal blood flow
(ml/min); BP, blood pressure (mmHg); RVR, renal vascular resistance (mmHg/ml/min); FEH2O and FENa, fractional excretion of water and sodium (%).
*P<0.001, † P<0.05 vs other groups, by ANOVA analysis.
Plasma magnesium concentrations were not significantly different among the three groups (Control:
1.71 ±0.28 mg/dl; IDV: 1.50 ±0.35 mg/dl; IDV+Mg:
1.51 ±0.14 mg/dl).
In the present study, the administered dose of LNAME (2.5 mg/l in drinking water), which
corresponds to a daily ingestion of 39 mg/kg BW, did
not change the blood pressure of the rats. As demonstrated in Table 1, GFR, RBF and RVR of both
IDV+Mg and IDV+Mg+L-NAME groups were similar,
suggesting that the protective effect of magnesium
supplementation was not mediated by NO. Plasma
magnesium of the IDV+Mg+L-NAME group was on
par with that of the other groups (1.55 ±0.18 mg/dl).
Discussion
In the present study, we investigated the mechanisms
by which IDV causes vasoconstriction in the rat. Also,
we studied protective procedures against IDV-induced
nephrotoxicity, using the vasodilator agents L-arginine,
nifedipine and magnesium.
Nitric oxide (NO) is a gas derived from the amino
acid L-arginine (LA) in the presence of nitric oxide
synthase (NOS). The synthesis of NO by endothelial
cells enables certain activities in neighbouring structures, such as the smooth muscle of the vascular wall,
where it can produce relaxation and, consequently,
vasodilation [11].
NO plays a critical role in multiple renal processes,
including regulation of renal plasma flow (RPF),
glomerular filtration rate (GFR), renin-angiotensin II
generation and sodium excretion [17]. The role of NO in
the regulation of RBF and GFR has been evaluated with
the use of several NOS inhibitors, including NG
monomethyl-L-arginine (L-NMMA), N-nitro-L-arginine
methylester (L-NAME) and N(omega)-nitro-L-arginine
(L-NNA).
In our study, IDV-treated rats had a lower urinary
excretion of the NO metabolite NO2 than did nontreated rats. Oral administration of LA to the
Antiviral Therapy 8:4
IDV-treated rats completely inhibited the haemodynamic effects of IDV. Taken together, these data suggest
that IDV-induced vasoconstriction may be mediated by
NO, directly or indirectly, by inhibition of NO
synthesis or availability. Our results also raise the
possibility of a direct effect of IDV on intrarenal NO
synthesis. More definitive experiments in this regard
should include direct assays of NOS expression levels
in renal tissue or isolated renal vessels exposed to IDV.
Studies have shown that LA administration has
protective effects in nephrotoxic acute renal failure.
Assis et al. showed that oral administration of LA
(1.5% in the drinking water) partially protected against
the fall in the GFR induced in rats by cyclosporine A
[9]. Andrade et al. demonstrated that hypercholesterolaemia aggravates radiocontrast nephrotoxicity, which
is attenuated by L-arginine administration [10].
Additionally, clinical studies have demonstrated that
oral administration of LA improves endothelial dysfunction in patients with essential hypertension and reduces
systemic blood pressure in type 2 diabetes [18,19].
Recent studies suggest a therapeutic potential for the
administration of L-arginine in acute renal failure and
kidney transplantation, at least in patients receiving
kidneys with shorter cold ischaemia time or receiving
kidneys from young patients [20].
Based on this evidence, it seems possible that LA administration can minimize haemodynamic alterations caused
by IDV and, consequently, ameliorate renal function in
AIDS patients to whom this drug is prescribed.
In this study, we have also shown that nifedipine
(NF) completely protected against IDV-induced
nephrotoxicity in rats. Several experimental studies
suggest that the vasodilator effects of the calcium
channel blockers are due, in part, to an endotheliumdependent mechanism. However, it is not clear if
calcium channel blockers increase the liberation of
endothelium-derived relaxing factors, such as NO [21].
Sanchez-Lozada et al. showed that NF prevented
declines in renal function in CsA-treated rats, with no
interference in the expression of NOS isoforms. This
297
M de Araujo & AC Seguro
Figure 1. Urinary nitrite (NO2) excretion in vehicle-treated
rats (Control) and in indinavir-treated rats for 15 days (IDV)
UN02 (µg/24 h)
1.5
1.0
0.5
P<0.001
0.0
Control
(n=6)
IDV
(n=7)
suggests that the changes in the NOS isoform pattern
of expression induced by CsA appear to be, at least in
part, mediated by the haemodynamic alterations
induced by this drug [22].
Travis et al. showed that the vasodilator responses
elicited by systemic injections of NO donors were
markedly attenuated by NF, suggesting that NO
relaxes resistance arteries ‘in vitro’ by inhibition of
Ca2+ channels [23].
Berkels et al. showed that the treatment of endothelial cells with NF significantly increased the NO release
not caused by altered expression of NOS mRNA and
protein, suggesting that there is an enhanced availability
of NO via antioxidative protection [24].
Thus, we could suggest that NF acts through Ca2+
channels to mobilize NO and to attenuate vasoconstriction induced by IDV.
The use of nifedipine in normotensive patients may
produce hypotension. However, our results suggest that
in hypertensive AIDS patients treated with IDV,
nifedipine should be the antihypertensive drug of choice.
In the present study, magnesium supplementation
also attenuated the decrease of the GFR induced by
IDV, by its vasodilator effect.
Magnesium is a predominantly intracellular cation,
involved in the maintenance of the ionic cellular
balance, Na-K-ATPase activity and in the modulation
of several ionic channels. Magnesium is also a calcium
channel blocker. Studies have shown that high Mg
concentrations inhibit Ca2+ flow through both intracellular-extracellular channels and from sarcoplasmic
reticulum [15].
Since it is a vasodilator agent, Mg benefical effects
have been demonstrated in the treatment of asthma,
preeclampsia and eclampsia, cardiac arrhythmias,
acute myocardial infarction and acute cerebral
ischaemia [15].
In the last two decades, it has been shown that
magnesium can modulate agonist actions and hormone
receptor binding on smooth muscle cells, and may be
required for the action of various relaxant vasodilator
298
substances, such as NO [25]. However, studies
concerning the vasodilator action of magnesium and
the mediation of that action, either by NO or by
blockage of Ca2+ channels, remain controversial.
Yang et al. showed that the extracellular magnesium
concentration produces both independent and
endothelium-dependent relaxation of ‘in vitro’ aortal
rings in rats. The primer is responsible for 40% of the
total observed relaxation and is mediated by NO,
whose release is calcium-dependent [25]. On the other
hand, Teragawa et al. showed that intracoronarian
infusion of magnesium caused vasodilation of coronary arteries in humans, but no NO-mediation was
observed [26].
In our study, we used L-NAME to verify if the Mginduced vasodilation was mediated by NO. The
administered doses of 2.5 mg/l (0.4 mg/BW/day) were
the highest doses of L-NAME that did not modify arterial blood pressure of the rats. The fact that L-NAME
did not block the improvement of GFR induced by Mg
supplementation suggests that this effect was not mediated by NO, but possibly due to a direct effect on the
glomerular ultrafiltration coefficient (Kf), which is
regulated by the contractile effect of mesangial cells.
Fandrey et al. investigated the effect of low and high
magnesium concentrations on the contractility of
mesangial cells pre-treated with CsA and observed that
in the absence of Mg, 80% of the cells contracted in
the presence of angiotensin II. On the other hand, there
was a decrease in the contractility in the presence of
Mg, suggesting that normal Mg levels can protect
against the fall of the GFR induced by CsA treatment
[27]. Pere et al. demonstrated that magnesium supplementation has beneficial effects on CsA toxicity in
spontaneously hypertensive rats, protecting against
mesangial thickening, necrosis of the arteriolar wall,
diffuse tubular atrophy and CsA-induced fibrosis [28].
Dietary intake of magnesium is a critical determinant of magnesium levels. Green leafy vegetables are
excellent sources of magnesium, as are unpolished
grains and nuts. The overall intake of magnesium seems
to be declining because of an increase in the use of
processed foods [15].
Magnesium deficiency is common in AIDS patients
due to low intake and to the use of drugs such as
amphotericin B, foscarnet and pentamidine, which
increase urinary excretion of magnesium [29].
Therefore, magnesium-rich diets (or even administration of oral magnesium supplements) should be
prescribed for AIDS patients receiving IDV.
Supplements may be helpful in reducing drug toxicity,
even if there is normal alimentary intake.
In conclusion, we have shown that renal vasoconstriction induced by IDV is probably mediated by NO
and that the vasodilator agents L-arginine, nifedipine and
©2003 International Medical Press
Vasodilators protect against indinavir nephrotoxicity
magnesium protect against IDV-induced nephrotoxicity in
rats. This study may have potential clinical implications
for prevention of IDV-induced nephrotoxicity.
Acknowledgements
This study was funded by the Instituto dos Laboratórios
de Investigação Médica HC/FMUSP/LIM-12, Fundação
Faculdade de Medicina, Fundação de Amparo à Pesquisa
do Estado de São Paulo and by the Universidade Federal
de São Paulo (UNIFESP/EPM). We thank Dr Francisco
RM Laurindo and Dr Laura IV Brandizzi for collaborative work. Portions of this work were presented at the
Annual Meetings of the American Society of Nephrology,
San Francisco, Calif., USA, November 2001 and
Philadelphia, Pa., USA, November 2002.
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Received 15 January 2003; accepted 26 February 2003
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