Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262
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ABSTRACT |
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Acute renal
failure (ARF) contributes substantially to the high morbidity and
mortality observed during endotoxemia. We hypothesized that selective
blockade of the renal nerves would be protective against ARF during the
early (16 h) stage of endotoxemia [5 mg lipopolysaccharide (LPS)/kg ip
in mice]. At 16 h after LPS, there was no change in mean arterial
pressure, but plasma epinephrine (4,604 ± 719 vs. 490 ± 152 pg/ml, P < 0.001), norepinephrine (2,176 ± 306 vs. 1,224 ± 218 pg/ml, P < 0.05), and plasma renin
activity (40 ± 5 vs. 27 ± 2 ng · ml1 · h
1,
P < 0.05) were higher in the LPS-treated vs. control
mice. The high plasma renin activity level decreased to the control
level with renal denervation in endotoxemic mice. After intravenous injection of phentolamine (200 µg/kg), the decrement in mean arterial pressure was significantly greater in LPS-treated vs. control mice
(19.4 ± 3.5 vs. 8.1 ± 1.5 mmHg, P < 0.01).
Sixteen hours after LPS administration, there were significant
decreases in glomerular filtration rate (52 ± 18 vs. 212 ± 23 µl/min, P < 0.01) and renal blood flow (0.58 ± 0.08 vs. 0.85 ± 0.06 ml/min, P < 0.01) in
sham-operated mice. The decrement in glomerular filtration rate during
endotoxemia was significantly attenuated in mice with denervated
kidneys (32 vs. 79%). Moreover, there was no change in renal blood
flow during endotoxemia in mice with renal denervation. The present
results therefore demonstrate a protective role of renal denervation
during normotensive endotoxemia-related ARF in mice, an effect that may
be, at least in part, due to a diminished activation of the
renin-angiotensin system.
glomerular filtration rate; renal blood flow; epinephrine; norepinephrine; sepsis
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INTRODUCTION |
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SEPSIS IS THE MOST FREQUENT CAUSE of acute renal failure (ARF) in intensive care units (1, 2, 9). When sepsis is associated with ARF, the mortality may be as high as 80%. The pathogenetic factors responsible for sepsis-related ARF, however, are incompletely defined. Although ARF may occur with septic shock, it is also clear that sepsis-related ARF can occur in the absence of hypotension (1, 11).
In recent studies from our laboratory, a mouse model of endotoxemia-related ARF has been studied. A significant decrease in glomerular filtration rate (GFR) and renal blood flow (RBF) occurs in the absence of a fall in blood pressure (7). We hypothesized that endotoxemia may be associated with normal blood pressure because of activation of the sympathetic nervous system and renin-angiotensin system (RAS), which secondarily causes renal vasoconstriction. The roles of renal nerves and the RAS in this early renal vasoconstriction during endotoxemia, however, have not been investigated. The present investigation was therefore undertaken in a normotensive mouse model of endotoxemia-induced ARF to examine the effect of selective renal denervation on this acute renal dysfunction.
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MATERIALS AND METHODS |
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Animals. The experimental protocol was approved by the Animal Ethics Review Committee at the University of Colorado Health Sciences Center. C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor, ME). Male mice aged 8-10 wk were used throughout the study. Mice were maintained on standard rodent chow and had free access to water.
Measurement of plasma epinephrine and norepinephrine levels,
plasma renin activity, and plasma nitric oxide.
Each plasma sample was pooled from three to four mice. Blood samples
were collected on ice in catecholamine tubes containing EGTA/glutathione preservative. Plasma was separated by centrifuging at
4°C and stored at 80°C before measurement. Plasma epinephrine (EPI) and norepinephrine (NE) levels were measured by HPLC using Dionex
instrumentation. The addition of washed alumina (Bio-Rad, Hercules, CA)
to each sample was used for selective adsorption of catecholamines onto
the alumina. An internal standard was prepared by adding appropriate
levels of dihydroxybenzylamine to 0.1 M phosphoric acid. The
sample/dihydroxybenzylamine solution was pH adjusted to 8.6 with 1.5 M
Tris buffer in 2% EDTA. The catecholamines were then selectively
desorbed from the alumina with 0.1 M phosphoric acid. An aliquot of the
sample was injected onto a reverse-phase HPLC column (HP Zorbax
300SB-C18) and eluted with mobile phase (acetonitrile carrier, pH 4.3).
The resulting chromatogram is analyzed by computer integration, and the
determination was on the basis of comparing the detector response (peak
area) generated by an unknown analyte to that for the same analyte
present at a known concentration in a standard solution. Plasma renin
activity (PRA) was measured by the RIA of generated angiotensin I using the GammaCoat [125I]RIA kit (Incstar, Stillwater, MN).
Plasma nitric oxide (NO) levels were determined by measuring plasma
NO2/NO3 levels with a nitrate/nitrite
colorimetric assay kit (Cayman Chemical, Ann Arbor, MI).
Renal denervation. Animals were anesthetized with 60 mg pentobarbital sodium/kg (Abbott Laboratories, North Chicago, IL). Kidneys were exposed by subcostal incision and were dissected free from perirenal tissue. Ten percent phenol in ethanol was applied to both kidney pedicles. In sham-operated mice, normal saline was applied instead of phenol solution. RBF was checked before and after the operation, and denervation was confirmed by a significant increase in RBF. Mice were allowed to recover for 7 days before lipopolysaccharide (LPS) administration.
Measurement of RBF, GFR, and mean arterial pressure.
The animals were anesthetized with 60 mg pentobarbital sodium/kg and
placed on a thermostatically controlled surgical table. A tracheotomy
was performed, at which time a steady stream of 95% O2-5%
CO2 was blown toward the tracheal tube throughout the experiment. Catheters (custom pulled from PE-250) were placed in the
jugular vein for maintenance infusion and in the carotid artery for
blood pressure determinations. Kidneys were exposed by a left subcostal
incision and were dissected free from perirenal tissue, and renal
arteries were isolated for the determination of RBF with a Transonic
Systems blood flow meter and probe (0.5v) as described by Traynor and
Schnermann (18). Mean arterial pressure (MAP) was
constantly measured by means of a carotid artery catheter connected to
a Transpac IV transducer and monitored continuously with Windaq
Waveform recording software (Dataq Instruments). An intravenous
maintenance infusion of 2.25% BSA in normal saline at a rate of 0.25 µl · g1 · min
1 was
started 1 h before experimentation. FITC-inulin (0.75%; Sigma, St
Louis, MO) was added to the infusion solution for the determination of
GFR, as described by Lorenz and Gruenstein (10). A bladder catheter (PE-10) was used to collect urine. Two 30-min collections of
urine were obtained, collected under oil, and weighed for volume determination. Blood for plasma inulin was drawn between urine collections. FITC in plasma and urine samples was measured with a
CytoFluorplate reader (PerSeptive Biosystems, Foster City, CA).
Statistical analysis. Values are expressed as means ± SE. Multiple comparisons were assessed by ANOVA. A P value of <0.05 was considered statistically significant.
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RESULTS |
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Normotensive endotoxemic ARF model in mice. Mice were injected with 5 mg LPS/kg ip (Sigma), a dose that permitted surgery and physiological measurements without an excessive mortality. With this dose of LPS, there was no significant change in MAP [82 ± 0.8 vs. 82 ± 2.2 mmHg, n = 6, P = not significant (NS)], thus allowing measurement of renal function in the absence of hypotension. Sixteen hours after the intraperitoneal injection, there were significant decreases in GFR (52 ± 18 vs. 212 ± 23 µl/min, n = 8, P < 0.01), RBF (0.58 ± 0.08 vs. 0.85 ± 0.06 ml/min, n = 8, P < 0.01), renal plasma flow (RPF; 0.29 ± 0.05 vs. 0.44 ± 0.03 ml/min, n = 8, P < 0.01), and filtration fraction (FF; 0.20 ± 0.05 vs. 0.48 ± 0.05, P < 0.01) compared with sham-operated controls.
PRA, plasma EPI, NE, and NO levels in endotoxemic ARF.
PRA, plasma EPI, NE, and NO levels were measured at 16 h after
intraperitoneal injection of LPS (5 mg/kg). At this time point, PRA was
higher in LPS-treated mice than in controls [40 ± 5 (n = 6) vs. 27 ± 2 ng · ml1 · h
1
(n = 5), P < 0.05; Fig.
1A]. This high PRA level
decreased to the control level with renal denervation in
endotoxemic mice [29 ± 4 (n = 7) vs. 27 ± 2 ng · ml
1 · h
1
(n = 5), P = NS]. There was a large
induction in NO production in the blood with the treatment of LPS in
control mice [227 ± 16 (n = 6) vs. 2.5 ± 0.4 µM (n = 12), P < 0.01]. Plasma
NO levels were significantly less in renal denervated mice compared
with sham-operated mice in endotoxemia [128 ± 21 (n = 9) vs. 227 ± 16 µM (n = 6), P < 0.01]. EPI and NE concentrations were also significantly higher in the LPS-treated than control animals
[4,604 ± 719 (n = 10) vs. 490 ± 152 pg/ml
(n = 9), P < 0.01 (Fig.
1B), and 2,176 ± 306 (n = 10) vs.
1,224 ± 218 pg/ml (n = 9), P < 0.05 (Fig. 1C), respectively].
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Effect of phentolamine on MAP change in endotoxemic mice.
To examine the hemodynamic role of sympathetic vasoconstriction on MAP
at 16 h in LPS (5 mg/kg ip) and vehicle-treated controls with
comparable baseline MAP, the effect of -adrenergic blockade with
phentolamine was studied. A single 200 µg/kg dose of phentolamine (Ben Venue Labs, Bedford, OH) was administered intravenously to the
control and LPS mice. There was a significantly greater decrease in MAP
in the LPS compared with the vehicle-treated mice [19.4 ± 3.5 mmHg (n = 7) vs. 8.1 ± 1.5 mmHg
(n = 7), P < 0.01].
Effect of renal denervation on GFR, RBF, RPF, and FF in endotoxemic
mice.
Renal denervation was confirmed by a significant acute increase in RBF
compared with baseline RBF (1.16 ± 0.05 vs. 0.85 ± 0.06 ml/min, n = 20, P < 0.001). When GFR
and RBF were measured 1 wk after renal denervation, there were no
significant changes compared with sham controls with renal innervation
(GFR: 223 ± 14 vs. 212 ± 23 µl/min, P = NS; and RBF: 0.87 ± 0.01 vs. 0.85 ± 0.06 ml/min,
P = NS). At 16 h after LPS injection (5 mg/kg ip), there were significant decreases in GFR (52 ± 18 vs. 212 ± 23 µl/min, n = 8, P < 0.01) and RBF
(0.58 ± 0.08 vs. 0.85 ± 0.06 ml/min, n = 8, P < 0.01) in sham-operated mice. The results were similar with RPF (0.29 ± 0.04 vs. 0.44 ± 0.03 ml/min,
P < 0.05) and FF (0.20 ± 0.05 vs. 0.48 ± 0.05, P < 0.01) with the treatment of LPS. In mice
with renal denervation, RBF and RPF were not changed during endotoxemic
compared with controls [RBF: 0.81 ± 0.05 vs. 0.87 ± 0.01 ml/min, P = NS (Fig.
2B); and RPF: 0.40 ± 0.02 vs. 0.45 ± 0.01 ml/min, P = NS (Fig.
2C)]. Although there were decreases in GFR and FF in renal
denervated endotoxemic mice compared with denervated controls [GFR:
151 ± 20 vs. 223 ± 14 µm/min, P < 0.05 (Fig. 2A); and FF: 0.38 ± 0.05 vs. 0.49 ± 0.03 ml/min (Fig. 2D)], the decrements were much smaller than
that in sham-operated mice [72 ± 5 (32%) vs. 168 ± 20 µm/min (79%), P < 0.01; and 0.11 ± 0.01 (22%) vs. 0.28 ± 0.02 ml/min (58%), P < 0.01, respectively].
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DISCUSSION |
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Endotoxemia, a major component of sepsis, is associated with an increase in inducible NO synthase (6, 7, 8, 16). In the mouse model used in the present study, a significant rise in plasma NO has been demonstrated at 16 h, and this increase in NO does not occur in inducible NO synthase knockout mice (7). Despite the potent NO-mediated vasodilation, blood pressure remains normal in this endotoxemic model of ARF (7). Thus the role of hypotension can be dissociated from the resultant deterioration of renal function. It is well known that ARF occurs more frequently in patients with septic shock than in those septic patients without a decrease in blood pressure. However, 20% of septic patients develop ARF in the absence of hypotension (1). In the endotoxemic mouse model used in the present study, the ARF develops in the absence of hypotension, leukocyte infiltration, apoptosis, or morphological evidence of coagulation (7).
Although increased vasoconstrictive hormones have been demonstrated to rise in various species, including humans during septic shock, the role of these vasoconstrictors in normotensive endotoxemia in mice has not been studied. In the present study, the plasma concentrations of EPI, NE, and PRA were shown to be increased at 16 h, the time at which endotoxemia-related ARF occurred despite normal blood pressure.
The role of the sympathetic nervous system in maintaining normal
blood pressure in the presence of increased plasma NO was implicated by
comparing the effect of -adrenergic blockade with phentolamine in
control vs. endotoxemic mice. The significantly greater
decrease in blood pressure in the LPS-treated mice with the acute
parenteral administration of phentolamine raised the possibility that
increased renal adrenergic neural activity could be contributing to the
endotoxemia-induced ARF. Such an increase in renal nerve activity might
be particularly important during endotoxemia, a state in which renal
constitutive endothelial NO synthase (14) and cyclic
guanosine monophosphate (6) may be downregulated. In
addition to any effect of adrenergic blockade with phentolamine to
decrease systemic vascular resistance, a decrease in cardiac output may
also occur.
Acute renal denervation was associated with a rise in RBF, indicating baseline renal nerve activity. However, 1 wk after renal denervation, GFR and RBF returned to levels that were not different from sham-operated mice. At 16 h after intraperitoneal injection of LPS, GFR, RBF, RPF, and FF decreased by an average of 79, 32, 34, and 58%, respectively, in mice with intact renal nerves. In contrast, in the LPS-treated mice with denervated kidneys, there were no changes in RBF and RPF and significantly smaller changes in GFR and FF compared with innervated mice.
The interaction of increased renal nerve activity during normotensive
endotoxemia-related ARF with various hormones must be considered. For
example, an interaction between renal adrenergic activity and
angiotensin may occur (3, 19), and the RAS was demonstrated to be activated in our normotensive endotoxemic model of
ARF. The observed lower PRA in the renal denervated mice during endotoxemia implicates a decreased activity of the RAS as a component of the protective effect of renal denervation against endotoxemia. An
interaction with tumor necrosis factor- has also been proposed (12), and the soluble tumor necrosis factor-
receptor
has been shown to offer renal protection in this septic model of ARF
(7). In a dog model of sepsis, the role of renal nerves
was also demonstrated after prostacyclin inhibition (5),
suggesting an interaction between prostaglandins and renal nerve
stimulation. In that regard, it is known that both
angiotensin and increased renal nerve activity stimulate vasodilating
prostaglandins (PGI2 and PGE2) in the kidney (4). Previous studies have shown an upregulation of NO
synthase activity with
-adrenergic activity (15, 17).
In concert with these previous results, in the present study, plasma NO
levels during endotoxemia were lower in mice with renal denervation. Thus the protective effect of renal denervation on renal function in
endotoxemia was unlikely to be due to NO-mediated renal vasodilation.
In conclusion, the present results suggest an important role for increased sympathetic nerve activity in this normotensive endotoxemic mouse model of ARF, in both supporting systemic blood pressure and vasoconstricting the kidney. An early interaction between renal nerves and the RAS during sepsis is demonstrated. Thus early renal events in endotoxemia appear to be vascular and reversible, whereas proinflammatory events may dominate later in the course of sepsis (13).
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ACKNOWLEDGEMENTS |
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This research was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-52599.
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FOOTNOTES |
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Address for reprint requests and other correspondence: R. W. Schrier, Univ. of Colorado Health Sciences Ctr., Box C-178, 4200 East 9th Ave., Denver, CO 80262 (E-mail: robert.schrier{at}uchsc.edu).
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. Section 1734 solely to indicate this fact.
March 12, 2002;10.1152/ajprenal.00270.2001
Received 27 August 2001; accepted in final form 2 March 2002.
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