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Protective effect of renal denervation on normotensive endotoxemia-induced acute renal failure in mice

Wei Wang, Sandor A. Falk, Suparoek Jittikanont, Patricia E. Gengaro, Charles L. Edelstein, and Robert W. Schrier

Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 · ml-1 · 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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 · g-1 · 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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 · ml-1 · 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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Fig. 1.   Sixteen hours after lipopolysaccharide (LPS; 5 mg/kg) or vehicle injection (ip), plasma renin activity (PRA; A) was measured by RIA, and plasma epinephrine (EPI; B) and norepinephrine (NE; C) levels were measured by HPLC. Values are means ± SE.

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 alpha -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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Fig. 2.   LPS (5 mg/kg) was injected (ip) in sham-operated or bilateral renal-denervated mice. Glomerular filtration rate (GFR; A), renal blood flow (RBF; B), renal plasma flow (RPF; C), and filtration fraction (FF; D) were all measured at 16 h after LPS in sham-operated and denervated mice. GFR and RBF were measured by inulin clearance and blood flow meter, respectively. Values are means ± SE.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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-alpha has also been proposed (12), and the soluble tumor necrosis factor-alpha 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 alpha -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).


    ACKNOWLEDGEMENTS

This research was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-52599.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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