Multiple organ failure in association with sepsis often leads to considerable morbidity and mortality, which is enhanced in the presence of acute renal failure (1 2,3 4
An overproduction of nitric oxide (NO) has been implicated in the hypotension and circulatory failure associated with septic shock (5,6 7,8 9–11
The pathogenesis of acute renal failure in association with sepsis is as yet incompletely understood, and thus specific therapies are at present lacking. Recent clinical trials have failed to replicate the beneficial effects seen in experimental studies of ischemic renal injury, following interventions including the administration of atrial natriuretic peptide, insulin-like growth factor-1, superoxide dismutase and dopamine (12,13 14 15
MATERIALS AND METHODS
Surgical preparation
All experiments described in this article were performed in adherence to National Institutes of Health guidelines on the use of experimental animals and with the Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act 1986, published by HMSO, London. This study was conducted on 30 male Wistar rats weighing 248–364 g (Tuck, Rayleigh, Essex, UK). Rats were anesthetized with thiopentone sodium (100 mg/kg i.p.) and placed on a thermostatically controlled heating mat (Harvard Apparatus Ltd., Edenbridge, Kent, UK).
Clearance study
Under anesthesia, polyethylene cannulae (PP50, I.D. 0.58 mm, Portex, Hythe, Kent, UK) were placed in the carotid artery and both jugular veins, the bladder was cannulated (PP90, I.D. 0.76 mm), and a tracheostomy was fashioned to maintain airway patency and facilitate spontaneous respiration. The carotid arterial cannula was connected via a pressure transducer (Senso-Nor 840, Senso-Nor, Horten, Norway) to a data acquisition system (MacLab 8e, ADInstruments, Hastings, UK) installed on an Apple Macintosh computer (Apple Computer Inc., Cupertino, CA), thus permitting measurement of phasic and mean arterial pressure (MAP) and derivation of heart rate (HR) from the pulse waveform. At the end of the surgical preparation, a bolus of 3 H-inulin (5 μCi in NaCl 0.9% 0.5 mL/kg i.v.) was administered, followed by a constant infusion at a rate of 1.5 μCi/h in NaCl 0.9% at 1.5 mL/h. Rats also received a co-infusion of NaCl 0.9% at 1.5 mL/h; thus, the total volume of fluid administered was 3 mL/h. Following 1 hour's equilibration after surgical preparation, urine was collected continuously at 30- to 60-min intervals from the bladder cannula, and plasma samples were obtained at the midpoint of each clearance period. The activity of 3 H-inulin in plasma and urine (from both kidneys) was measured in a scintillation counter (Beckman Instrumentation, Fullerton, CA), and inulin clearance was calculated by using standard formulae.
Effect of carboxy-PTIO on LPS-induced alterations in systemic hemodynamics and renal function
Following equilibration, baseline hemodynamics, namely MAP and HR, were recorded. Urine was collected over 2 sequential 30-min periods with midpoint plasma samples to measure urine flow rate and calculate inulin clearance. Thereafter, lipopolysaccharide (LPS, 6 mg/kg in 0.45 mL NaCl 0.9%) was infused over 30 min. Hemodynamic parameters and inulin clearance were then recorded hourly for a further 6 h following the induction of endotoxemia. To address whether NO scavenging was efficacious when introduced after the induction of endotoxemia, carboxy-PTIO (10 mg/kg in NaCl 0.9% 0.45 mL, n = 7) or vehicle (n = 7) was administered over 60 min to LPS-treated rats commencing 30 min after the completion of the LPS infusion. Sham operated rats (n = 7) underwent the same surgical preparation, but they received vehicle instead of LPS or carboxy-PTIO. At the end of the experimental period, blood was sampled from the carotid arterial cannula for further biochemical analysis, and a lethal dose of thiopentone sodium was administered.
Systemic and intrarenal hemodynamics study
Under anesthesia, cannulae were placed in the carotid artery, jugular veins, and bladder as described above, and a tracheostomy was fashioned. Through a flank incision, the left kidney was mobilized and supported in a Perspex cup to eliminate respiratory movement. The left ureter was cannulated (PP25, I.D. 0.40 mm), tied, and divided distally. An ultrasonic flow probe was placed around the left renal artery and connected to a Doppler ultrasound flowmeter (T206, Transonic, Ithaca, NY). A surface laser Doppler probe (PF318, Perimed UK Ltd., Bury St. Edmunds, UK) was placed directly over the renal cortex, and a needle laser Doppler probe with an outside diameter of 0.45 mm (PF 302) was inserted through the cortex into the renal medulla to a depth of 3.0–4.0 mm from the renal surface. Both probes were connected to laser Doppler flowmeters (PF3, Perimed UK Ltd, Bury St. Edmunds, UK). MAP and HR were recorded as described above, and line outputs from the ultrasound and laser Doppler flowmeters were also connected to the MacLab. A constant infusion of NaCl 0.9% at 3 mL/h was commenced post-surgery, and a 60-min period for equilibration elapsed prior to the start of the experimental protocol.
Effect of co-administration of carboxy-PTIO on LPS-induced alterations in systemic and intrarenal hemodynamics
Following equilibration, baseline values for MAP, HR, urine flow, renal blood flow (RBF), and cortical and medullary laser Doppler flux were measured over 2 sequential 30-min periods. Thereafter, LPS 6 mg/kg was infused over 30 min. In previous studies we demonstrated an early corticomedullary redistribution of perfusion, occurring within the first 60 min of endotoxemia (14
At the end of the study, animals were killed by an overdose of thiopentone sodium, whereupon background flux values for cortex and medulla were measured, and then subtracted from previous recordings to eliminate nonspecific effects of laser reflection. The left kidney was then dissected, and the medullary position of the needle probe was verified by inspection.
Measurement of plasma nitrite and nitrate-nitrate reductase assay
Blood was collected into heparinized capillary tubes and centrifuged (6000 g for 5 min) to separate cells and plasma. Then, the nitrate in the plasma sample was enzymatically converted to nitrite according to the method of Schmidt et al. (16 17 550 ) was measured by using a microplate reader (Molecular Devices, Richmond, CA). Total nitrite-nitrate concentrations were calculated by comparison with OD550 of a standard solution of sodium nitrate (also stoichiometrically converted to nitrite) prepared in saline.
Materials
3 H-inulin was obtained from Amersham Life Sciences (Little Chalfont, Bucks, UK), and thiopentone sodium was obtained from Rhône Mérieux (Harlow, Essex, UK). Carboxy-PTIO was obtained from Alexis Biochemicals (Nottingham, UK). Nitrate reductase (from Aspergillus species ) and lactate dehydrogenase (from rabbit muscle) were obtained from Boehringer-Manheim (Nottingham, UK). Lipopolysaccharide (Escherichia Coli serotype 0127:B8) and all other chemicals or materials were from Sigma Chemical Company (Poole, Dorset, UK). All drugs were dissolved in nonpyrogenic saline (NaCl 0.9%, Baxter Healthcare Ltd., Thetford, Norfolk, UK).
Statistical analysis
All values in the figures and text are expressed as means ± SEM of n observations, where n is the number of rats in each experimental group. The data were analyzed by using factorial or repeated measures ANOVA, and a P value of <0.05 was considered to be statistically significant.
RESULTS
Effect of carboxy-PTIO on renal function following induction of endotoxemia
Infusion of LPS led to a prompt reduction in Cin (to 54% and 40% of baseline values) at 60 min, which remained reduced for 6 h in saline-treated rats. Administration of carboxy-PTIO commencing 60 min after LPS led to a prompt and sustained increase in Cin over 360 min post-LPS (Fig. 1 ). This improvement in renal function was supported by measurement of plasma urea and creatinine, which rose to 13.4 ± 1.6 mmol/L and 58.3 ± 8.4 μmol/L, respectively, 6 h after LPS (P < 0.05), compared to 2.5 ± 0.1 mmol/L and 38 ± 1.1 μmol/L in sham-operated rats. Administration of carboxy-PTIO attenuated the rise in urea and prevented the rise in creatinine (P < 0.05, Table 1 ). Plasma phosphate rose in the LPS group, and again this rise was prevented in the carboxy-PTIO group, whereas carboxy-PTIO had no effect on the fall in plasma albumin. Serum concentrations of nitrite-nitrate rose progressively between 180 and 360 min following induction of endotoxemia, and this rise was not affected by administration of carboxy-PTIO (Table 2 ).
Table 1: Plasma levels of urea, creatinine, phosphate, and albumin sampled 6 h after the induction of endotoxemia
Table 2: Serial values for nitrite-nitrate sampled at 0, 180, and 360 min after the induction of endotoxemia
Fig. 1: Serial values for inulin clearance (Cin ) after infusion of LPS 6 mg/kg i.v. over 30 min commencing at 0 min (hatched columns). Sham operated rats received vehicle (saline) instead of LPS (open columns). Carboxy-PTIO (CPTIO, 10 mg/kg i.v.) was infused over 60 min commencing 60 min after the LPS infusion (filled columns). Endotoxaemia led to a prompt and sustained reduction in Cin , which was completely reversed by the NO scavenger carboxy-PTIO. * P < 0.05 when compared by ANOVA to baseline values (−30 min); † P < 0.05 when compared to rats that received LPS alone; ‡ P < 0.01 when compared to rats that received LPS alone.
Effect of co-administration of carboxy-PTIO on LPS-induced alterations in systemic and intrarenal hemodynamics
Infusion of LPS led to a prompt but transient fall in MAP from 113 ± 5 to 81 ± 8 mmHg, recovering by 60 min after the start of the infusion. HR rose from 334 ± 11 to 390 ± 7 bpm within 60 min and remained elevated above baseline for the duration of the study period. There were only minor fluctuations in renal blood flow from the baseline value of 10.7 ± 0.5 mL/min (Fig. 2 ). Cortical flux fell by 28 ± 9% (P < 0.05 vs. baseline) at 30 min and remained depressed for 180 min. In contrast, medullary flux rose by 71 ± 11% (P < 0.001 vs. baseline) at 30 min, thereafter progressively falling to baseline by 180 min (Fig. 3 ).
Fig. 2: Serial changes in (A) mean arterial pressure and (B) renal blood flow in rats receiving LPS 6 mg/kg over 30 min commencing at 0 min (open squares, solid line). The NO scavenger carboxy-PTIO (CPTIO, 10 mg/kg/h, filled triangles, dashed line) was co-infused over 60 min commencing at 0 min. No significant differences between the two groups were observed.
Fig. 3: Serial changes in (A) cortical and (B) medullary laser Doppler flux in rats receiving LPS 6 mg/kg over 30 min commencing at 0 min (open squares, solid line). Endotoxaemia led to a prompt and sustained reduction in cortical flux, and a transient increase in medullary flux, returning to baseline by 150 min post-LPS. Co-infusion of the NO scavenger carboxy-PTIO 10 mg/kg over 60 min commencing at 0 min (A) did not affect the changes in cortical flux, but (B) completely prevented the increase in medullary flux (filled triangles, dashed line). * P < 0.05 compared by ANOVA to baseline values; † P < 0.05 compared by ANOVA to LPS alone; ‡ P < 0.01 compared by ANOVA to LPS alone.
Co-administration of carboxy-PTIO had no effect on the changes in MAP, HR, and RBF following LPS infusion (P > 0.05;Fig. 2 ). The reduction in cortical flux was modestly accentuated in the carboxy-PTIO-treated group compared with those receiving LPS alone, whereas co-administration of carboxy-PTIO completely prevented the increase in medullary flux observed following LPS infusion (Fig. 3 ).
DISCUSSION
Imidazolineoxyl-N-oxides , such as 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) and its water soluble derivative carboxy-PTIO, are stable radicals that oxidize NO to generate nitrogen dioxide, NO2 , and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl (PTI) (18,19 2 may either react further with NO to form N2 O3 , which reacts rapidly with water to yield NO2 − , or NO2 may dimerize to form N2 O4 , which spontaneously dismutates to yield NO2 − and NO3 − . Hence, nitrite and nitrate levels are not a useful index to assess the degree of NO scavenging by carboxy-PTIO; moreover, the compound interferes with the Griess reaction (20 G -monomethyl-L-arginine (L-NMMA), implying that the enhanced conversion reflects scavenging of excess NO produced during endotoxemia (21
It has long been recognized that fundamental differences in structure and function exist between the cortical and medullary vasculature (22 23 imidazolineoxyl-N-oxides for NO (18 24,25
Although this study suggests a significant role for NO in the regulation of intrarenal hemodynamics, it is likely that changes in other vasoactive mediators also contribute, including endothelin and prostanoids. Plasma levels of endothelin are elevated in sepsis and correlate with mortality (26 27 28 29 30,31 1 receptor activation and post-glomerular vasodilation via A2 receptors. Inhibition of adenosine has been shown to have beneficial effects in models of acute renal failure, including radiocontrast nephropathy and glycerol-induced rhabdomyolysis, and theophylline, a competitive non-selective adenosine antagonist, ameliorated renal dysfunction following endotoxemia (32
Complete occlusion of one or both renal arteries has commonly been used to induce experimental acute renal failure, and many important insights into the mechanisms underlying acute renal failure have been gained by using this model. However, in clinical practice, acute, complete, and reversible renal artery occlusion only occurs in the setting of vascular surgery, urological surgery, and renal transplantation, and the relevance of the latter setting is further confounded by additional factors such as cryopreservation and immunological injury. More commonly, acute renal failure develops as a result of a sustained reduction in perfusion pressure in the setting of systemic hypotension and often in the presence of sepsis. Furthermore, the injury sustained by the kidney at risk is often exacerbated by the administration of nephrotoxic drugs such as aminoglycosides, or non-steroidal anti-inflammatory drugs. Focal alterations in perfusion may thus play an important role in determining renal function, and models of acute renal failure comprising hypotension and/or multiple insults may approximate the clinical syndrome more closely (33,34
In ischemic acute renal failure, the obstruction of renal tubules by casts and cellular debris has been shown to contribute to the injury (35
Clinical experience with vasopressors, such as noradrenaline and dopamine, has demonstrated the ease with which excessive vasoconstriction can worsen the outcome, and invasive hemodynamic monitoring techniques are needed to guide accurate dosing. Our experience with anti-NO interventions is much more limited; thus, it may be that more precise physiological targets are needed to better tailor the judicious use of all vasoactive drugs, including strategies to attenuate the overproduction of NO that occurs in sepsis and the systemic inflammatory response syndrome. The results of this study suggest that, in the acute renal dysfunction following the infusion of lipopolysaccharide, an appreciation of the intrarenal hemodynamic alterations following the induction of endotoxemia provides an important target for manipulation and to guide the development of new therapeutic approaches.
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