ORIGINAL ARTICLE
Renal haemodynamic response to amino acids infusion in an experimental porcine model of septic shock O. Vassal1,2, J.-M. Bonnet3, A. Barthelemy3, B. Allaouchiche1,2, I. Goy-Thollot3, V. Louzier3, C. Paquet3, J.-Y. Ayoub3, O. Dauwalder4, M. Jacquet-Lagrèze1,2 and S. Junot3 1
Service d’Anesthésie–Réanimation, Hospices Civils de Lyon, Hôpital Edouard-Herriot, Lyon, France Université Claude-Bernard, Lyon, France 3 Université de Lyon, EA 4174 Hémostase Inflammation Sepsis, VetAgro Sup – Campus Vétérinaire de Lyon, Marcy l’Etoile, France 4 Université de Lyon, Laboratory of Microbiology, Groupement Hospitalier Est, Lyon, France 2
Correspondence S. Junot, Université de Lyon, EA 4174 Hémostase Inflammation Sepsis, VetAgro Sup – Campus Vétérinaire de Lyon, F-69280, Marcy l’Etoile, France E-mail: [email protected] Conflict of interest The authors declare that they have no competing interests. Funding This study was supported by an unrestricted operating grant from the Scientific Council of Vetagro Sup, an internal grant from Agriculture Ministry (EA 4174) and an internal grant from Hospices Civiles de Lyon. Submitted 31 January 2015; accepted 3 February 2015; submission 15 July 2014. Citation Vassal O, Bonnet J-M, Barthelemy A, Allaouchiche B, Goy-Thollot I, Louzier V, Paquet C, Ayoub J-Y, Dauwalder O, Jacquet-Lagrèze M, Junot S. Renal haemodynamic response to amino acids infusion in an experimental porcine model of septic shock. Acta Anaesthesiologica Scandinavica 2015
Background: Acute kidney injury (AKI) is common in sepsis. Treatments allowing maintenance of renal blood flow (RBF) could help to prevent AKI associated with renal hypoperfusion. Amino acids (AA) have been associated with an increase of RBF and glomerular filtration rate (GFR) in several species. The aim of this study was to evaluate the effects of an AA infusion on RBF and GFR in a porcine model of septic shock. Methods: A total of 17 piglets were randomly assigned into three groups: Sham (Sham, n = 5), sepsis without AA (S-NAA, n = 6), sepsis treated with AA (S-AA, n = 6). Piglets preparation included the placement of ultrasonic transit time flow probes around left renal artery for continuous RBF measurement; ureteral catheters for GFR and urine output evaluation; pulmonary artery catheter for cardiac output (CO) and pulmonary arterial pressure measurements. Mean arterial pressure (MAP) and renal vascular resistance (RVR) were also determined. Septic shock was induced with a live Pseudomonas aeruginosa infusion. Crystalloids, colloids and epinephrine infusion were used to maintain and restore MAP > 60 mmHg and CO > 80% from baseline. Results: Renal haemodynamic did not change significantly in the Sham group, whereas RBF increased slightly in the S-NAA group. Conversely, a significant increase in RVR and a decrease in RBF and GFR were observed in the S-AA group. AA infusion was associated with a higher requirement of epinephrine [340.0 (141.2; 542.5) mg vs. 32.5 (3.8; 65.0) mg in the S-NAA group P = 0.044]. Conclusion: An infusion of amino acids impaired renal haemodynamics in this experimental model of septic shock.
doi: 10.1111/aas.12507
Editorial comment: what this article tells us An amino acid infusion was associated with an impairment of renal haemodynamics in a septic porcine model. The results do not support the administration of amino acid in the early phase of septic shock.
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Acute kidney injury (AKI) is a common complication of sepsis associated with an increased morbidity and mortality, which has been reported in 40–50% of patients with severe sepsis.1–3 Recently, AKI has been confirmed as a powerful predictor of hospital mortality in severe sepsis and septic shock.4 The pathophysiology of AKI during sepsis remains a matter of debate,5 with both haemodynamic and inflammatory disorders contributing to the development of kidney functional impairment.6,7 Renal hypoperfusion and increased renal vascular resistance (RVR) during haemodynamic failure in severe sepsis have been suggested as central mechanisms for the development of septic AKI.8–10 Prevention of sepsis-induced AKI is complicated, but maintenance of renal blood flow (RBF) remains recommended as a protective strategy. Pharmacologic anti-inflammatory treatments have not shown any significant efficacy;11,12 macrocirculation support such as fluids, vasopressors or diuretics have failed to prevent the occurrence of the disease.10,12 High doses of catecholamines may cause excessive renal vasoconstriction, and thus precipitating the occurrence of renal failure.12 A treatment allowing renal vasodilation without any systemic effect could be promising, particularly in conditions of haemodynamic alterations associated with vasopressors requirement. Amino acids (AA) infusions have been associated in several species with an increase of RBF and glomerular filtration rate (GFR) without any systemic arterial pressure variation.13–16 These renal effects of AA could be used as a means of maintaining renal perfusion in sepsis condition associated with increased RVR. The aim of this study was to evaluate the effects of an AA infusion on renal haemodynamics and function in a porcine model of septic shock. Our hypothesis was that the administration of AA would prevent a decrease in RBF or an increase in renal resistance during sepsis.
Animals A total of 18 Landrace female piglets (2.5–3.5 month old) weighing between 20 and 25 kg were fasted for 12 h prior the experiment, with free access to water. Animals were pre-medicated with an intramuscular 1 : 1 mixture of tiletamine and zolazepam (3 and 3 mg/kg respectively). An intravenous catheter was then inserted in the auricular vein for administration of buprenorphine (0.02 mg/kg) and infusion of Ringer’s lactate solution at a rate of 10 ml/kg/h to replace fluid losses. Piglets were orally intubated after induction of general anaesthesia with intravenous injection of propofol (3 mg/kg). Anaesthesia was maintained by inhalation of isoflurane in 100% oxygen. Animals were ventilated with a volumecontrolled ventilator (Alpha100, Minerve, Esternay, France) with a tidal volume of 8 ml/kg. Body temperature was maintained above 37°C by using an electric warming blanket.
Surgical preparation The left and right internal jugular veins were isolated and catheterised. The left jugular catheter was devoted to fluid and drug administration. A 7CH pulmonary artery catheter (Edwards Lifesciences, Guyancourt, France) was inserted through the right jugular vein into the pulmonary artery for measurement of pulmonary artery pressure (PAP), pulmonary capillary wedge pressure (PCWP) and cardiac output (CO). A catheter was placed in the left carotid artery to allow arterial pressure measurement and blood sampling. Following a paravertebral laparotomy, a 4-mm ultrasonic Doppler flow probe (Transonic System, Ithaca, NY, USA) was positioned around the left renal artery for continuous measurement of RBF. A midline laparotomy was then performed. The bladder was emptied and the ureters were insulated and cannulated for continuous collection of urine.
Methods
Study design
This study was approved by the Animal Care Committee of VetAgro Sup, Marcy l’Etoile, France (n°1180) in order to satisfy the criteria of European regulation (Directive EU 86/609).
Animals were randomly allocated into three groups (Fig. 1): Sham (Sham, n = 6), sepsis without AA (S-NAA, n = 6) or sepsis treated with AA (S-AA, n = 6) (Fig. 1).
Acta Anaesthesiologica Scandinavica 59 (2015) 598–608 ª 2015 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd
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Fig. 1. Experimental design. Sham group (n = 5): non-septic piglet receiving no amino acids, S-NAA group (n = 6): septic piglets receiving no amino acids, S-AA group (n = 6): septic piglets receiving amino acids (P. aeruginosa: Pseudomonas aeurigona). AA, amino acids.
Following surgical preparation, piglets were stabilised for 30 min. Haemodynamic, respiratory and renal parameters were recorded thereafter every 20 min before treatment (T20 and T0) and 160 min after treatment, divided into 8 20-min periods (T20–T160). After baseline measurements (T20 and T0), septic shock was induced (S-NAA and S-AA groups) with an intravenous infusion of Pseudomonas aeruginosa (5.108 UFC/ml – 0.3 ml/20 kg/min). The P. aeruginosa strain (ATCC 27853) used in this study was obtained from a patient at the hospital with a standardised method in order to avoid variations of virulence.17 The infusion was stopped when the systolic PAP reached 45 mmHg to avoid a possible right heart failure. Animals in the Sham group received Ringer’s lactate alone. Forty minutes after the beginning of P. aeruginosa infusion (end of T40), a continuous infusion of AA (Vintene®, Baxter, Maurepas, France) was administered at a rate of 6 mg/kg/ min (0.05 ml/kg/min) and maintained until the end of experiment. Piglets were resuscitated by standardised combination of fluids and inotropes (Fig. 2): crystalloid boli (NaCl 0.9%) (two boli of 10 ml/kg) were first administered in case of hypotension (MAP < 60 mmHg), then colloids boli (6% hydroxyethyl starch (130/0.4), (two boli of 10 ml/
kg) if hypotension persisted or recurred and finally epinephrine infusion starting at 0.02 mg/kg/min and given to effect in case of fluid unresponsiveness. These treatments were administered in order to maintain and restore mean arterial pressure (MAP) > 60 mmHg and CO > 80% of their baseline values. Concomitantly, Ringer’s lactate infusion rate was reduced by 3 ml/kg/h so that every pig received the same volume expansion. At the end of experiment, animals were euthanised with a solution of pentobarbital.
Monitoring Heart rate, electrocardiogram and pulse oximetry were continuously monitored. Every 20 min, temperature, urinary output (UO), PCWP and CO (by thermodilution) were measured. Mean, diastolic and systolic arterial pressure as well as PAP and RBF were continuously measured and processed using an acquisition software (AcqKnowledge version 3.7.3 for Windows 95/98, Biopac Systems Inc, Goleta, CA , USA). From these records, values were averaged for each 20-min period of the experimental procedure. Systemic vascular resistances (SVR) were calculated as followed: MAP × 80/CO. RVR was calculated according to Acta Anaesthesiologica Scandinavica 59 (2015) 598–608
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If MAP < 60 mmHg CO < 80% baseline
1st and 2nd bolus Ringer’s Lactate
Stop resuscitation
If MAP > 60 mmHg CO > 80% baseline
If MAP < 60 mmHg CO < 80% baseline After 2nd bolus Fig. 2. Resuscitation protocol. Piglets were resuscitated following a standardised algorithm: crystalloid boli (two boli of 10 ml/kg) were first administered in case of hypotension or decreased cardiac output (CO) [mean arterial pressure (MAP) < 60 mmHg, CO < 80% of its initial value], then colloids boli (two boli of 10 ml/kg) if hypotension or decreased CO persisted or recurred; finally epinephrine infusion starting at 0.02 mg/kg/min and given to effect in case of fluid unresponsiveness. HES, hydroxyethyl starch.
1st and 2nd bolus of HES
If MAP > 60 mmHg CO > 80% baseline
If MAP < 60 mmHg CO < 80% baseline After 2nd bolus Epinephrine infusion
the following formula: MAP/RBF. GFR was estimated by endogenous creatinine clearance over a 20-min period. Sampling Blood samples were drawn every 20 min for biochemical analyses including: arterial blood gas (included pH, pCO2, pO2 and bicarbonate), glucose, lactate, total protein, serum osmolality, creatinine, electrolytes, glucagone and insulin. Urine samples were also collected every 20 min for urine sodium and creatinine measurements. Statistical analysis All analyses were two-sided with a threshold of 0.05 using a statistical software program (GraphPad Prism, v6; GraphPad Software, San Diego, CA, USA). Normality of distribution was assessed using an Agostino–Pearson test. As most haemodynamic and renal variables were not normally distributed, data are presented as median ± interquartile range (IQR). Incomplete data or values
If MAP > 60 mmHg CO > 80% baseline
outside the 95% confidence interval were excluded from statistical analyses. Differences within each group before and after induction of sepsis were tested using a Friedman analysis of variance on ranks and, subsequently, a Dunn’s multiple comparisons post hoc test. A Kruskal– Wallis rank sum test was performed to compare data between treatment groups with a Dunn’s multiple comparisons post hoc test. For comparison of the amount of epinephrine and fluids administered between groups, unpaired t-tests were used. Sample size was estimated according to preliminary data; we hypothesised a difference of 50% of primary outcome (RBF) with alpha = 0.05 and beta = 0.20, which gave a sample size of six animals per group.
Results One piglet of the NS-NAA group was withdrawn due to technical defect of the RBF probe. No animals died during the experiment. Results are summarised in Table 1 for haemodynamic data and Table 2 for biochemical data.
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Table 1 Main haemodynamic variables.
Parameter MAP (mmHg)
Cardiac output (l/min)
Heart rate (bpm)
SVR (dyn • s/cm5)
PAP (mmHg)
PCWP (mmHg)
Urine output (ml/min)
GFR (ml/min)
RVR (mmHg • min/ml)
RBF (ml/min)
Sham S-NAA S-AA Sham S-NAA S-AA Sham S-NAA S-AA Sham S-NAA S-AA Sham S-NAA S-AA Sham S-NAA S-AA Sham S-NAA S-AA Sham S-NAA S-AA Sham S-NAA S-AA Sham S-NAA S-AA
Baseline
Bacterial infusion
Amino acids
End of experiment
T0
T40
T80
T120
T160
63 (55;73) 64 (62;67) 73 (64;80) 1.9 (1.6;2.1) 2.1 (1.8;2.3) 2.1 (1.7;2.2) 92 (83;101) 88 (83;90) 77 (73;98) 3200 (2360;3404) 2527 (2110;3331) 2528 (2253;2851) 16 (16;20) 21 (15;26) 19 (14;22) 14 (10;14) 10 (8;16) 15 (14;15) 0.77 (0.49;2.11) 0.65 (0.20;1.14) 0.82 (0.48;1.18) 67.9 (54.4;72.2) 63.6 (24.1;73.0) 54.9 (40.7;63.1) 0.35 (0.31;0.37) 0.35 (0.34;0.49) 0.34 (0.31;0.44) 193 (175;195) 189 (172;205) 208 (184;210)
63 (55;76) 64 (57;71) 64 (59;66) 2.0 (1.7;2.5) 1.7 (1.2;2.4) 1.7 (1.4;2.0) 93 (85;100) 107 (92;118) 121 (97;136) 3047 (2283;3144) 2932 (1976;4143) 4043 (3152;4095) 20 (17;23) 36 (28;40) 30 (27;34) 13 (10;14) 13 (11;16) 19 (13;20) 1.52 (0.76;3.74) 0.45 (0.22;0.79) 0.66 (0.50;1.24) 52.3 (33.5;76.1) 44.6 (30.5;61.9) 39.2 (27.2;43.1) 0.42 (0.34;0.49) 0.34 (0.31;0.49) 0.45 (0.43;0.52) 179 (168;189) 166 (146;203) 135 (124;179)*
71 (66;79) 66 (62;70) 64 (58;69) 2.2 (2.1;2.5) 2.3 (2.1;3.1) 1.5 (1.0;1.9) 96 (92;99) 88 (83;102) 109 (78;112) 2578 (2514;2998) 2345 (1636;2606) 4601 (4028;4790) 20 (18;25) 30 (29;33) 31 (27;34) 13 (9;14) 17 (9;19) 14 (12;14) 1.47 (0.85;3.31) 2.09 (1.12;3.42) 1.66 (0.21;3.68) 70.7 (58.3;78.8) 48.4 (44.4;57.5) 39.1 (7.2;55.6) 0.42 (0.38;0.52) 0.32 (0.27;0.35) 0.46 (0.40;0.50) 171 (169;173) 215 (214;218) 131 (117;157)
68 (61;80) 53 (50;56) 73 (62;76) 2.3 (2.0;2.3) 2.0 (1.8;2.4) 2.4 (1.9;2.7) 107 (101;117) 112 (102;115) 97 (87;100) 2242 (2109;2862) 2027 (1684;2485) 2287 (1763;3215) 20 (16;25) 26 (23;28) 30 (23;33) 13 (10;14) 13 (9;17) 10 (9;12) 1.41 (0.71;2.44) 0.23 (0.05;1.51) 1.08 (0.46;1.48) 58.4 (35.9;74.5) 10.4 (1.1;48.3) 39.3 (12.8;49.6) 0.38 (0.34;0.47) 0.26 (0.23;0.30) 0.38 (0.29;0.49) 176 (170;177) 212 (204;240) 210 (178;233)
65 (58;78) 54 (50;61) 66 (58;71) 2.2 (2.1;2.6) 2.2 (2.0;2.3) 1.7 (1.3;2.0) 109 (102;128) 123 (108;138) 93 (82;98) 2365 (2135;2702) 1993 (1918;2686) 2469 (2279;3161) 19 (15;27) 25 (24;30) 25 (23;26) 13 (10;13) 13 (8;20) 12 (8;18) 0.96 (0.59;2.24) 0.14 (0.02;0.86) 1.34 (0.34;2.77) 52.7 (37.4;76.6) 12.0 (1.7;31.1) 17.9 (10.5;40.8) 0.40 (0.33;0.49) 0.28 (0.22;0.29) 0.36 (0.27;0.46) 172 (170;179) 226 (191;256)* 156 (153;198)
Sham group: non-septic piglet receiving no amino acids, S-NAA group: septic piglets receiving no amino acids, S-AA group: septic piglets receiving amino acids. *Significant intragroup difference (P < 0.05) in comparison with baseline values. GFR, glomerular filtration rate; MAP, mean arterial pressure; PAP, pulmonary artery pressure; PCWP, pulmonary capillary wedge pressure; RBF, renal blood flow; RVR, renal vascular resistance; SVR, systemic vascular resistances.
Effect of bacterial infusion Fluid loading and epinephrine requirements during the experiment are shown in Fig. 3. A significant decrease in pH and increase in blood lactate was observed in both septic groups (P = 0.04 for S-NAA group, P = 0,001 for S-AA group). Following bacterial infusion, PAP increased significantly over time reaching a maximal value at T60: 40 (33; 42) mmHg in S-NAA group (P < 0,001) and 43 (28; 46) in S-AA group (P < 0.001). In comparison, no significant variation of PAP was observed in Sham group.
Amines and colloid requirements were significantly higher in septic groups in comparison with non-septic group (P = 0.04) (Fig. 3).
Effects of amino acids infusion on haemodynamic parameters Except PAP, which increased significantly following bacterial infusion, no significant variation was found over time in Sham, S-NAA and S-AA groups for CO, MAP, SVR, PCWP. There was no significant difference between groups. Acta Anaesthesiologica Scandinavica 59 (2015) 598–608
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Table 2 Main biochemical variables.
Parameters Lactate (mmol/l)
PaO2 (kPa)
PaCO2 (kPa)
pH
Bicarbonate (mmol/l)
Serum osmolality (mOsm/kg) Creatininemia (mmol/l)
Natremia (mmol/l)
Glycaemia (g/l)
Insulinemia (mU/l)
Glucagonemia (pg/ml)
Sham S-NAA S-AA Sham S-NAA S-AA Sham S-NAA S-AA Sham S-NAA S-AA Sham S-NAA S-AA Sham S-NAA S-AA Sham S-NAA S-AA Sham S-NAA S-AA Sham S-NAA S-AA Sham S-NAA S-AA Sham S-NAA S-AA
Baseline
Start bacterial infusion
Start of amino acids
End of experiment
T0
T40
T80
T120
T160
1.6 (1.1;2.6) 2.5 (2.0;3.2) 2.1 (1.5;2.4) 68.4 (61.2;68.4) 61.5 (60.4;66.6) 67.1 (64.4;67.9) 6.0 (5.3;6.0) 6.1 (5.8;6.4) 6.7 (6.2;6.8) 7.31 (7.31;7.42) 7.42 (7.40;7.43) 7.42 (7.39;7.43) 29.0 (29.0;29.8) 30.7 (27.7;31.7) 31.1 (26.9;32.8) 286 (284;291) 295 (284;299) 294 (287;297) 115 (81;130) 112 (96;126) 85 (76;96) 141 (139;144) 142 (142;143) 141 (139;143) 0.7 (0.5;0.8) 0.7 (0.5;1.0) 0.9 (0.8;1.0) 6.5 (2.8;8.0) 14.0 (8.3;19.0) 25.0 (17.0;27.8) 60 (38;80) 71 (65;87) 87 (67;101)
2.2 (1.5;4.1) 2.6 (2.2;3.7) 3.2 (2.4;4.3) 65.3 (62.1;65.3) 62.5 (61.9;67.9) 58.3 (56.8;65.9) 6.4 (5.4;6.4) 6.5 (6.0;7.0) 6.9 (6.6;7.4) 7.40 (7.33;7.42) 7.37 (7.33;7.42) 7.40 (7.35;7.40) 29.8 (29.0;29.8) 29.6 (26.7;30.7) 26.8 (26.2;31.9) 287 (284;289) 296 (293;312) 300 (291;304)* 117 (82;128) 105 (91;121) 80 (77;99) 142 (141;142) 144 (143;145) 143 (140;144) 0.8 (0.8;1.0) 0.8 (0.6;1.2) 1.3 (1.0;1.6) 9.0 (8.0;16.8) 14.5 (8.0;18.8) 18.0 (15.3;23.8) 76 (46;98) 67 (65;72) 82 (68;150)
1.9 (1.4;2.6) 2.9 (2.3;3.5) 5.9 (4.4;7.2)*† 63.1 (59.1;63.5) 55.9 (49.1;60.7) 59.7 (55.0;62.8) 5.5 (5.3;5.5) 5.9 (5.9;6.1) 6.4 (5.9;6.7) 7.44 (7.28;7.45) 7.34 (7.33;7.36) 7.33 (7.31;7.36) 28.8 (28.6;28.8) 26.5 (25.4;28.1) 27.7 (26.5;28.4) 292 (289;293) 302 (294;305)* 301 (295;310)* 118 (87;127) 105 (89;121) 98 (87;115) 141 (139;142) 145 (144;145) 143 (142;143) 0.8 (0.7;1.0) 1.0 (0.7;1.3) 1.8 (1.7;2.1)* 10.0 (6.3;10.0) 13.0 (9.3;16.0) 18.5 (14.0;24.5) 83 (70;105) 112 (99;121) 387 (213;400)*
1.9 (1.3;2.0) 3.1 (2.2;4.1) 4.5 (4.2;5.3)† 59.3 (59.3;64.8) 55.6 (47.2;63.6) 54.9 (47.3;64.4) 5.8 (5.1;5.9) 6.8 (6.1;7.1) 6.9 (6.3;7.0) 7.40 (7.25;7.41) 7.32 (7.31;7.32)* 7.32 (7.29;7.39) 29.9 (27.9;29.9) 26.3 (23.6;27.2) 24.5 (23.0;29.3) 290 (289;292) 301 (296;303)* 300 (298;310)* 118 (113 : 129) 96 (85;111) 102 (94;125) 141 (139;142) 145 (144;146) 144 (142;144) 0.7 (0.6;1.1) 0.8 (0.5;1.0) 1.5 (1.5;1.5) 8.5 (5.0;12.0) 14.0 (8.8;16.3) 19.0 (15.3;25.0) 100 (75;137) 101 (77;137) 132 (103;334)
1.7 (1.4;2.5) 4.0 (2.8;5.9)* 4.1 (3.8;5.9)† 65.2 (56.3;65.2) 55.3 (41.2;62.4) 54.0 (48.4;65.2) 5.6 (4.9;5.6) 7.3 (6.6;8.3) 7.2 (6.6;7.7) 7.42 (7.26;7.42) 7.29 (7.25;7.32)* 7.28 (7.25;7.32)* 27.6 (26.0;28.6) 23.9 (21.8;26.3) 23.2 (19.6;27.6) 293 (291;294) 298 (295;308)*† 303 (299;305)*† 128 (116.150) 99 (86;110) 103 (81;115)* 142 (141;142) 145 (144;145) 144 (141;144) 0.8 (0.6;1.2) 0.4 (0.4;0.9) 1.2 (1.1;1.3) 6.0 (5.3;9.8) 11.0 (9.3;14.3) 27.0 (18.8;27.0) 122 (76;340) 146 (142;161)* 131 (111;218)
Sham group: non -septic piglet receiving no amino acids, S-NAA group: septic piglets receiving no amino acids, S-AA group: septic piglets receiving amino acids. *Significant intragroup difference (P < 0.05) in comparison with baseline values. †Significant intergroup difference (P < 0.05) at a given period.
Effects of amino acids infusion on renal parameters No significant change in renal parameters over time was noticed in the Sham group. RBF and GFR decreased significantly, and RVR increased significantly over time in the S-AA group (P = 0.03 and P = 0.01, P = 0.02 respectively). Conversely, RBF increased slightly over time in the S-NAA group in comparison with baseline values (+19% at T160, P = 0.009), with no signifi-
cant concomitant variation of GFR and RVR. A significant intergroup difference was found between the S-NAA and S-AA groups at T60 for RBF (P = 0.03).
Effects of amino acids infusion on biochemical parameters Blood lactate concentration was significantly higher in S-AA group than in the Sham group from T60 to T160 (P = 0.04), whereas there was no
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Total ml
Total mg
1000
2000
900
*
800
*†
1800
*
1600
700
1400
600
1200
500
1000
400
800
300
600
200
400
100
200
0
0 Sham
S-NAA NaCl 0.9%
S-AA
Sham
S-NAA
S-AA
Colloids
Sham
S-NAA
S-AA
Epinephrine
Fig. 3. Amines and fluids requirement. Sham group: non-septic piglet receiving no amino acids, S-NAA group: septic piglets receiving no amino acids, S-AA group: septic piglets receiving amino acids. *P < 0.05, significant intergroup difference between S-NAA and Sham or S-AA and Sham; †P < 0.05, significant intergroup difference between S-NAA and S-AA group.
significant difference between the SNAA group and the Sham group. Creatinine level increased significantly over time in the S-AA group (P = 0.01), but remained within normal range. Levels of glucose and glucagon increased significantly over time in the S-AA group from T60 to T100 (P = 0.03 and P = 0.006 respectively), whereas only glucagon level increased significantly and late in the S-NAA group (P = 0.005) at T160. Plasma osmolality increased significantly in the S-AA group (P = 0.001) with a significant intergroup difference at T160 (P = 0.04). Sodium level increased significantly over time in the S-NAA group (P = 0.04, Friedman test), but remaining within normal range. Effects of amino acids infusion on fluid and amines administration Animals in the S-AA group tended to receive more isotonic saline and colloids, but there was no significant difference between these animals and those in the S-NAA group. An AA infusion
was significantly associated with a higher total requirement of epinephrine [340.0 (141.2–542.5) mg vs. 32.5 (3.8–65.0) mg in S-NAA group P = 0.04, and no epinephrine was used in the Sham group P = 0.01]. Discussion In our experimental septic model, AA infusion did not result in a preservation of renal haemodynamic. Conversely, we noticed a significant decrease in RBF and a significant increase in RVR, and this was also associated with a higher requirement of epinephrine in the AA-treated septic group. Our study aimed to evaluate renal haemodynamics in the time-course of sepsis. To meet this objective, a large animal model was chosen as it allows better haemodynamic monitoring, equipment and frequent blood sampling. In order to better reproduce human physiopathology of sepsis, we opted for an animal model that has been previously validated and described as hyperkinetic with an increase of CO and a concomitant decrease in systemic vascular resisActa Anaesthesiologica Scandinavica 59 (2015) 598–608
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tances.17 As these haemodynamic alterations appeared early in this model, a shorter follow up of the animals than the original description was chosen. We also opted for an early goal directed resuscitation to fulfil human recommendation for the treatment of sepsis. Despite this shorter experimental duration, we observed haemodynamic alterations, but the severity of shock, based on biochemical markers, was less than expected and described in the original description of the model. The development of sepsis AKI is a major concern because it is associated with a high mortality rate. Even though a decrease in RBF following excessive renal vasoconstriction has been mentioned,8,12 cases of AKI with a normal RBF have been reported.7,10,18 Based on magnetic resonance imaging and Doppler echography, increased RVR have also been implicated.19 Maintaining systemic haemodynamic parameters while preventing increased renal resistance may thus appear as a potential interesting strategy to prevent sepsis-induced AKI. As AA infusion has been reported to increase RBF and GFR and decrease RVR in healthy animal and patients,13,20,21 their use in sepsis condition in order to prevent AKI appeared attractive. However, renal effects of AA infusion in critical condition have not been evaluated to our knowledge. A meta-analysis supported their use in intensive care, with parenteral nutrition reducing mortality,22 but this study was not focused on kidney injury. Moreover, when examining in detail AA solutions, they contain several AA of potential interest during sepsis, which may exert a positive action on immunity or on signalling pathways altered in the time-course of septic shock such as nitric oxide (NO) pathway.13–16 Glutamic acid, arginine, lysine, phenylalanine and tyrosine are for example present in Vintene and have been associated with interesting immunomodulatory effect during sepsis.23–28 The AA dose infusion of 0.05 ml/kg/min (6 mg/ kg/min) was used originally to demonstrate the renal functional reserve in piglets.13 We used the same dose in our experimental setting, even though the AA solution was different. At this dosage, we observed an increase of plasma osmolality in our experimental condition (septic piglets), but also in non-septic piglets (personal data), presumably due to the hypertonicity of the
solution (1140 mOsm/L). However, this increase was slight and not significantly different with Sham group. More important was the rise in glucagon level noticed in the S-AA group in comparison with Sham and S-NAA groups. Such an increase has been described following AA infusion in healthy animals and patients,13,15,29 but might be undesirable during sepsis because hyperglycaemia or poor glycaemic control have been associated with an increased mortality.30 Regarding renal effects of AA, the increased RBF and decreased RVR in healthy patient remain to be explained: NO, prostaglandins and glucoregulatory hormones15,21 have been proposed as a putative mechanism. An alteration of renal haemodynamic has been reported in human patients with hyperkinetic shock.12 This was not found in our experimental model: no significant alteration of renal parameters was found in the non-AA septic group; conversely, a slight increase of RBF without any significant change in RVR and GFR was noticed at the end of the experiment for this group. This discrepancy between clinical data and our experimental data can be explained not only by the short time of follow up of the animals, but also by the early resuscitation criteria, based on human guidelines,31 which allowed prompt restoration of systemic haemodynamic parameters. Paradoxically, AA infusion altered renal function in our experimental critical conditions, whereas we were expecting at least its stabilisation or improvement. These results are also in accordance with those of an experimental study on rodents: the use of AA at therapeutic dosage worsened an ischaemic renal injury created by renal artery ligation. In this experiment, no putative mechanism was given to explain these AA deleterious effects.32 Whether the increased renal resistances are due to loss of renal autoregulation following severe hypotension or to direct effect of AA is difficult to elucidate in our experiment. It must be emphasised that many AA contained in the solution have opposite actions on NO pathway or immunity, some of them may have different effects depending if the patient is in septic shock or not.27 The diversity of AA contained in the solution renders difficult the interpretation and anticipation of its biological properties. It is thus possible that in critical condition, AA may worsen sys-
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temic and renal haemodynamic parameters. We cannot exclude that the timing of administration of AA may also influence their effect. At least, our results preclude their use in the early phase of sepsis as the shock appeared more severe in the S-AA group than in the S-NAA group, with a higher epinephrine requirement. Recently, a meta-analysis reported a decrease rate of infection with glutamine supplementation, but questioned the appropriate timing and patient selection to meet these positive effects.33 The greater severity of shock observed in the S-AA group is in accordance with results of a prospective study that reported an increased risk of death associated with the use of AA in septic shock in human patients.34 The intensity of shock observed during bacterial and AA infusions limits the interpretation of our results regarding renal function. It is difficult to conclude whether the alterations of renal parameters are due to the own systemic and renal effects of AA or due to this higher catecholamine requirement. Indeed, the use of epinephrine is associated with a major increase of RVR,35,36 which may have dampened the potential beneficial renal vasodilation of AA. Other factors may have contributed to the significant difference between groups in blood lactate and epinephrine requirements. It is possible that the high dose of epinephrine has precipitated the rise of blood lactate observed in the AA group, as hyperlactataemia is a welldescribed consequence of epinephrine administration.37,38 The kidney metabolises 20–30% of lactate; thus, alteration of renal function in the AA group may also have played a role in the increase blood lactate. Metabolic acidosis decreases myocardial sensitivity to catecholamines,39 which may have worsen the requirement of epinephrine in the time-course of shock. However, the clinical relevance of these factors can hardly be estimated in our experimental conditions. We acknowledge some limitations in our study. The small sample size of the groups may have dampened the differences between groups and decrease the power of our statistical analysis. Moreover, we decided to apply resuscitation criteria based on human guidelines, but early in the time-course of septic shock, which may have limited the development of AKI. Similar to the original description of the model, we used an inhalant anaesthetic agent that may have inter-
fered with vascular resistance and renal autoregulation. The Sham group was included in the experimental design in order to assess the influence of the anaesthetic protocol on haemodynamic and renal parameters. GFR was estimated with creatinine clearance, a method that requires steady state and may overestimate GFR in case of AKI.40 Due to the onset of shock, a steady state could not be achieved in our experimental setting, but urine was collected every 20 min directly from ureters, which is more accurate than a collection from the bladder over 24 h. Clearance of inulin or of a radiolabelled compound would have been more precise, but were not feasible in our experimental setting. As previously mentioned, we used epinephrine to resuscitate the animal, as it was described in the original model, but norepinephrine is recommended as the first-choice vasopressor in the early management of sepsis31 and would have probably been more appropriate regarding renal resistances. The development of septic-induced AKI is probably not solely related to alteration of RBF and renal vasoconstriction.5 The kidney may be injured at multiple levels: RBF, glomerular tubular cell function and structure, microcirculation, mitochondria and proteome. Particularly, sepsis is characterised with overproduction of proinflammatory mediators, which may lead to intrarenal inflammation and AKI.41,42 However, our experimental setting was designed to evaluate the potential interest of AA on renal resistance during sepsis and did not focus on renal inflammation. Conclusion In a piglet model of sepsis induced by P. aeruginosa infusion, an AA infusion significantly impaired renal haemodynamics. Further studies are needed to draw definite conclusions and clinical implications. Acknowledgement The authors would like to thank Sarah HetierJaccoud for her help in data collection and analysis. References 1. Oppert M, Engel C, Brunkhorst F-M, Bogatsch H, Reinhart K, Frei U, Eckardt K-U, Loeffler M, Acta Anaesthesiologica Scandinavica 59 (2015) 598–608
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John S, German Competence Network Sepsis (SepNet). Acute renal failure in patients with severe sepsis and septic shock-a significant independent risk factor for mortality: results from the German Prevalence Study. Nephrol Dial Transplant 2008; 23: 904–9. Bagshaw SM, Lapinsky S, Dial S, Arabi Y, Dodek P, Wood G, Ellis P, Guzman J, Marshall J, Parrillo JE, Skrobik Y, Kumar A, Cooperative Antimicrobial Therapy of Septic Shock (CATSS) Database Research Group. Acute kidney injury in septic shock: clinical outcomes and impact of duration of hypotension prior to initiation of antimicrobial therapy. Intensive Care Med 2009; 35: 871–81. Uchino S, Kellum JA, Bellomo R, Doig GS, Morimatsu H, Morgera S, Schetz M, Tan I, Bouman C, Macedo E, Gibney N, Tolwani A, Ronco C, Beginning FT, Investigators ESTFTKBK. Acute renal failure in critically ill patients: a multinational, multicenter study. JAMA 2005; 294: 813–8. White LE, Hassoun HT, Bihorac A, Moore LJ, Sailors RM, McKinley BA, Valdivia A, Moore FA. Acute kidney injury is surprisingly common and a powerful predictor of mortality in surgical sepsis. J Trauma Acute Care Surg 2013; 75: 432–8. Wan L, Bagshaw SM, Langenberg C, Saotome T, May C, Bellomo R. Pathophysiology of septic acute kidney injury: what do we really know? Crit Care Med 2008; 36: S198–203. Lipcsey M, Bellomo R. Septic acute kidney injury: hemodynamic syndrome, inflammatory disorder, or both? Crit Care 2011; 15: 1–2. Langenberg C, Wan L, Egi M, May CN, Bellomo R. Renal blood flow in experimental septic acute renal failure. Kidney Int 2006; 69: 1996–2002. Badr KF. Sepsis-associated renal vasoconstriction: potential targets for future therapy. Am J Kidney Dis 1992; 20: 207–13. Zarjou A, Agarwal A. Sepsis and acute kidney injury. J Am Soc Nephrol 2011; 22: 999–1006. De Vriese AS. Prevention and treatment of acute renal failure in sepsis. J Am Soc Nephrol 2003; 14: 792–805. Reinhart K, Karzai W. Anti-tumor necrosis factor therapy in sepsis: update on clinical trials and lessons learned. Crit Care Med 2001; 29: S121–5. Schrier RW, Wang W. Acute renal failure and sepsis. N Engl J Med 2004; 351: 159–69. Poulsen EU, Frøkiaer J, Jørgensen TM, Pedersen EB, Rehling M. Renal functional reserve in pigs: renal haemodynamics, renal tubular
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function and salt and water homeostatic hormones during amino acid and dopamine stimulation. Clin Physiol 1997; 17: 57–69. Denis N, Tebot I, Bonnet J-M, Cirio A, Boivin R. Effects of intravenous perfusion of glucagon on renal blood flow in conscious sheep. Exp Physiol 2003; 88: 575–80. Kuhara T, Ikeda S, Ohneda A, Sasaki Y. Effects of intravenous infusion of 17 amino acids on the secretion of GH, glucagon, and insulin in sheep. Am J Physiol 1991; 260: E21–6. Tebot I, Bonnet J-M, Paquet C, Ayoub J-Y, Da Silva SM, Louzier V, Cirio A. The effect of intravenous insulin infusion on renal blood flow in conscious sheep is partially mediated by nitric oxide but not by prostaglandins. J Anim Sci 2012; 90: 1192–200. Rimmelé T, Assadi A, Benatir F, Boselli E, Kaminski C, Arnal F, Lambert C, Goudable J, Chassard D, Allaouchiche B. Validation of a Pseudomonas aeruginosa porcine model of septic shock. J Infect 2006; 53: 199–205. Di Giantomasso D, May CN, Bellomo R. Vital organ blood flow during hyperdynamic sepsis. Chest 2003; 124: 1053–9. Bouglé A, Duranteau J. Pathophysiology of sepsis-induced acute kidney injury: the role of global renal blood flow and renal vascular resistance. Contrib Nephrol 2011; 174: 89–97. Lee KE, Summerill RA. Glomerular filtration rate following administration of individual amino acids in conscious dogs. Q J Exp Physiol 1982; 67: 459–65. Livi R, Teghini L, Pignone A, Generini S, Matucci-Cerinic M, Cagnoni M. Renal functional reserve is impaired in patients with systemic sclerosis without clinical signs of kidney involvement. Ann Rheum Dis 2002; 61: 682–6. Simpson F, Doig GS. Parenteral vs. enteral nutrition in the critically ill patient: a meta-analysis of trials using the intention to treat principle. Intensive Care Med 2004; 31: 12–23. Déchelotte P, Hasselmann M, Cynober L, Allaouchiche B, Coëffier M, Hecketsweiler B, Merle V, Mazerolles M, Samba D, Guillou YM, Petit J, Mansoor O, Colas G, Cohendy R, Barnoud D, Czernichow P, Bleichner G. L-alanyl-L-glutamine dipeptide-supplemented total parenteral nutrition reduces infectious complications and glucose intolerance in critically ill patients: the French controlled, randomized, double-blind, multicenter study. Crit Care Med 2006; 34: 598–604.
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24. Goeters C, Wenn A, Mertes N, Wempe C, Van Aken H, Stehle P, Bone H-G. Parenteral L-alanyl-L-glutamine improves 6-month outcome in critically ill patients. Crit Care Med 2002; 30: 2032–7. 25. Newsholme P, Brennan L, Rubi B, Maechler P. New insights into amino acid metabolism, beta-cell function and diabetes. Clin Sci 2005; 108: 185–94. 26. Wu G, Flynn NE, Flynn SP, Jolly CA, Davis PK. Dietary protein or arginine deficiency impairs constitutive and inducible nitric oxide synthesis by young rats. J Nutr 1999; 129: 1347–54. 27. Liaudet L, Gnaegi A, Rosselet A, Markert M, Boulat O, Perret C, Feihl F. Effect of L-lysine on nitric oxide overproduction in endotoxic shock. Br J Pharmacol 1997; 122: 742–8. 28. Li P, Yin Y-L, Li D, Kim SW, Wu G. Amino acids and immune function. Br J Nutr 2007; 98: 237–52. 29. Woods LL. Mechanisms of renal hemodynamic regulation in response to protein feeding. Kidney Int 1993; 44: 659–75. 30. Egi M, Finfer S, Bellomo R. Glycemic control in the ICU. Chest 2011; 140: 212–20. 31. Dellinger RP, Levy MM, Rhodes A, Annane D, Gerlach H, Opal SM, Sevransky JE, Sprung CL, Douglas IS, Jaeschke R, Osborn TM, Nunnally ME, Townsend SR, Reinhart K, Kleinpell RM, Angus DC, Deutschman CS, Machado FR, Rubenfeld GD, Webb SA, Beale RJ, Vincent J-L, Moreno R, Surviving Sepsis Campaign Guidelines Committee including the Pediatric Subgroup. Surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock: 2012. Crit Care Med 2013; 41: 580–637. 32. Zager RA, Venkatachalam MA. Potentiation of ischemic renal injury by amino acid infusion. Kidney Int 1983; 24: 620–5. 33. Chen QH, Yang Y, He HL, Xie JF, Cai SX, Liu AR, Wang HL, Qiu HB. The effect of glutamine therapy on outcomes in critically ill patients: a meta-analysis of randomized controlled trials. Crit Care 2014; 18: R8. 34. Elke G, Schädler D, Engel C, Bogatsch H, Frerichs I, Ragaller M, Scholz J, Brunkhorst FM,
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Löffler M, Reinhart K, Weiler N, German Competence Network Sepsis (SepNet). Current practice in nutritional support and its association with mortality in septic patients – Results from a national, prospective, multicenter study. Crit Care Med 2008; 36: 1762–7. Moyer JH, Handley CA. Norepinephrine and epinephrine effect on renal hemodynamics with particular reference to the possibility of vascular shunting and decreasing the active glomeruli. Circulation 1952; 5: 91–7. Gombos EA, Hulet WH, Bopp P, Goldring W, Baldwin DS, Chasis H. Reactivity of renal and systemic circulations to vasoconstrictor agents in normotensive and hypertensive subjects. J Clin Invest 1962; 41: 203–17. Levy B, Mansart A, Bollaert P-E, Franck P, Mallie J-P. Effects of epinephrine and norepinephrine on hemodynamics, oxidative metabolism, and organ energetics in endotoxemic rats. Intensive Care Med 2003; 29: 292–300. Gjedsted J, Buhl M, Nielsen S, Schmitz O, Vestergaard ET, Tønnesen E, Møller N. Effects of adrenaline on lactate, glucose, lipid and protein metabolism in the placebo controlled bilaterally perfused human leg. Acta Physiol 2011; 202: 641–8. Campbell GS, Houle DB, Crisp NW, Weil MH, Brown EB. Depressed response to intravenous sympathicomimetic agents in humans during acidosis. Chest 1958; 33: 18–22. Bragadottir G, Redfors B, Ricksten S-E. Assessing glomerular filtration rate (GFR) in critically ill patients with acute kidney injury – true GFR versus urinary creatinine clearance and estimating equations. Crit Care 2013; 17: R108. Jo SK, Cha DR, Cho WY, Kim HK, Chang KH, Yun SY, Won NH. Inflammatory cytokines and lipopolysaccharide induce Fas-mediated apoptosis in renal tubular cells. Nephron 2002; 91: 406–15. Lerolle N, Nochy D, Guérot E, Bruneval P, Fagon J-Y, Diehl J-L, Hill G. Histopathology of septic shock induced acute kidney injury: apoptosis and leukocytic infiltration. Intensive Care Med 2010; 36: 471–8.
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Renal haemodynamic response to amino acids infusion in an experimental porcine model of septic shock O. Vassal1,2, J.-M. Bonnet3, A. Barthelemy3, B. Allaouchiche1,2, I. Goy-Thollot3, V. Louzier3, C. Paquet3, J.-Y. Ayoub3, O. Dauwalder4, M. Jacquet-Lagrèze1,2 and S. Junot3 1
Service d’Anesthésie–Réanimation, Hospices Civils de Lyon, Hôpital Edouard-Herriot, Lyon, France Université Claude-Bernard, Lyon, France 3 Université de Lyon, EA 4174 Hémostase Inflammation Sepsis, VetAgro Sup – Campus Vétérinaire de Lyon, Marcy l’Etoile, France 4 Université de Lyon, Laboratory of Microbiology, Groupement Hospitalier Est, Lyon, France 2
Correspondence S. Junot, Université de Lyon, EA 4174 Hémostase Inflammation Sepsis, VetAgro Sup – Campus Vétérinaire de Lyon, F-69280, Marcy l’Etoile, France E-mail: [email protected] Conflict of interest The authors declare that they have no competing interests. Funding This study was supported by an unrestricted operating grant from the Scientific Council of Vetagro Sup, an internal grant from Agriculture Ministry (EA 4174) and an internal grant from Hospices Civiles de Lyon. Submitted 31 January 2015; accepted 3 February 2015; submission 15 July 2014. Citation Vassal O, Bonnet J-M, Barthelemy A, Allaouchiche B, Goy-Thollot I, Louzier V, Paquet C, Ayoub J-Y, Dauwalder O, Jacquet-Lagrèze M, Junot S. Renal haemodynamic response to amino acids infusion in an experimental porcine model of septic shock. Acta Anaesthesiologica Scandinavica 2015
Background: Acute kidney injury (AKI) is common in sepsis. Treatments allowing maintenance of renal blood flow (RBF) could help to prevent AKI associated with renal hypoperfusion. Amino acids (AA) have been associated with an increase of RBF and glomerular filtration rate (GFR) in several species. The aim of this study was to evaluate the effects of an AA infusion on RBF and GFR in a porcine model of septic shock. Methods: A total of 17 piglets were randomly assigned into three groups: Sham (Sham, n = 5), sepsis without AA (S-NAA, n = 6), sepsis treated with AA (S-AA, n = 6). Piglets preparation included the placement of ultrasonic transit time flow probes around left renal artery for continuous RBF measurement; ureteral catheters for GFR and urine output evaluation; pulmonary artery catheter for cardiac output (CO) and pulmonary arterial pressure measurements. Mean arterial pressure (MAP) and renal vascular resistance (RVR) were also determined. Septic shock was induced with a live Pseudomonas aeruginosa infusion. Crystalloids, colloids and epinephrine infusion were used to maintain and restore MAP > 60 mmHg and CO > 80% from baseline. Results: Renal haemodynamic did not change significantly in the Sham group, whereas RBF increased slightly in the S-NAA group. Conversely, a significant increase in RVR and a decrease in RBF and GFR were observed in the S-AA group. AA infusion was associated with a higher requirement of epinephrine [340.0 (141.2; 542.5) mg vs. 32.5 (3.8; 65.0) mg in the S-NAA group P = 0.044]. Conclusion: An infusion of amino acids impaired renal haemodynamics in this experimental model of septic shock.
doi: 10.1111/aas.12507
Editorial comment: what this article tells us An amino acid infusion was associated with an impairment of renal haemodynamics in a septic porcine model. The results do not support the administration of amino acid in the early phase of septic shock.
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Acute kidney injury (AKI) is a common complication of sepsis associated with an increased morbidity and mortality, which has been reported in 40–50% of patients with severe sepsis.1–3 Recently, AKI has been confirmed as a powerful predictor of hospital mortality in severe sepsis and septic shock.4 The pathophysiology of AKI during sepsis remains a matter of debate,5 with both haemodynamic and inflammatory disorders contributing to the development of kidney functional impairment.6,7 Renal hypoperfusion and increased renal vascular resistance (RVR) during haemodynamic failure in severe sepsis have been suggested as central mechanisms for the development of septic AKI.8–10 Prevention of sepsis-induced AKI is complicated, but maintenance of renal blood flow (RBF) remains recommended as a protective strategy. Pharmacologic anti-inflammatory treatments have not shown any significant efficacy;11,12 macrocirculation support such as fluids, vasopressors or diuretics have failed to prevent the occurrence of the disease.10,12 High doses of catecholamines may cause excessive renal vasoconstriction, and thus precipitating the occurrence of renal failure.12 A treatment allowing renal vasodilation without any systemic effect could be promising, particularly in conditions of haemodynamic alterations associated with vasopressors requirement. Amino acids (AA) infusions have been associated in several species with an increase of RBF and glomerular filtration rate (GFR) without any systemic arterial pressure variation.13–16 These renal effects of AA could be used as a means of maintaining renal perfusion in sepsis condition associated with increased RVR. The aim of this study was to evaluate the effects of an AA infusion on renal haemodynamics and function in a porcine model of septic shock. Our hypothesis was that the administration of AA would prevent a decrease in RBF or an increase in renal resistance during sepsis.
Animals A total of 18 Landrace female piglets (2.5–3.5 month old) weighing between 20 and 25 kg were fasted for 12 h prior the experiment, with free access to water. Animals were pre-medicated with an intramuscular 1 : 1 mixture of tiletamine and zolazepam (3 and 3 mg/kg respectively). An intravenous catheter was then inserted in the auricular vein for administration of buprenorphine (0.02 mg/kg) and infusion of Ringer’s lactate solution at a rate of 10 ml/kg/h to replace fluid losses. Piglets were orally intubated after induction of general anaesthesia with intravenous injection of propofol (3 mg/kg). Anaesthesia was maintained by inhalation of isoflurane in 100% oxygen. Animals were ventilated with a volumecontrolled ventilator (Alpha100, Minerve, Esternay, France) with a tidal volume of 8 ml/kg. Body temperature was maintained above 37°C by using an electric warming blanket.
Surgical preparation The left and right internal jugular veins were isolated and catheterised. The left jugular catheter was devoted to fluid and drug administration. A 7CH pulmonary artery catheter (Edwards Lifesciences, Guyancourt, France) was inserted through the right jugular vein into the pulmonary artery for measurement of pulmonary artery pressure (PAP), pulmonary capillary wedge pressure (PCWP) and cardiac output (CO). A catheter was placed in the left carotid artery to allow arterial pressure measurement and blood sampling. Following a paravertebral laparotomy, a 4-mm ultrasonic Doppler flow probe (Transonic System, Ithaca, NY, USA) was positioned around the left renal artery for continuous measurement of RBF. A midline laparotomy was then performed. The bladder was emptied and the ureters were insulated and cannulated for continuous collection of urine.
Methods
Study design
This study was approved by the Animal Care Committee of VetAgro Sup, Marcy l’Etoile, France (n°1180) in order to satisfy the criteria of European regulation (Directive EU 86/609).
Animals were randomly allocated into three groups (Fig. 1): Sham (Sham, n = 6), sepsis without AA (S-NAA, n = 6) or sepsis treated with AA (S-AA, n = 6) (Fig. 1).
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Fig. 1. Experimental design. Sham group (n = 5): non-septic piglet receiving no amino acids, S-NAA group (n = 6): septic piglets receiving no amino acids, S-AA group (n = 6): septic piglets receiving amino acids (P. aeruginosa: Pseudomonas aeurigona). AA, amino acids.
Following surgical preparation, piglets were stabilised for 30 min. Haemodynamic, respiratory and renal parameters were recorded thereafter every 20 min before treatment (T20 and T0) and 160 min after treatment, divided into 8 20-min periods (T20–T160). After baseline measurements (T20 and T0), septic shock was induced (S-NAA and S-AA groups) with an intravenous infusion of Pseudomonas aeruginosa (5.108 UFC/ml – 0.3 ml/20 kg/min). The P. aeruginosa strain (ATCC 27853) used in this study was obtained from a patient at the hospital with a standardised method in order to avoid variations of virulence.17 The infusion was stopped when the systolic PAP reached 45 mmHg to avoid a possible right heart failure. Animals in the Sham group received Ringer’s lactate alone. Forty minutes after the beginning of P. aeruginosa infusion (end of T40), a continuous infusion of AA (Vintene®, Baxter, Maurepas, France) was administered at a rate of 6 mg/kg/ min (0.05 ml/kg/min) and maintained until the end of experiment. Piglets were resuscitated by standardised combination of fluids and inotropes (Fig. 2): crystalloid boli (NaCl 0.9%) (two boli of 10 ml/kg) were first administered in case of hypotension (MAP < 60 mmHg), then colloids boli (6% hydroxyethyl starch (130/0.4), (two boli of 10 ml/
kg) if hypotension persisted or recurred and finally epinephrine infusion starting at 0.02 mg/kg/min and given to effect in case of fluid unresponsiveness. These treatments were administered in order to maintain and restore mean arterial pressure (MAP) > 60 mmHg and CO > 80% of their baseline values. Concomitantly, Ringer’s lactate infusion rate was reduced by 3 ml/kg/h so that every pig received the same volume expansion. At the end of experiment, animals were euthanised with a solution of pentobarbital.
Monitoring Heart rate, electrocardiogram and pulse oximetry were continuously monitored. Every 20 min, temperature, urinary output (UO), PCWP and CO (by thermodilution) were measured. Mean, diastolic and systolic arterial pressure as well as PAP and RBF were continuously measured and processed using an acquisition software (AcqKnowledge version 3.7.3 for Windows 95/98, Biopac Systems Inc, Goleta, CA , USA). From these records, values were averaged for each 20-min period of the experimental procedure. Systemic vascular resistances (SVR) were calculated as followed: MAP × 80/CO. RVR was calculated according to Acta Anaesthesiologica Scandinavica 59 (2015) 598–608
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If MAP < 60 mmHg CO < 80% baseline
1st and 2nd bolus Ringer’s Lactate
Stop resuscitation
If MAP > 60 mmHg CO > 80% baseline
If MAP < 60 mmHg CO < 80% baseline After 2nd bolus Fig. 2. Resuscitation protocol. Piglets were resuscitated following a standardised algorithm: crystalloid boli (two boli of 10 ml/kg) were first administered in case of hypotension or decreased cardiac output (CO) [mean arterial pressure (MAP) < 60 mmHg, CO < 80% of its initial value], then colloids boli (two boli of 10 ml/kg) if hypotension or decreased CO persisted or recurred; finally epinephrine infusion starting at 0.02 mg/kg/min and given to effect in case of fluid unresponsiveness. HES, hydroxyethyl starch.
1st and 2nd bolus of HES
If MAP > 60 mmHg CO > 80% baseline
If MAP < 60 mmHg CO < 80% baseline After 2nd bolus Epinephrine infusion
the following formula: MAP/RBF. GFR was estimated by endogenous creatinine clearance over a 20-min period. Sampling Blood samples were drawn every 20 min for biochemical analyses including: arterial blood gas (included pH, pCO2, pO2 and bicarbonate), glucose, lactate, total protein, serum osmolality, creatinine, electrolytes, glucagone and insulin. Urine samples were also collected every 20 min for urine sodium and creatinine measurements. Statistical analysis All analyses were two-sided with a threshold of 0.05 using a statistical software program (GraphPad Prism, v6; GraphPad Software, San Diego, CA, USA). Normality of distribution was assessed using an Agostino–Pearson test. As most haemodynamic and renal variables were not normally distributed, data are presented as median ± interquartile range (IQR). Incomplete data or values
If MAP > 60 mmHg CO > 80% baseline
outside the 95% confidence interval were excluded from statistical analyses. Differences within each group before and after induction of sepsis were tested using a Friedman analysis of variance on ranks and, subsequently, a Dunn’s multiple comparisons post hoc test. A Kruskal– Wallis rank sum test was performed to compare data between treatment groups with a Dunn’s multiple comparisons post hoc test. For comparison of the amount of epinephrine and fluids administered between groups, unpaired t-tests were used. Sample size was estimated according to preliminary data; we hypothesised a difference of 50% of primary outcome (RBF) with alpha = 0.05 and beta = 0.20, which gave a sample size of six animals per group.
Results One piglet of the NS-NAA group was withdrawn due to technical defect of the RBF probe. No animals died during the experiment. Results are summarised in Table 1 for haemodynamic data and Table 2 for biochemical data.
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Table 1 Main haemodynamic variables.
Parameter MAP (mmHg)
Cardiac output (l/min)
Heart rate (bpm)
SVR (dyn • s/cm5)
PAP (mmHg)
PCWP (mmHg)
Urine output (ml/min)
GFR (ml/min)
RVR (mmHg • min/ml)
RBF (ml/min)
Sham S-NAA S-AA Sham S-NAA S-AA Sham S-NAA S-AA Sham S-NAA S-AA Sham S-NAA S-AA Sham S-NAA S-AA Sham S-NAA S-AA Sham S-NAA S-AA Sham S-NAA S-AA Sham S-NAA S-AA
Baseline
Bacterial infusion
Amino acids
End of experiment
T0
T40
T80
T120
T160
63 (55;73) 64 (62;67) 73 (64;80) 1.9 (1.6;2.1) 2.1 (1.8;2.3) 2.1 (1.7;2.2) 92 (83;101) 88 (83;90) 77 (73;98) 3200 (2360;3404) 2527 (2110;3331) 2528 (2253;2851) 16 (16;20) 21 (15;26) 19 (14;22) 14 (10;14) 10 (8;16) 15 (14;15) 0.77 (0.49;2.11) 0.65 (0.20;1.14) 0.82 (0.48;1.18) 67.9 (54.4;72.2) 63.6 (24.1;73.0) 54.9 (40.7;63.1) 0.35 (0.31;0.37) 0.35 (0.34;0.49) 0.34 (0.31;0.44) 193 (175;195) 189 (172;205) 208 (184;210)
63 (55;76) 64 (57;71) 64 (59;66) 2.0 (1.7;2.5) 1.7 (1.2;2.4) 1.7 (1.4;2.0) 93 (85;100) 107 (92;118) 121 (97;136) 3047 (2283;3144) 2932 (1976;4143) 4043 (3152;4095) 20 (17;23) 36 (28;40) 30 (27;34) 13 (10;14) 13 (11;16) 19 (13;20) 1.52 (0.76;3.74) 0.45 (0.22;0.79) 0.66 (0.50;1.24) 52.3 (33.5;76.1) 44.6 (30.5;61.9) 39.2 (27.2;43.1) 0.42 (0.34;0.49) 0.34 (0.31;0.49) 0.45 (0.43;0.52) 179 (168;189) 166 (146;203) 135 (124;179)*
71 (66;79) 66 (62;70) 64 (58;69) 2.2 (2.1;2.5) 2.3 (2.1;3.1) 1.5 (1.0;1.9) 96 (92;99) 88 (83;102) 109 (78;112) 2578 (2514;2998) 2345 (1636;2606) 4601 (4028;4790) 20 (18;25) 30 (29;33) 31 (27;34) 13 (9;14) 17 (9;19) 14 (12;14) 1.47 (0.85;3.31) 2.09 (1.12;3.42) 1.66 (0.21;3.68) 70.7 (58.3;78.8) 48.4 (44.4;57.5) 39.1 (7.2;55.6) 0.42 (0.38;0.52) 0.32 (0.27;0.35) 0.46 (0.40;0.50) 171 (169;173) 215 (214;218) 131 (117;157)
68 (61;80) 53 (50;56) 73 (62;76) 2.3 (2.0;2.3) 2.0 (1.8;2.4) 2.4 (1.9;2.7) 107 (101;117) 112 (102;115) 97 (87;100) 2242 (2109;2862) 2027 (1684;2485) 2287 (1763;3215) 20 (16;25) 26 (23;28) 30 (23;33) 13 (10;14) 13 (9;17) 10 (9;12) 1.41 (0.71;2.44) 0.23 (0.05;1.51) 1.08 (0.46;1.48) 58.4 (35.9;74.5) 10.4 (1.1;48.3) 39.3 (12.8;49.6) 0.38 (0.34;0.47) 0.26 (0.23;0.30) 0.38 (0.29;0.49) 176 (170;177) 212 (204;240) 210 (178;233)
65 (58;78) 54 (50;61) 66 (58;71) 2.2 (2.1;2.6) 2.2 (2.0;2.3) 1.7 (1.3;2.0) 109 (102;128) 123 (108;138) 93 (82;98) 2365 (2135;2702) 1993 (1918;2686) 2469 (2279;3161) 19 (15;27) 25 (24;30) 25 (23;26) 13 (10;13) 13 (8;20) 12 (8;18) 0.96 (0.59;2.24) 0.14 (0.02;0.86) 1.34 (0.34;2.77) 52.7 (37.4;76.6) 12.0 (1.7;31.1) 17.9 (10.5;40.8) 0.40 (0.33;0.49) 0.28 (0.22;0.29) 0.36 (0.27;0.46) 172 (170;179) 226 (191;256)* 156 (153;198)
Sham group: non-septic piglet receiving no amino acids, S-NAA group: septic piglets receiving no amino acids, S-AA group: septic piglets receiving amino acids. *Significant intragroup difference (P < 0.05) in comparison with baseline values. GFR, glomerular filtration rate; MAP, mean arterial pressure; PAP, pulmonary artery pressure; PCWP, pulmonary capillary wedge pressure; RBF, renal blood flow; RVR, renal vascular resistance; SVR, systemic vascular resistances.
Effect of bacterial infusion Fluid loading and epinephrine requirements during the experiment are shown in Fig. 3. A significant decrease in pH and increase in blood lactate was observed in both septic groups (P = 0.04 for S-NAA group, P = 0,001 for S-AA group). Following bacterial infusion, PAP increased significantly over time reaching a maximal value at T60: 40 (33; 42) mmHg in S-NAA group (P < 0,001) and 43 (28; 46) in S-AA group (P < 0.001). In comparison, no significant variation of PAP was observed in Sham group.
Amines and colloid requirements were significantly higher in septic groups in comparison with non-septic group (P = 0.04) (Fig. 3).
Effects of amino acids infusion on haemodynamic parameters Except PAP, which increased significantly following bacterial infusion, no significant variation was found over time in Sham, S-NAA and S-AA groups for CO, MAP, SVR, PCWP. There was no significant difference between groups. Acta Anaesthesiologica Scandinavica 59 (2015) 598–608
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Table 2 Main biochemical variables.
Parameters Lactate (mmol/l)
PaO2 (kPa)
PaCO2 (kPa)
pH
Bicarbonate (mmol/l)
Serum osmolality (mOsm/kg) Creatininemia (mmol/l)
Natremia (mmol/l)
Glycaemia (g/l)
Insulinemia (mU/l)
Glucagonemia (pg/ml)
Sham S-NAA S-AA Sham S-NAA S-AA Sham S-NAA S-AA Sham S-NAA S-AA Sham S-NAA S-AA Sham S-NAA S-AA Sham S-NAA S-AA Sham S-NAA S-AA Sham S-NAA S-AA Sham S-NAA S-AA Sham S-NAA S-AA
Baseline
Start bacterial infusion
Start of amino acids
End of experiment
T0
T40
T80
T120
T160
1.6 (1.1;2.6) 2.5 (2.0;3.2) 2.1 (1.5;2.4) 68.4 (61.2;68.4) 61.5 (60.4;66.6) 67.1 (64.4;67.9) 6.0 (5.3;6.0) 6.1 (5.8;6.4) 6.7 (6.2;6.8) 7.31 (7.31;7.42) 7.42 (7.40;7.43) 7.42 (7.39;7.43) 29.0 (29.0;29.8) 30.7 (27.7;31.7) 31.1 (26.9;32.8) 286 (284;291) 295 (284;299) 294 (287;297) 115 (81;130) 112 (96;126) 85 (76;96) 141 (139;144) 142 (142;143) 141 (139;143) 0.7 (0.5;0.8) 0.7 (0.5;1.0) 0.9 (0.8;1.0) 6.5 (2.8;8.0) 14.0 (8.3;19.0) 25.0 (17.0;27.8) 60 (38;80) 71 (65;87) 87 (67;101)
2.2 (1.5;4.1) 2.6 (2.2;3.7) 3.2 (2.4;4.3) 65.3 (62.1;65.3) 62.5 (61.9;67.9) 58.3 (56.8;65.9) 6.4 (5.4;6.4) 6.5 (6.0;7.0) 6.9 (6.6;7.4) 7.40 (7.33;7.42) 7.37 (7.33;7.42) 7.40 (7.35;7.40) 29.8 (29.0;29.8) 29.6 (26.7;30.7) 26.8 (26.2;31.9) 287 (284;289) 296 (293;312) 300 (291;304)* 117 (82;128) 105 (91;121) 80 (77;99) 142 (141;142) 144 (143;145) 143 (140;144) 0.8 (0.8;1.0) 0.8 (0.6;1.2) 1.3 (1.0;1.6) 9.0 (8.0;16.8) 14.5 (8.0;18.8) 18.0 (15.3;23.8) 76 (46;98) 67 (65;72) 82 (68;150)
1.9 (1.4;2.6) 2.9 (2.3;3.5) 5.9 (4.4;7.2)*† 63.1 (59.1;63.5) 55.9 (49.1;60.7) 59.7 (55.0;62.8) 5.5 (5.3;5.5) 5.9 (5.9;6.1) 6.4 (5.9;6.7) 7.44 (7.28;7.45) 7.34 (7.33;7.36) 7.33 (7.31;7.36) 28.8 (28.6;28.8) 26.5 (25.4;28.1) 27.7 (26.5;28.4) 292 (289;293) 302 (294;305)* 301 (295;310)* 118 (87;127) 105 (89;121) 98 (87;115) 141 (139;142) 145 (144;145) 143 (142;143) 0.8 (0.7;1.0) 1.0 (0.7;1.3) 1.8 (1.7;2.1)* 10.0 (6.3;10.0) 13.0 (9.3;16.0) 18.5 (14.0;24.5) 83 (70;105) 112 (99;121) 387 (213;400)*
1.9 (1.3;2.0) 3.1 (2.2;4.1) 4.5 (4.2;5.3)† 59.3 (59.3;64.8) 55.6 (47.2;63.6) 54.9 (47.3;64.4) 5.8 (5.1;5.9) 6.8 (6.1;7.1) 6.9 (6.3;7.0) 7.40 (7.25;7.41) 7.32 (7.31;7.32)* 7.32 (7.29;7.39) 29.9 (27.9;29.9) 26.3 (23.6;27.2) 24.5 (23.0;29.3) 290 (289;292) 301 (296;303)* 300 (298;310)* 118 (113 : 129) 96 (85;111) 102 (94;125) 141 (139;142) 145 (144;146) 144 (142;144) 0.7 (0.6;1.1) 0.8 (0.5;1.0) 1.5 (1.5;1.5) 8.5 (5.0;12.0) 14.0 (8.8;16.3) 19.0 (15.3;25.0) 100 (75;137) 101 (77;137) 132 (103;334)
1.7 (1.4;2.5) 4.0 (2.8;5.9)* 4.1 (3.8;5.9)† 65.2 (56.3;65.2) 55.3 (41.2;62.4) 54.0 (48.4;65.2) 5.6 (4.9;5.6) 7.3 (6.6;8.3) 7.2 (6.6;7.7) 7.42 (7.26;7.42) 7.29 (7.25;7.32)* 7.28 (7.25;7.32)* 27.6 (26.0;28.6) 23.9 (21.8;26.3) 23.2 (19.6;27.6) 293 (291;294) 298 (295;308)*† 303 (299;305)*† 128 (116.150) 99 (86;110) 103 (81;115)* 142 (141;142) 145 (144;145) 144 (141;144) 0.8 (0.6;1.2) 0.4 (0.4;0.9) 1.2 (1.1;1.3) 6.0 (5.3;9.8) 11.0 (9.3;14.3) 27.0 (18.8;27.0) 122 (76;340) 146 (142;161)* 131 (111;218)
Sham group: non -septic piglet receiving no amino acids, S-NAA group: septic piglets receiving no amino acids, S-AA group: septic piglets receiving amino acids. *Significant intragroup difference (P < 0.05) in comparison with baseline values. †Significant intergroup difference (P < 0.05) at a given period.
Effects of amino acids infusion on renal parameters No significant change in renal parameters over time was noticed in the Sham group. RBF and GFR decreased significantly, and RVR increased significantly over time in the S-AA group (P = 0.03 and P = 0.01, P = 0.02 respectively). Conversely, RBF increased slightly over time in the S-NAA group in comparison with baseline values (+19% at T160, P = 0.009), with no signifi-
cant concomitant variation of GFR and RVR. A significant intergroup difference was found between the S-NAA and S-AA groups at T60 for RBF (P = 0.03).
Effects of amino acids infusion on biochemical parameters Blood lactate concentration was significantly higher in S-AA group than in the Sham group from T60 to T160 (P = 0.04), whereas there was no
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Total ml
Total mg
1000
2000
900
*
800
*†
1800
*
1600
700
1400
600
1200
500
1000
400
800
300
600
200
400
100
200
0
0 Sham
S-NAA NaCl 0.9%
S-AA
Sham
S-NAA
S-AA
Colloids
Sham
S-NAA
S-AA
Epinephrine
Fig. 3. Amines and fluids requirement. Sham group: non-septic piglet receiving no amino acids, S-NAA group: septic piglets receiving no amino acids, S-AA group: septic piglets receiving amino acids. *P < 0.05, significant intergroup difference between S-NAA and Sham or S-AA and Sham; †P < 0.05, significant intergroup difference between S-NAA and S-AA group.
significant difference between the SNAA group and the Sham group. Creatinine level increased significantly over time in the S-AA group (P = 0.01), but remained within normal range. Levels of glucose and glucagon increased significantly over time in the S-AA group from T60 to T100 (P = 0.03 and P = 0.006 respectively), whereas only glucagon level increased significantly and late in the S-NAA group (P = 0.005) at T160. Plasma osmolality increased significantly in the S-AA group (P = 0.001) with a significant intergroup difference at T160 (P = 0.04). Sodium level increased significantly over time in the S-NAA group (P = 0.04, Friedman test), but remaining within normal range. Effects of amino acids infusion on fluid and amines administration Animals in the S-AA group tended to receive more isotonic saline and colloids, but there was no significant difference between these animals and those in the S-NAA group. An AA infusion
was significantly associated with a higher total requirement of epinephrine [340.0 (141.2–542.5) mg vs. 32.5 (3.8–65.0) mg in S-NAA group P = 0.04, and no epinephrine was used in the Sham group P = 0.01]. Discussion In our experimental septic model, AA infusion did not result in a preservation of renal haemodynamic. Conversely, we noticed a significant decrease in RBF and a significant increase in RVR, and this was also associated with a higher requirement of epinephrine in the AA-treated septic group. Our study aimed to evaluate renal haemodynamics in the time-course of sepsis. To meet this objective, a large animal model was chosen as it allows better haemodynamic monitoring, equipment and frequent blood sampling. In order to better reproduce human physiopathology of sepsis, we opted for an animal model that has been previously validated and described as hyperkinetic with an increase of CO and a concomitant decrease in systemic vascular resisActa Anaesthesiologica Scandinavica 59 (2015) 598–608
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tances.17 As these haemodynamic alterations appeared early in this model, a shorter follow up of the animals than the original description was chosen. We also opted for an early goal directed resuscitation to fulfil human recommendation for the treatment of sepsis. Despite this shorter experimental duration, we observed haemodynamic alterations, but the severity of shock, based on biochemical markers, was less than expected and described in the original description of the model. The development of sepsis AKI is a major concern because it is associated with a high mortality rate. Even though a decrease in RBF following excessive renal vasoconstriction has been mentioned,8,12 cases of AKI with a normal RBF have been reported.7,10,18 Based on magnetic resonance imaging and Doppler echography, increased RVR have also been implicated.19 Maintaining systemic haemodynamic parameters while preventing increased renal resistance may thus appear as a potential interesting strategy to prevent sepsis-induced AKI. As AA infusion has been reported to increase RBF and GFR and decrease RVR in healthy animal and patients,13,20,21 their use in sepsis condition in order to prevent AKI appeared attractive. However, renal effects of AA infusion in critical condition have not been evaluated to our knowledge. A meta-analysis supported their use in intensive care, with parenteral nutrition reducing mortality,22 but this study was not focused on kidney injury. Moreover, when examining in detail AA solutions, they contain several AA of potential interest during sepsis, which may exert a positive action on immunity or on signalling pathways altered in the time-course of septic shock such as nitric oxide (NO) pathway.13–16 Glutamic acid, arginine, lysine, phenylalanine and tyrosine are for example present in Vintene and have been associated with interesting immunomodulatory effect during sepsis.23–28 The AA dose infusion of 0.05 ml/kg/min (6 mg/ kg/min) was used originally to demonstrate the renal functional reserve in piglets.13 We used the same dose in our experimental setting, even though the AA solution was different. At this dosage, we observed an increase of plasma osmolality in our experimental condition (septic piglets), but also in non-septic piglets (personal data), presumably due to the hypertonicity of the
solution (1140 mOsm/L). However, this increase was slight and not significantly different with Sham group. More important was the rise in glucagon level noticed in the S-AA group in comparison with Sham and S-NAA groups. Such an increase has been described following AA infusion in healthy animals and patients,13,15,29 but might be undesirable during sepsis because hyperglycaemia or poor glycaemic control have been associated with an increased mortality.30 Regarding renal effects of AA, the increased RBF and decreased RVR in healthy patient remain to be explained: NO, prostaglandins and glucoregulatory hormones15,21 have been proposed as a putative mechanism. An alteration of renal haemodynamic has been reported in human patients with hyperkinetic shock.12 This was not found in our experimental model: no significant alteration of renal parameters was found in the non-AA septic group; conversely, a slight increase of RBF without any significant change in RVR and GFR was noticed at the end of the experiment for this group. This discrepancy between clinical data and our experimental data can be explained not only by the short time of follow up of the animals, but also by the early resuscitation criteria, based on human guidelines,31 which allowed prompt restoration of systemic haemodynamic parameters. Paradoxically, AA infusion altered renal function in our experimental critical conditions, whereas we were expecting at least its stabilisation or improvement. These results are also in accordance with those of an experimental study on rodents: the use of AA at therapeutic dosage worsened an ischaemic renal injury created by renal artery ligation. In this experiment, no putative mechanism was given to explain these AA deleterious effects.32 Whether the increased renal resistances are due to loss of renal autoregulation following severe hypotension or to direct effect of AA is difficult to elucidate in our experiment. It must be emphasised that many AA contained in the solution have opposite actions on NO pathway or immunity, some of them may have different effects depending if the patient is in septic shock or not.27 The diversity of AA contained in the solution renders difficult the interpretation and anticipation of its biological properties. It is thus possible that in critical condition, AA may worsen sys-
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temic and renal haemodynamic parameters. We cannot exclude that the timing of administration of AA may also influence their effect. At least, our results preclude their use in the early phase of sepsis as the shock appeared more severe in the S-AA group than in the S-NAA group, with a higher epinephrine requirement. Recently, a meta-analysis reported a decrease rate of infection with glutamine supplementation, but questioned the appropriate timing and patient selection to meet these positive effects.33 The greater severity of shock observed in the S-AA group is in accordance with results of a prospective study that reported an increased risk of death associated with the use of AA in septic shock in human patients.34 The intensity of shock observed during bacterial and AA infusions limits the interpretation of our results regarding renal function. It is difficult to conclude whether the alterations of renal parameters are due to the own systemic and renal effects of AA or due to this higher catecholamine requirement. Indeed, the use of epinephrine is associated with a major increase of RVR,35,36 which may have dampened the potential beneficial renal vasodilation of AA. Other factors may have contributed to the significant difference between groups in blood lactate and epinephrine requirements. It is possible that the high dose of epinephrine has precipitated the rise of blood lactate observed in the AA group, as hyperlactataemia is a welldescribed consequence of epinephrine administration.37,38 The kidney metabolises 20–30% of lactate; thus, alteration of renal function in the AA group may also have played a role in the increase blood lactate. Metabolic acidosis decreases myocardial sensitivity to catecholamines,39 which may have worsen the requirement of epinephrine in the time-course of shock. However, the clinical relevance of these factors can hardly be estimated in our experimental conditions. We acknowledge some limitations in our study. The small sample size of the groups may have dampened the differences between groups and decrease the power of our statistical analysis. Moreover, we decided to apply resuscitation criteria based on human guidelines, but early in the time-course of septic shock, which may have limited the development of AKI. Similar to the original description of the model, we used an inhalant anaesthetic agent that may have inter-
fered with vascular resistance and renal autoregulation. The Sham group was included in the experimental design in order to assess the influence of the anaesthetic protocol on haemodynamic and renal parameters. GFR was estimated with creatinine clearance, a method that requires steady state and may overestimate GFR in case of AKI.40 Due to the onset of shock, a steady state could not be achieved in our experimental setting, but urine was collected every 20 min directly from ureters, which is more accurate than a collection from the bladder over 24 h. Clearance of inulin or of a radiolabelled compound would have been more precise, but were not feasible in our experimental setting. As previously mentioned, we used epinephrine to resuscitate the animal, as it was described in the original model, but norepinephrine is recommended as the first-choice vasopressor in the early management of sepsis31 and would have probably been more appropriate regarding renal resistances. The development of septic-induced AKI is probably not solely related to alteration of RBF and renal vasoconstriction.5 The kidney may be injured at multiple levels: RBF, glomerular tubular cell function and structure, microcirculation, mitochondria and proteome. Particularly, sepsis is characterised with overproduction of proinflammatory mediators, which may lead to intrarenal inflammation and AKI.41,42 However, our experimental setting was designed to evaluate the potential interest of AA on renal resistance during sepsis and did not focus on renal inflammation. Conclusion In a piglet model of sepsis induced by P. aeruginosa infusion, an AA infusion significantly impaired renal haemodynamics. Further studies are needed to draw definite conclusions and clinical implications. Acknowledgement The authors would like to thank Sarah HetierJaccoud for her help in data collection and analysis. References 1. Oppert M, Engel C, Brunkhorst F-M, Bogatsch H, Reinhart K, Frei U, Eckardt K-U, Loeffler M, Acta Anaesthesiologica Scandinavica 59 (2015) 598–608
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