Angiotensin Ii in Septic Shock: Effects on Tissue Perfusion, Organ Function and Mitochondrial Respiration in a Porcine Model Of

ANGIOTENSIN II IN SEPTIC SHOCK: EFFECTS ON TISSUE PERFUSION,

ORGAN FUNCTION AND MITOCHONDRIAL RESPIRATION IN A PORCINE

MODEL OF FECAL PERITONITIS

Supplemental Digital Content

Authors:

Thiago D Corrêa, MD, PhD; Victor Jeger, MD; Adriano José Pereira, MD, PhD;

Jukka Takala, MD, PhD; Siamak Djafarzadeh, PhD; Stephan M Jakob, MD, PhD.

Supplemental Digital Content

Word text file containing additional information about the methods, along with related references and the Figure S1 which describes the hemodynamic protocol.

The results section contains additional 6 Tables and 7 Figures. Table S1 describes hemodynamics of non-septic sham controls; Table S2 gives an overview about the effect of untreated peritonitis; Table S3 gives the acid-base-balance parameters and respiratory system variables; Table S4 describes systemic and regional hemodynamics and arterial lactate level; Table S5 summarizes electrolytes and blood glucose analysis and Table S6 describes the main results of thrombelastography. Figure S1 depicts the hemodynamic protocol; Figure S2 shows the administered infusion rate of angiotensin II to non-septic sham controls; Figure S3 shows individual mean arterial blood pressure and vasopressor infusion rates; Figure S4 describes the stability of arterial PAH concentrations; Figure S5 represents respiration rates of isolated heart mitochondria;

Figure S6 shows respiration rates of isolated liver mitochondria and Figure S7 shows respiration rates of permeabilized heart fibers. METHODS

SEDATION, ANALGESIA AND VENTILATION

The pigs were sedated with intramuscular ketamine (20 mg/kg) and xylazine

(2 mg/kg) and a catheter was inserted in an ear vein for administration of fluids and drugs. Anesthesia was induced with midazolam (0.5 mg/kg) and atropine (0.02 mg/kg). Orotracheal and gastric tubes were inserted, and anesthesia maintained with propofol (4 mg/kg/h) and fentanyl (2-10 µg/kg/h). Additional fentanyl (50 µg) or midazolam (5 mg) were administered as needed. Volume-controlled ventilation with a positive end-expiratory pressure (PEEP) of 5 cm H2O, a fractional inspiratory oxygen concentration (FiO2) of 30%, and a tidal volume of 8 ml/kg (Servo-i®; Maquet

Critical Care, Solna, Sweden) was adjusted to maintain an arterial carbon dioxide partial pressure (PaCO2) between 35 and 45 mmHg.

HEMODYNAMIC SUPPORT

Throughout the resuscitation period, the volume status was evaluated clinically every hour. If signs of hypovolemia became evident, alternating boluses of 150 mL

Ringer’s lactate (RL) and 6% hydroxyethyl starch (HES 130/0.4) were given (Figure

S1). Fluid boluses were repeated as long as the stroke volume was increased by

10% or more after fluid administration. The maximum dose of HES administered was

30 ml/kg. After this maximal dose was reached, only boluses of Ringer’s lactate were given. If the mean arterial blood pressure was lower than 75 mmHg, vasopressors

(norepinephrine or angiotensin II) were administrated. If the mixed venous oxygen saturation (SvO2) was less than 50%, dobutamine administration was started at a dose of 5.0 mg per hour. This dose was increased by 5.0 mg per hour every 30 minutes until the SvO2 was 50% or higher or until a maximal dose of 20 mg per hour was given (Figure S1). 2

Further treatment consisted of antibiotics (piperacillin-tazobactam (Tazobac®)

2.25 g/8h, i.v.), tight blood glucose control and deep vein thrombosis prophylaxis

(continuous i.v. infusion of 10,000 IU of non-fractionated heparin/24 hours). Ringer’s lactate and Glucose 50% were infused adding up to 1.5 mL/kg/h (observation period), and to 3.0 mL/kg/h (resuscitation period), adjusted to keep blood glucose in the range of 3.5 to 5.0 mmol/L.

MONITORING

Hemodynamics, temperature and respiratory parameters (S/5 Critical Care

Monitor®; Datex-Ohmeda, GE Healthcare, Helsinki, Finland) thermodilution cardiac output, mixed venous oxygen saturation (SvO2;Vigilance®; Edwards Lifesciences

LLC, Irvine, CA, USA), and carotid and femoral artery blood flows (Transonic

Systems Inc., Ithaca, NY, USA) were continuously measured and recorded

(Soleasy™; National Instruments Corp., Austin, TX, USA Centricity Clinisoft®; GE

Healthcare, Helsinki, Finland). Systemic VO2 was calculated based on the Fick principle (3).

BLOOD SAMPLING

Blood from the carotid artery was withdrawn at baseline and every six hours from the indwelling catheter and immediately analyzed in a blood gas analyzer (GEM

Premier 3000 analyzer; Bedford, MA, USA) for PaO2, PaCO2 (adjusted to central body temperature), pH, lactate (mmol/L), base excess (BE), sodium, potassium and calcium. Arterial oxygen saturation and total hemoglobin concentration were measured at baseline and every six hours using a separate analyzer (OSM 3;

Radiometer, Copenhagen, Denmark, porcine mode).

Plasma interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) were determined using a porcine immunoassay kit (R&D Systems Europe Ltd; Abingdon,

Oxon, UK). Hemoglobin, platelets, white blood cell count, serum creatinine, total bilirubin, IL-6 and TNF-alpha were measured with blood samples from the carotid artery at baseline, the end of the observation period, and at the end of the study (48 hours of resuscitation or before death, for animals that died before completion of the study).

Blood from the pulmonary artery was withdrawn at baseline and every 6 hours for mixed oxygen saturation measurement using a Radiometer OSM 3 blood gas analyzer (OSM 3; Radiometer, Copenhagen, Denmark, porcine mode).

Blood from the kidney vein was withdrawn at baseline, end of the observation period, and at the end of the study (48 hours of resuscitation or before death, for animals that died before completion of the study) for kidney vein oxygen saturation measurement using a Radiometer OSM 3 blood gas analyzer (OSM 3; Radiometer,

Copenhagen, Denmark, porcine mode).

THROMBELASTOGRAPHY

Thrombelastography (TEG) was performed by a TEG® thrombelastograph

5000 (Haemonetics Corporation, Braintree, MA, USA). Temperature was set to 37°C for all samples. Citrated whole blood samples (340 µl) for TEG assays were recalcified by adding 0.2M CaCl2 (20 µl) and coagulation was activated by kaolin.

Heparinase coated cups were used to prevent any heparin effect. TEG instruments were tested for quality control before the measurement of baseline parameters using standardised samples provided by the manufacturer and all measured quality control parameters were always within the range. TEG samples were analysed at induction of anaesthesia, at the end of observation period and at the end of the experiment 4

(Figure 1, main manuscript). Four main TEG parameters were analyzed in this study:

R (reaction time, time from start of test till begin of clot formation), K (time from activation of coagulation till 20 mm of amplitude), α-angle and maximal amplitude

(MA).

ESTIMATION OF RENAL BLOOD FLOW AND FUNCTION

Renal blood flow was estimated using primed, continuous infusion of para- amininohippurate (PAH; Aminohippurate sodium; Merck & CO., INC, Whitehouse

Station, NJ). After a priming bolus (8 mg/kg), para-amininohippurate (PAH;

Aminohippurate sodium; Merck & CO., INC, Whitehouse Station, NJ) of 20% paraaminohippurate [PAH; was continuously at 8mg/min for 95 minutes. Urine samples were collected at 30, 60 and 90 minutes. Blood samples from carotid artery and kidney vein were collected at 30, 60, 85, 90 and 95 minutes. PAH concentration was measured using the colorimetric PAH assay kit from BioVision (Milpitas, CA,

USA).

PAH clearance was calculated as PAH infusion rate/arterial-renal vein PAH concentration difference (1, 2) at time of highest renal PAH extraction PAH extraction

<0.20, was disregarded. The reproducibility of PAH measurements was assessed from 244 duplicate blood samples. Colorimetric PAH assay were performed in duplicates, and measurements were repeated if the coefficient of variation (standard deviation of measurements divided by their average) was >10%.

Six hours urine samples were collected before baseline, end of observation period and end of the experiment. Creatinine, sodium and potassium concentrations were measured using standard analysis from these samples and fractional excretions of sodium and potassium were calculated following the formula: FE = (urineelectrolyte x serumcreatinine x 100) / (serumelectrolyte x urinecreatinine). Sodium results “<10 mmol/L” = 5 below detection threshold (51/120 measurements, 43%), were replaced by 10 in order to calculate FENa+ in all animals.

Acute kidney injury (AKI) was defined according to the AKIN criteria as an absolute increase in serum creatinine ≥0.30 mg/dl (≥26.4 µmol/L) or a percentage increase in serum creatinine of more than or equal to 50% (1.5-fold from baseline) (3,

4).

MITOCHONDRIAL FUNCTION ANALYSIS

Tissue samples were taken from the kidney, liver and heart at the end of the experiment. In animals that died earlier, the final samples were taken when the animals were still alive, receiving the maximal vasopressor dose (5000 mcg/h of norepinephrine or 1000 ng/kg/min of angiotensin II), and when mean arterial blood pressure approached 30 mmHg.

Complex I-, II- and IV-dependent respiration rates were measured as described previously using high-resolution respirometry (Oxygraph-2k; Oroboros

Instruments, Innsbruck, Austria) (5, 6) and is expressed as pmol/second/mg mitochondrial protein. For heart fibers, state 3 respiration was measured using glutamate/malate, succinate and ascorbate/TMPD as substrates for complex I, II and

IV, respectively. Maximal electron transport system capacity was also measured in the presence of substrates for both complex I and complex II.

State 3 represents active respiration after addition of ADP and State 4 represents the respiration after depletion of ADP. The respiratory control ratio (RCR) is calculated by dividing the state 3 by the state 4 respiration rate.

ISOLATION OF LIVER AND KIDNEY MITOCHONDRIA

Isolation of liver or kidney mitochondria was performed immediately after tissue harvesting at 4°C using a standard procedure based on differential centrifugation. All centrifugations were performed at 4°C. The samples of liver or kidney (15 g) excised at the end of the experiment were rapidly immersed in ice-cold liver isolation buffer (mannitol 220 mmol/l, sucrose 70 mmol/l, morpholinopropane sulfonic acid 5 mmol/l, pH 7.4), minced with scissors, and homogenized with an additional 10 ml/g of homogenization media (liver isolation buffer plus EDTA 2 mmol/l) in a Potter Elvehjem homogenizer with a loose-fitting Teflon pestle (four strokes). The homogenate was then centrifuged for 10 minutes at 700 g. The supernatant was collected and centrifuged again for 10 minutes at 7,000 g. The supernatant was discarded at this time; the pellet was then resuspended in isolation buffer and centrifuged twice for 10 minutes at 7,000 g for further purification of the mitochondria. The pellets were then suspended in buffer at a final concentration of

50–100 mg mitochondrial protein per milliliter.

PREPARATION OF PERMEABILIZED HEART FIBERS

Heart fibers were isolated by dissection of the muscle tissue in relaxing

2+ solution (in mM: 2.77 CaK2EGTA, 7.23 K2EGTA [free Ca concentration 0.1 µM], 20

imidazole, 20 taurine, 6.56 MgCl2, 5.77 ATP, 15 phosphocreatine, 0.5 dithiothreitol, and 50 K-MES, pH 7.1) on ice and permeabilized by gentle agitation for 30 min on ice in the relaxing solution supplemented with 50 µg/ml saponin (7). Fibers were washed in ice-cold respiration medium (110 mM sucrose, 60 mM K-lactobionate, 0.5 mM

EGTA, 1 g/L bovine serum albumin essentially fatty acid-free, 3 mM MgCl2, 20 mM

taurine, 10 mM KH2PO4, 20 mM HEPES, pH 7.1) (8) by agitation for 20 min on ice and were kept in this medium until respirometric assay. 7

CARDIAC MUSCLE MITOCHONDRIAL ISOLATION

Mitochondria were isolated from left ventricular heart muscle immediately after tissue harvesting at 4°C using a trypsin digestion procedure (9, 10). Tissue (4-5 g) was minced, washed, and suspended in 30 ml of ice cold isolation medium (0.3 M sucrose, 10 mM sodium HEPES, pH 7.2, and 0.2 mM EDTA). The tissue was subjected to mild trypsin digestion (1.25 mg) for 15 min at 4°C and then diluted with

10 ml of isolation medium (pH 7.4) containing 1 mg/ml BSA and 6.5 mg of trypsin inhibitor. The suspension was stirred, and the supernatant was discarded. The partially digested tissue was resuspended in 10 ml of isolation medium containing

1 mg/ml albumin and homogenized briefly with a Teflon-glass homogenizer. The homogenate was centrifuged for 10 min at 600 g (4°C). The resulting supernatant was centrifuged for 15 min at 8,000 g (4°C). The supernatant was discarded, and the pellet was resuspended twice in 10 ml of isolation medium containing 1 mg/ml albumin and centrifuged each time for 15 min at 8,000 g (4°C). The final washed pellet was suspended in 1 ml of isolation medium without albumin.

SKELETAL MUSCLE ATP CONTENT

Samples from quadriceps femoris muscle were taken at the end of the experiment and frozen immediately in liquid nitrogen and stored for further analysis at

-80°C. Skeletal muscle ATP content (µmol/g tissue wet weight) was quantified using a luciferase-based ATP determination kit (Molecular Probes; Eugene, OR, USA).

ANGIOTENSIN II DOSE-RESPONSE ANALYSIS

The first three hours after start of angiotensin II infusion were selected to compare the dose-response in sham controls and septic animals. All animals 8 received the same dose of angiotensin II in the beginning (1000 ng/min), which corresponds to 24 ng/kg/min (mean body weight 41 kg). The infusion rate was further adjusted following the study protocol described in the method section. Medians from

2-min-median mean arterial blood pressure were plotted for both groups and assessed qualitatively.

RESULTS

PAH-CLEARANCE QUALITY CONTROL

Due to technical problems/low PAH extraction, PAH clearance could not be calculated for 2 out of 24 animals at baseline and at study end, and for 4 animals at

EOP (renal vein oxygen saturation data from the same time points were also excluded from further analysis). Arterial paraaminohippurate (PAH) concentrations were stable after 30-60 minutes of PAH infusion (Figure S2). The inter-assay PAH concentration coefficient of variation (CV, 244 pairs of measurements) was 0.07 ±

0.07. The CV for PAH concentrations of duplicate blood samples (mean of 60 and 90 minutes samples) was 0.13 ± 0.13 (n=83, arterial) and 0.16 ± 0.22 (n=79, venous) respectively.

EFFECTS OF ANGIOTENSIN CONVERTING ENZYME INHIBITION

The maintenance dose of enalapril during the resuscitation was reduced in all animals due to refractory hypotension from initially 0.02 mg/kg/h to a minimum predefined dose of 0.005 mg/kg/h.

Mean pulmonary artery pressure, cardiac output, systemic oxygen consumption and arterial lactate levels did not differ between the groups in the end of the study (Figure 2 main article; Table S4). All enalapril pretreated animals required glucose infusion to maintain blood glucose concentration during the course of the study, but the amount required was smaller compared to the Group NE (Table 1 main article).

Mitochondrial function

State 3 and 4 of complex I dependent respiration of isolated renal mitochondria were lower in group E than in group NE (Figure 4 main article), without 10 affecting its efficiency (RCR, respiratory control ratio). Complex II RCR was higher, but complex IV dependent respiration smaller (Figure 4 main article). In contrast,

RCR of complex II dependent respiration in heart mitochondria was lower in group E

(p=0.021; Figure S5), and liver mitochondria were not affected (Figure S6).

Skeletal muscle ATP content

The ATP content in skeletal muscle samples at the end of the experiment was higher in group E as compared to group NE (p=0.007; Figure 5 main article).

Table S1. Hemodynamics of non-septic sham control group (n=4) during highest angiotensin II infusion at maximal mean arterial blood pressure. Values are medians

(range).

Before angiotensin II Max angiotensin II Variables % change infusion infusion MAP (mm Hg) 76 (74 - 77) 131 (96 - 154) 72 (30 - 102) MPAP (mm Hg) 13 (12 - 15) 19 (15 - 22) 44 (15 - 73) Heart rate (beats/min) 76 (60 - 103) 88 (64 - 101) -3 (-9 - 68) Cardiac output (ml/kg/min) 93 (71 - 162) 91 (74 - 130) -2 (-20 - 3)

SvO2 (%) 52 (47 - 58) 57 (52 - 69) 8 (-6 - 44) CVP (mm Hg) 2.2 (1.7 - 3.2) 3.0 (2.3 - 4.3) 40 (-5 - 83)

Legend: Angiotensin II infusion rate: 24.4 ng/kg/min (23.3 – 27.0, median and range).

MAP: mean arterial blood pressure, MPAP: mean pulmonary artery pressure, SvO2: mixed venous oxygen saturation, CVP: central venous pressure.

Table S2. Effect of untreated peritonitis. Values are medians with interquartile ranges.

NE + AT-II (n=16) E (n=8) NE + AT-II E

§ § delta EOP- delta EOP- # Baseline EOP p-value Baseline EOP p-value p-value Baseline Baseline MAP (mm Hg) 65 (62-70) 73 (62-80) 0.461 63 (60-73) 64 (56-73) 0.844 5 (-5-13) -1 (-12-10) 0.620

HR (beats/min) 91 (84-95) 175 (161-185) <0.001 83 (67-87) 166 (134-178) 0.008 87 (76-106) 76 (67-95) 0.481

MPAP (mm Hg) 12 (10-14) 15 (14-18) <0.001 13 (11-15) 16 (13-18) 0.008 3 (1-6) 3 (2-3) 0.964

CVP (mm Hg) 2 (1-3) 1 (0-1) 0.003 1 (0-2) 0 (-1-2) 0.023 -1 (-2-0) -1 (-2-0) 0.467

PAOP (mm Hg) 3 (2-3) 1 (0-2) <0.001 3 (3-4) 1 (1-2) 0.031 -2 (-3- -1) -2 (-3- -1) 0.933

Cardiac output 89 (82-97) 66 (62-85) 0.002 109 (95-111) 88 (80-104) 0.078 -18 (-33- -4) -9 (-24- 1) 0.417 (ml/kg/min)

SvO2 (%) 48 (44-52) 50 (47-56) 0.110 55 (50-59) 51 (49-62) 0.742 3 (-1-10) -1 (-7-4) 0.257

PAH Clearance 19 (15-21) 12 (11-16) <0.001 16 (14-21) 14 (13-18) 0.461 -6 (-8- -3) 0 (-6-2) 0.082 (ml/kg/min) Systemic VO2 240 (219-266) 263 (239-292) 0.074 252 (208-262) 300 (274-319) 0.008 18 (-5-59) 65 (13-86) 0.093 (ml/min)

Arterial lactate 0.8 (0.7-0.9) 1.5 (1.0-2.0) 0.003 0.8 (0.6-0.9) 1.2 (1.1-1.4) 0.023 0.5 (0.1-1.3) 0.5 (0.3-0.8) 0.869 (mmol/L) FE Sodium (%) 0.18 (0.09-0.30) 0.04 (0.03-0.06) <0.001 0.09 (0.06-0.11) 0.05 (0.03-0.06) 0.008 -0.13 (-0.25- -0.05) -0.04 (-0.07- -0.03) 0.034

FE Potassium (%) 17 (12-25) 6 (5-9) <0.001 17 (15-20) 10 (6-11) 0.008 -11 (-20- -5) -8 (-12- -5) 0.265

Legend: NE: norepinephrine group, AT-II: angiotensin II group, E: enalapril +

norepinephrine group. EOP: end of observation period. MAP: mean arterial blood

pressure, HR: heart rate, MPAP: mean pulmonary artery pressure, CVP: central

venous pressure, PAOP: pulmonary artery occlusion pressure, SvO2: mixed venous

oxygen saturation, PAH: paraaminohippurate, VO2: oxygen consumption. NE + AT-

II: effect of untreated peritonitis in 16 animals, which were later randomized to either

norepinephrine or angiotensin II vasopressor treatment. Delta: difference of variables

at EOP – baseline. p-values for §: Wilcoxon signed rank test and #: Mann-Whitney U-

test.

Table S3. Arterial blood gases analysis and respiratory parameters. Values are

medians with interquartile ranges.

Variables Group BL EOP RP 12h RP 24h RP 36h RP 48h P value ¶ pH NE 7.52 (7.49-7.53) 7.43 (7.42-7.44) 7.46 (7.42-7.50) 7.47 (7.44-7.50) 7.47 (7.41-7.49) 7.48 (7.41-7.49) 0.219 a

AT-II 7.49 (7.47-7.51) 7.46 (7.41-7.46) 7.47 (7.45-7.48) 7.45 (7.40-7.48) 7.44 (7.42-7.47) 7.41 (7.40-7.43) 0.052 b

E 7.48 (7.47-7.51) 7.43 (7.42-7.45) 7.42 (7.39-7.43) 7.47 (7.44-7.49) 7.46 (7.43-7.50) 7.49 (7.42-7.51) 0.094 c

Bicarbonate NE 33.1 (31.1-34.3) 26.0 (24.2-27.7) 27.0 (25.3-28.0) 29.1 (28.3-31.1) 29.5 (29.1-31.4) 30.6 (27.4-32.7) <0.001 a

(mmol/L) AT-II 31.1 (30.3-32.3) 26.5 (25.8-27.5) 27.4 (25.2-31.1) 30.3 (28.0-31.0) 30.7 (29.4-31.3) 30.6 (28.9-32.0) 0.001 b

E 30.8 (29.7-31.7) 25.7 (24.9-26.7) 23.6 (21.7-25.1) 25.4 (24.7-26.8) 25.0 (24.7-25.9) 24.7 (23.1-28.0) 0.227 c

BE NE 10.2 (7.6-11.5) 2.9 (1.4-3.8) 4.3 (2.3-5.1) 5.9 (4.6-8.3) 6.6 (4.7-8.2) 7.3 (3.8-9.0) 0.001 a

(mmol/L) AT-II 7.8 (7.0-8.5) 3.7 (2.8-4.7) 4.5 (3.1-7.6) 6.3 (5.3-6.9) 6.8 (5.4-7.4) 5.8 (3.1-7.7) 0.139 b

E 7.7 (6.0-8.4) 2.6 (2.0-3.5) 0.8 (-2.0-2.0) 3.3 (2.3-3.6) 2.7 (1.1-3.4) 1.1 (-0.6-5.5) 0.133 c

a PaCO2 NE 41. (39.5-41.5) 40.0 (38.5-42.0) 39.0 (36.0-41.0) 42.0 (41.0-43.0) 43.5 (40.0-50.5) 45.5 (41.5-47.5) 0.030 (mm Hg) AT-II 41.5 (40.0-45.5) 40.5 (38.5-45.0) 40.0 (37.0-42.5) 43.0 (40.0-50.0) 45.5 (43.5-48.0) 50.0 (46.0-50.0) <0.001 b E 42.0 (39.5-43.5) 40.0 (38.5-42.0) 38.0 (36.0-39.0) 37.0 (34.0-39.0) 38.0 (31.0-41.0) 37.0 (34.0-39.0) 0.205 c

MV NE 8.0 (7.3-8.5) 8.8 (8.2-9.8) 9.8 (8.7-10.7) 10.1 (9.3-11.1) 10.5 (9.6-10.9) 10.2 (9.6-11.7) 0.019 a

(L/min) AT-II 8.0 (7.5-8.3) 8.1 (7.8-8.8) 8.1 (7.7-9.6) 10.3 (9.3-10.9) 10.1 (9.4-11.6) 10.6 (9.0-11.2) 0.002 b

E 8.3 (7.4-8.6) 8.8 (8.3-9.5) 10.6 (9.4-11.6) 10.8 (10.2-11.3) 10.2 (10.0-11.3) 10.5 (10.1-10.8) 0.006 c

a PO2/FiO2 NE 458 (452-473) 440 (423-455) 422 (405-437) 320 (300-358) 259 (213-293) 195 (173-256) <0.001 AT-II 452 (423-475) 430 (412-455) 392 (344-453) 195 (170-332) 183 (149-226) 177 (102-203) <0.001 b

E 470 (437-488) 452 (432-472) 300 (224-372) 198 (172-272) 196 (170-318) 197 (162-300) <0.001 c a PEEP NE 5 (5-5) 5 (5-5) 5 (5-5) 5 (5-5) 7 (6-8) 8 (6-10) <0.001 5 (5-5) 5 (5-5) 5 (5-5) 7 (5-11) 10 (9-12) 10 (10-12) <0.001 b (cm H20) AT-II c E 5 (5-5) 5 (5-5) 5 (5-8) 8 (6-8) 10 (8-12) 10 (8-12) <0.001

Legend: NE: fecal peritonitis control group, AT-II: angiotensin II group, E: enalapril +

norepinephrine group, BL: baseline, EOP: end of observation period, RP:

resuscitation period, pH: arterial pH, BE: arterial base excess, PaCO2: arterial carbon

dioxide partial pressure, MV: expired minute volume, PaO2/FiO2: arterial oxygen

partial pressure and fraction of inspired oxygen relationship PEEP: positive end-

expiratory pressure. ¶: p values: time effect with Friedman’s test including EOP, Rp

12h, Rp 24h, Rp 36h and Rp 48h for (A): NE, (B): AT-II and (C): E.

Table S4. Systemic and regional hemodynamics and arterial lactate level. Values are

medians with interquartile ranges.

Variables Group BL EOP RP 12h RP 24h RP 36h RP 48h P value ¶

Heart rate NE 87 (82-98) 176 (169-206) 177 (160-187) 170 (163-186) 139 (133-159) 125 (113-136) <0.001 a

(beats/min) AT-II 92 (91-95) 171 (157-181) 159 (144-164) 165 (147-170) 137 (99-167) 124 (110-158) 0.009 b

E 83 (67-87) 166 (134-178) 168 (154-194) 168 (158-172) 144 (116-200) 157 (130-159) 0.432 c

a SvO2 NE 49 (45-54) 52 (48-58) 58 (56-63) 64 (60-68) 63 (59-71) 64 (58-67) 0.038 (%) AT-II 46 (44-50) 50 (47-54) 59 (53-63) 59 (56-62) 58 (56-60) 55 (46-56) 0.008 b

E 55 (50-59) 51 (49-62) 69 (63-72) 76 (75-80) 72 (69-76) 77 (63-77) 0.007 c

CVP NE 2 (1-3) 1 (0-1) 2 (1-3) 3 (2-5) 5 (3-7) 6 (3-9) <0.001 a

(mm Hg) AT-II 2 (1-3) 1 (0-2) 2 (1-4) 4 (2-7) 8 (5-10) 10 (6-11) <0.001 b

E 2 (0-3) 1 (-2-3) 4 (0-7) 3 (2-5) 5 (4-6) 5 (2-8) <0.001 c

PAOP NE 3 (2-5) 1 (0-2) 3 (2-4) 3 (2-5) 6 (4-7) 6 (5-9) <0.001 a

(mm Hg) AT-II 3 (2-3) 0 (0-2) 3 (2-4) 5 (4-6) 8 (6-8) 9 (7-10) <0.001 b

E 3 (3-4) 1 (1-2) 4 (2-5) 5 (3-6) 7 (6-8) 8 (6-9) <0.001 c

Arterial lactate NE 0.8 (0.6-0.8) 1.5 (1.1-2.8) 1.5 (1.4-1.6) 1.2 (1.1-1.6) 1.2 (1.0-1.2) 0.9 (0.8-1.1) <0.001 a

(mmol/L) AT-II 0.9 (0.8-1.1) 1.4 (1.0-1.8) 1.3 (1.0-1.4) 0.8 (0.8-1.1) 0.6 (0.6-0.9) 0.9 (0.5-1.2) <0.001 b

E 0.8 (0.6-0.9) 1.2 (1.1-1.4) 1.8 (1.4-3.1) 1.0 (0.9-1.3) 0.8 (0.7-1.1) 0.8 (0.6-1.0) 0.006 c

d Systemic VO2 NE 240 (229-258) 264 (237-286) 256 (227-294) 0.641 (ml/min) AT-II 223 (213-280) 263 (241-309) 296 (242-328) 0.461 e

E 252 (208-262) 300 (274-319) 250 (238-284) 0.070 f

Legend: NE: fecal peritonitis control group, AT-II: angiotensin II group, E: enalapril +

norepinephrine group, BL: baseline, EOP: end of observation period, RP:

resuscitation period, SvO2: mixed venous oxygen saturation, CVP: central venous

pressure, PAOP: pulmonary artery occlusion pressure, VO2: oxygen consumption, ¶:

p values: time effect with Friedman’s test including EOP, Rp 12h, Rp 24h, Rp 36h

and Rp 48h for (A): NE, (B): AT-II and (C): E; time effect with Wilcoxon signed rank

test including EOP and END for (d): NE, (e): AT-II and (f): E.

Table S5. Electrolytes and arterial blood glucose analysis. Values are medians with

interquartile ranges.

Variables Group BL EOP RP 12 h RP 24 h RP 36 h RP 48 h P value ¶

Sodium NE 138 (135-140) 135 (134-137) 134 (133-135) 135 (133-136) 132 (131-133) 134 (131-135) 0.093 a

(mmol/L) AT-II 137 (136-138) 135 (135-137) 135 (134-136) 137 (135-137) 136 (134-137) 136 (135-138) 0.479 b

E 137 (136-138) 135 (134-137) 135 (133-135) 134 (133-135) 134 (134-135) 134 (132-135) 0.050 c

Potassium NE 4.3 (4.1-4.3) 4.8 (4.5-5.4) 4.8 (4.6-5.0) 4.5 (4.2-4.7) 4.6 (4.2-4.9) 4.3 (4.0-5.8) 0.425 a

(mmol/L) AT-II 4.6 (4.1-4.8) 5.3 (4.9-5.5) 4.6 (4.5-4.7) 4.5 (4.3-4.9) 4.5 (4.1-5.0) 4.6 (4.1-5.2) 0.019 b

E 4.3 (4.1-4.4) 4.5 (4.3-5.0) 4.3 (4.2-5.5) 4.8 (4.6-5.5) 4.7 (4.6-5.5) 4.9 (4.6-5.1) 0.480 c

Calcium NE 1.26 (1.25-1.29) 1.17 (1.15-1.21) 1.17 (1.15-1.17) 1.21 (1.20-1.23) 1.24 (1.23-1.25) 1.26 (1.25-1.27) 0.001 a

(mmol/L) AT-II 1.29 (1.24-1.33) 1.17 (1.12-1.19) 1.20 (1.19-1.23) 1.21 (1.19-1.27) 1.30 (1.29-1.30) 1.31 (1.30-1.34) <0.001 b

E 1.25 (1.22-1.27) 1.12 (1.07-1.13) 1.11 (1.07-1.14) 1.17 (1.13-1.19) 1.19 (1.16-1.20) 1.20 (1.11-1.25) <0.001c

BG NE 4.9 (4.5-5.1) 3.3 (3.2-3.5) 3.3 (3.1-3.9) 3.7 (3.7-3.8) 4.2 (3.8-4.6) 5.0 (4.6-5.5) 0.001 a

(mmol/L) AT-II 6.3 (5.4-6.6) 3.3 (3.2-3.8) 3.6 (3.5-3.6) 3.8 (3.5-3.8) 3.9 (3.6-4.2) 4.7 (4.4-6.2) <0.001b

E 5.2 (4.6-5.8) 3.8 (3.5-4.1) 4.0 (3.8-4.2) 3.8 (3.4-3.9) 4.9 (4.2-6.2) 5.4 (3.8-5.6) 0.020 c

Legend: NE: fecal peritonitis control group, AT-II: angiotensin II group, E: enalapril +

norepinephrine group, BL: baseline, EOP: end of observation period, RP:

resuscitation period, BG: arterial blood glucose. ¶: p values: time effect with

Friedman’s test including EOP, Rp 12h, Rp 24h, Rp 36h and Rp 48h for (A): NE, (B):

AT-II and (C): E.

Table S6. Main results of thrombelastography. Values are medians with interquartile ranges.

Variables Group BL EOP End P value¶

r NE 6.0 (5.0-7.0) 5.0 (4.0-6.0) 4.5 (4.0-5.0) 1.000 a

(minutes) AT-II 7.5 (7.0-8.0) 6.0 (6.0-6.0) 5.5 (4.5-7.0) 0.813 b

E 7.0 (6.5-8.0) 6.5 (6.0-7.0) 5.0 (3.5-6.0) 0.031 c

0.055 d

k NE 1.0 (1.0-1.5) 1.0 (1.0-2.0) 1.0 (1.0-1.0) 1.000 a

(minutes) AT-II 2.0 (1.0-2.0) 2.0 (1.0-2.0) 1.5 (1.0-2.0) 1.000 b

E 2.0 (1.0-2.0) 2.0 (1.0-2.0) 1.0 (1.0-1.5) 0.250 c

0.180 d

α angle NE 73 (70-76) 70 (67-75) 73 (72-75) 1.000 a

(deg) AT-II 67 (62-73) 69 (65-72) 71 (65-74) 0.836 b

E 70 (66-72) 59 (56-68) 73 (68-76) 0.023 c

0.052 d

MA NE 71 (65-73) 67 (64-73) 67 (62-71) 0.625 a

(mm) AT-II 74 (72-77) 73 (71-75) 73 (69-77) 0.844 b

E 72 (70-73) 73 (70-76) 70 (69-72) 0.016 c

0.115 d

Legend: BL: baseline, EOP: end of observation period, End: end of the experiment

(at 48 hours of resuscitation or before death if earlier). r: reaction time (time from start of test till begin of clot formation, K: time from activation of coagulation till 20 mm of amplitude) and MA: maximal amplitude. ¶: p values: time effect with Wilcoxon signed rank test including EOP and END for (a): NE, (b): AT-II, (c): E and (d): conducted on the pooled data from all groups comparing EOP to End .

Figure S1. Hemodynamic protocol.

Legend: MAP = mean arterial blood pressure, SvO2 = mixed venous oxygen saturation, SV = stroke volume, CVP = central venous pressure, PAOP = pulmonary artery occlusion pressure, SVR = systemic vascular resistance, RL = Ringer’s lactate, HES = 6% hydroxyethyl starch (130/0.4), ↑ = increasing, ↓ = decreasing, a =

= alternating boluses of 150 mL Ringer’s lactate and 6% hydroxyethyl starch (HES

130/0.4) until the maximum dose of 30 ml/Kg of HES is reached. Then, only boluses of Ringer’s lactate were given, b = norephinephrine was administrated to

18 norepinephrine group (NE) and to enalapril + norepinephrine group (E). Angiotensin

II was administrated to angiotensin II (AT-II) and to sham control groups. The maximal doses of vasopressors allowed were 5000 mcg/h for norepinephrine and

1000 ng/kg/min for angiotensin II.

Figure S2. Administered angiotensin II to non-septic sham controls (n=4).

Legend: Values indicate median (range) of angiotensin II infusion rate during the 48 hours of protocolized resuscitation.

Figure S3. Individual mean arterial blood pressure and vasopressor infusion rates.

Legend: NE: norepinephrine group; AT-II: angiotensin II group; E: enalapril group.

Red lines (and red circles) represent mean arterial blood pressure of each animal; black dotted lines (and black squares) represent vasopressor (norepinephrine or angiotensin II) infusion rates. MAP: mean arterial blood pressure; BL: baseline; EOP: end of observation period (untreated sepsis); RP(12h, 24h, 36h, 48h): resuscitation period 12h, 24h, 36h, 48h after start.

Figure S4. Stability of arterial paraaminohippurate (PAH) concentrations in blood samples from the carotid artery.

Legend: Stability of PAH concentrations. EOP: end of observation period, End: end of the experiment (at 48 hours of resuscitation or before death if earlier). Outlier in

EOP and at study end: same animal, which was anuric at EOP.

Figure S5. Isolated heart mitochondrial respiration.

Legend: NE: norepinephrine group, AT-II: angiotensin II group, E: enalapril + norepinephrine group. State 3 and 4 oxygen consumption is expressed as pmol/second/mg protein. State 3: active respiration after addition of ADP. State 4: respiration after consumption of ADP. RCR: respiratory control ratio (oxygen consumption of state 3/state 4). Horizontal lines represent median values. Filled circles represent animals that died early. p values: Mann-Whitney U-test comparing

(A) AT-II vs. NE and (B) E vs. NE.

Figure S6. Isolated liver mitochondrial respiration.

(A) AT-II vs. NE and (B) E vs. NE.

Figure S7. Oxygen consumption of permeabilized heart fibers.

Legend: NE: norepinephrine group, AT-II: angiotensin II group, E: enalapril + norepinephrine group. State 2: respiration after addition of substrates, before ADP supplementation. State 3: active respiration after addition of ADP. p values: Mann-

Whitney U-test comparing (a) AT-II vs. NE and (b) E vs. NE.

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