INTRODUCTION
Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection. Septic shock is characterized by a vasopressor requirement to maintain a mean arterial blood pressure of 65 mm Hg in the absence of hypovolemia (1) (2–4) (5) (6) (7)
Adrenomedullin (ADM), a vasoactive peptide of 52 amino acids, is secreted by various tissues into the bloodstream and exerts a stabilizing effect on the endothelial barrier. In experimental animal studies, application of ADM in septic shock led to improvement of organ function and better survival by reducing the vascular hyperpermeability and inflammation (8–12) (13) (14–17)
A potential new therapeutic option in sepsis may be Adrecizumab, a humanized non-neutralizing anti-ADM antibody (Adrenomed AG, Germany), which is directed against the N-terminal part of ADM. In several septic rodent models, this antibody and its murine parent antibody significantly improved various readouts including hemodynamic parameters, vascular and kidney barrier function and survival (18–21) (18, 19)
After the successful use of Adrecizumab in rodent sepsis, we used Adrecizumab in this trial for the first time in large animal sepsis in our well-established porcine two-hit model consisting of a hemorrhagic and subsequent septic shock (22, 23) (24) (25–27)
According to previous animal and human phase I studies (13, 28, 29)
For this reason, in our study we investigated the effects of Adrecizumab on adrenomedullin concentration, hemodynamic parameters, and renal injury in a porcine two-hit model of hemorrhagic and septic shock.
MATERIALS AND METHODS
Animals
We anesthetized and ventilated 12 female German Landrace pigs (33 ± 1.5 kg body weight (BW) (mean ± standard deviation (SD)) and followed the standard procedures for laboratory animal care. The institutional and local committee on the care and use of animals (Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen, Germany, 84–02.04.2015.A037) approved this study.
General anesthesia and catheterization
Animals were premedicated with azaperone (1–2 mg/kg BW) and ketamine (10 mg/kg BW), and we induced general anesthesia by intravenous injection of propofol (1–2 mg/kg BW). The animals were orally intubated and placed in supine position. General anesthesia was maintained with infusions of propofol (5–10 mg/kgBW/h) and fentanyl (4–10 μg/kgBW/h). Controlled pressure mode ventilation was chosen to ventilate the animals with an inspiratory oxygen fraction of 0.5, an inspiratory/expiratory ratio of 1:1.5, PEEP set to 5 cm H2 O, and a tidal volume of 8 to 10 mL/kg BW. The respiratory rate was set to maintain a PaCO2 of 4.7 to 5.9 kPa. The body core temperature was maintained at a minimum of 37.5°C with a warming blanket. Two central venous catheters (Arrow International, Inc, Pa) were inserted into the external jugular vein and the femoral vein and a PiCCO (Pulse index Contour Cardiac Output) arterial thermodilution catheter (PULSION Medical Systems, Feldkirchen, Germany) was inserted into the femoral artery by transcutaneous puncture. At the end of the study, we euthanized the animals in the presence of a veterinarian with a lethal dose (80–160 mg/kgBW) of pentobarbital (Narcoren, Merial, Hallbergmoos, Germany) while they were still under deep anesthesia.
Escherichia coli fibrin clotIn this model, we used an E coli -laden clot with 7–9 × 1011 colony-forming units (CFUs) per kg/BW to induce septic shock. The clot consists of a sterile solution of porcine fibrinogen (Sigma-Aldrich Inc., St. Louis, Mo) (final volume was adjusted to the pig's weight) and E coli , to which thrombin from bovine plasma was added. After that, the clot was incubated for 30 min at room temperature (22)
Hemodynamic measurements
All intravascular pressure measurements were referenced to the mid-chest level, and values were obtained at end-expiration. We recorded heart rate, mean arterial pressure (MAP), central venous pressure (CVP), and stroke volume variation continuously. Cardiac output was measured performing transpulmonary thermodilution using the PICCO system. Extravascular lung water (EVLW), intrathoracic blood volume, and global end-diastolic volume were calculated using standard formula.
Laboratory
We conducted blood gas analyses using a standard blood gas oximetry system (ABL 800; Radiometer, Copenhagen, Denmark) with a co-oximeter. The oxygenation index was calculated using the standard formula (oxygenation index = PaO2 /FiO2 ; PaO2 = arterial oxygen partial pressure; FiO2 fraction of oxygen in the inhaled air). Blood count, electrolytes, creatinine, urea, and liver enzymes were determined using standard laboratory techniques. Creatinine clearance was measured as an estimate of the glomerular filtration rate (ClCrea = Ucrea × Uvol/Pcrea × duration of urine collection period; Ucrea = urine creatinine concentration; Uvol = urine volume during the collection period; Pcrea = serum creatinine concentration). Levels of cytokines (interleukin (IL)-6 and tumor-necrosis-factor (TNF)-alpha) in plasma were assessed by an enzyme-linked immunosorbent assay using commercially available kits specific for pigs (R&D Systems, Inc, Minneapolis, Minn). Measurements were made according to the manufacturer's guidelines.
Renal histopathology
The histopathological examination of the kidneys was performed by an experienced nephropathologist. He was not a part of the study team and he was not involved in the experiments. He was blinded to the study groups.
The left kidney of each pig was collected at the end of the experiment and pieces of 1 cm2 from renal cortex and medulla were separated and fixed overnight using 4% formalin. After dehydration and embedding in paraffin tissue was cut into sections of 2 μm thickness, followed by deparaffination and staining with perjodat acid Schiff's staining. Renal injury was examined in 10 high power fields from the cortex and 10 more from the medulla at ×200 magnification using a score grading acute tubular necrosis and inflammation: Score 0= intact tubules, no signs of inflammation; Score 1=acute tubular necrosis (ATN) and interstitial inflammatory cells affecting 1% to 25%; Score 2= ATN and interstitial inflammatory cells affecting 26% to 50%; ATN and interstitial inflammatory cells in 51% to 75%; ATN and interstitial inflammation in more than 76%. Granulocytes were also counted in 10 high power fields from the cortex and 10 more from the medulla at ×400 magnification and shown as number per mm2 . The amount of glomerular thrombi in each animal was evaluated in 15 glomerular cross sections using a semiquantitative score: Score 0=no thrombi; Score 1=thrombi in 1% to 25% of the glomerular cross section (gcs); Score 2=thrombi in 26% to 50% of the gcs; Score 3=thrombi in 51% to 75% of the gcs; Score 4=thrombi in more than 76%. Grading was performed for each high powerfield separately and means were calculated for all cases.
Adrenomedullin
Adrecizumab has been shown to be fully cross-reactive against adrenomedullin from different species including pigs. Bioactive adrenomedullin (bio-ADM) was measured using a novel chemiluminescence immunoassay (sphingotest bio-ADM) provided by Sphingotec GmbH (Hennigsdorf, Germany) as previously described (30) (31)
Experimental protocol
During catheterization animals received 10 mL/kg BW/h of a balanced crystalloid solution (Sterofundin Iso, B.Braun, Melsungen, Germany). Hemorrhagic shock was induced by bleeding the animals via the femoral vein catheter (35 ± 5 mL/kgBW blood loss) over 15 min until half of the baseline MAP was reached. This hemorrhagic shock was maintained for 45 min, followed by fluid resuscitation with balanced crystalloid solution in order to restore baseline mean arterial pressure. Two hours after hemorrhagic shock the blood collected during hemorrhagic shock was retransfused. As second hit, sepsis was induced using the E coli -laden clot placed into the abdominal cavity 6 h after hemorrhagic shock. Animals were randomly assigned to receive the ADM antibody (n = 6) or vehicle solution (n = 6). The solutions were delivered in neutral bags marked with A or B and the investigators were blinded to the solutions. Unblinding was realized after study end of the last animal. The therapy test solution started immediately after the induction of sepsis. 2 mg/kg BW of the antibody/vehicle solution were infused over a period of 30 min. Four hours after sepsis induction, therapy of septic shock started using balanced crystalloids and noradrenaline titrated to maintain a CVP of 8 mm Hg to 12 mm Hg, an MAP above 65 mm Hg and a central venous oxygen saturation of 70%. Sepsis therapy continued for another 8 h. Measurements were performed before, during, and after hemorrhagic shock, before sepsis induction and 1, 2, 3, 4, 6, 8, 10, and 12 h after sepsis induction.
Statistical analysis
According to the result of a power analysis we chose the group size n = 6. Group sample sizes of n = 6 per group achieve 80% power to reject the null hypothesis of equal means when the standardized population mean difference is 2 with a standard deviation for both groups of 1.0 and with a significance level (alpha) of 0.05 using a two-sided two-sample equal-variance t test. Values are expressed as means and standard deviations, or counts and percentages. To test for a treatment effect, an interaction test using generalized linear models for repeated measures (GLM interaction test), testing if, after sepsis induction, the two groups developed differently, is applied to the serial data from sepsis induction to end of observation, 12 h after induction (SI+12 h). If treatment has an effect, the test for interaction should be significant as both groups should be equal at time SL (=time of treatment), but different later on.
In cases were the data suggests that change is only present at single time points, specifically for 12 h after sepsis induction, t test for these time points is applied. For categorical data, the chi2 test for 2 × 2 tables was applied instead.
All statistical tests were 2-tailed and a two-sided P value of 0.05 was considered for significance. P values were not adjusted for multiple testing. The statistical analyses were performed using Statistical Package for the Social Sciences (SPSS) version 22.0 (SPSS Inc, Chicago, Ill).
RESULTS
Hemodynamic
Hemorrhagic shock has occurred to the same extent in both groups and the animals of both groups have recovered equally from this hemorrhagic shock until sepsis induction. After sepsis induction in both groups the target parameters of hemodynamic could be maintained over the whole study period. In order to achieve these values, a significantly lower amount of volume resuscitation was needed in the antibody group than in the vehicle group (5,300 ± 482 vs. 6,654 ± 1,308 mL, P = 0.039, t test, SI+12 h) (Fig. 1 ). The number of animals in septic shock, defined by vasopressor need after sepsis-1 criteria (32) P = 0.014, chi2 test, SI+12 h) (Fig. 2 ). Heart rate (P = 0.097, GLM interaction test), stroke volume variation (P = 0.324, GLM interaction test), and cardiac output (P = 0.055, t test; P = 0.275, GLM interaction test) showed no significant differences between the two groups over the study period (Table 1 ).
Fig. 1: Cumulated fluid input.
Fig. 2: Frequency of noradrenaline requirement.
Table 1 -
Hemodynamic parameters, fluid input and pulmonary function
Adrecizumab
Vehicle
MAP, mm Hg
Baseline
74 ± 5
72 ± 5
HS
37 ± 3
36 ± 3
1 h after HS
74 ± 5
72 ± 5
Before sepsis
80 ± 7
82 ± 13
4 h after sepsis
73 ± 9
76 ± 4
8 h after sepsis
77 ± 9
69 ± 6
12 h after sepsis
69 ± 5
67 ± 1
CVP, mm Hg
Baseline
11 ± 1
10 ± 1
HS
7 ± 1
6 ± 1
1 h after HS
10 ± 2
10 ± 2
Before sepsis
11 ± 1
10 ± 2
4 h after sepsis
10 ± 2
8 ± 1
8 h after sepsis
11 ± 1
10 ± 1
12 h after sepsis
11 ± 1
11 ± 1
SvO2 , %
Baseline
72 ± 7
76 ± 8
HS
49 ± 10
60 ± 5
1 h after HS
68 ± 16
78 ± 7
Before sepsis
74 ± 7
75 ± 5
4 h after sepsis
51 ± 13
55 ± 9
8 h after sepsis
58 ± 10
56 ± 25
12 h after sepsis
68 ± 9
72 ± 11
Cumulated fluid input, mL
Baseline
0 ± 0
0 ± 0
HS
0 ± 0
0 ± 0
1 h after HS
2,121 ± 378
2,325 ± 360
Before sepsis
2,121 ± 378
2,325 ± 360
4 h after sepsis
2,121 ± 378
2,325 ± 360
8 h after sepsis
3,829 ± 584
4,300 ± 994
12 h after sepsis
5,300 ± 482∗
6,654 ± 1,308
Norepinephrine, μg/min
Baseline
0 ± 0
0 ± 0
HS
0 ± 0
0 ± 0
1 h after HS
0 ± 0
0 ± 0
Before sepsis
0 ± 0
0 ± 0
4 h after sepsis
0 ± 0
0 ± 0
8 h after sepsis
1.17 ± 2.04
1.93 ± 3.39
12 h after sepsis
4.22 ± 7.09
8.42 ± 12.28
Animals requiring Norepinephrin, n (%)
Baseline
0 (0)
0 (0)
HS
0 (0)
0 (0)
1 h after HS
0 (0)
0 (0)
Before sepsis
0 (0)
0 (0)
4 h after sepsis
0 (0)
0 (0)
8 h after sepsis
2 (33)
2 (33)
12 h after sepsis
2 (33)∗
6 (100)
HR, bpm
Baseline
53 ± 9
62 ± 9
HS
160 ± 35
158 ± 37
1 h after HS
104 ± 22
125 ± 14
Before sepsis
61 ± 8
65 ± 11
4 h after sepsis
79 ± 20
103 ± 28
8 h after sepsis
102 ± 17
112 ± 19
12 h after sepsis
98 ± 23
125 ± 32
Stroke volume variation, %
Baseline
9 ± 2
8 ± 1
HS
26 ± 5
30 ± 6
1 h after HS
9 ± 2
9 ± 2
Before sepsis
8 ± 2
7 ± 1
4 h after sepsis
12 ± 3
14 ± 3
8 h after sepsis
9 ± 2
8 ± 2
12 h after sepsis
8 ± 2
12 ± 11
CO, L/min
Baseline
2.7 ± 0.3
3.4 ± 0.4
HS
1.6 ± 0.2
2.1 ± 0.3
1 h after HS
4.2 ± 0.7
5.8 ± 1.0
Before sepsis
3.1 ± 0.4
3.6 ± 0.6
4 h after sepsis
1.9 ± 0.5
2.6 ± 0.5
8 h after sepsis
2.9 ± 0.7
3.6 ± 0.7
12 h after sepsis
3.5 ± 0.7
4.9 ± 1.7
Lactate, mmol/L
Baseline
1.5 ± 0.7
1.1 ± 0.4
HS
6.0 ± 3.5
5.9 ± 1.4
1 h after HS
4.8 ± 3.2
6.6 ± 2.5
Before sepsis
0.7 ± 0.2
0.6 ± 0.1
4 h after sepsis
1.8 ± 0.6
1.9 ± 0.5
8 h after sepsis
1.2 ± 0.3
1.2 ± 0.3
12 h after sepsis
1.4 ± 0.4
1.5 ± 0.5
Oxygenation index, mm Hg
Baseline
490 ± 30
495 ± 19
HS
458 ± 49
438 ± 40
1 h after HS
493 ± 28
500 ± 14
Before sepsis
470 ± 37
466 ± 26
4 h after sepsis
365 ± 77
284 ± 102
8 h after sepsis
370 ± 77
264 ± 135
12 h after sepsis
405 ± 58
299 ± 125
EVLW, mL
Baseline
297 ± 37
309 ± 79
HS
325 ± 84
337 ± 142
1 h after HS
283 ± 28
349 ± 81
Before sepsis
330 ± 72
388 ± 42
4 h after sepsis
419 ± 117
442 ± 100
8 h after sepsis
435 ± 167
444 ± 113
12 h after sepsis
387 ± 123
459 ± 166
∗ P ≤ 0.05 vs. vehicle (t test of chi2 test, as appropriate).CVP indicates central venous pressure; HS, hemorrhagic shock; MAP, mean arterial pressure; SvO2, central venous oxygen.
Pulmonary function
The oxygenation index (P = 0.088, t test, SI+12 h) and the calculated EVLW (P = 0.410, t test, SI+12 h) showed no significant differences after sepsis induction until study end (Table 1 ).
Renal function and extravasation
Serum creatinine (P = 0.442, t test, SL+12) and urine excretion (P = 0.961, GLM interaction test) did not differ between both groups after sepsis induction until study end. The histological examination of renal biopsies showed significantly lower granulocytes invasion both in cortex (30.9 ± 18.9 vs. 65.7 ± 19.9 n/mm2 ; P = 0.011, t test) and medulla (36.9 ± 19.2 vs. 92.6 ± 22.6 n/mm2 ; P = 0.001, t test) in kidneys of antibody treated animals (Fig. 3 ). The cortical (P = 0.417, t test) and medullary (P = 0.073, t test) injury as assessed by semiquantitative scoring of acute tubulus necrosis and inflammation, was lower in the kidneys of the antibody group compared to the vehicle group, but the significance level could not be achieved (Table 2 ).
Fig. 3: Granulocytes invasion in renal cortex and medulla.
Table 2 -
Renal function and renal histopathology
Adrecizumab
Vehicle
Serum creatinine, μmol/L
Baseline
131 ± 9
124 ± 11
Before sepsis
155 ± 14
144 ± 15
4 h after sepsis
165 ± 23
167 ± 10
8 h after sepsis
168 ± 24
192 ± 21
12 h after sepsis
203 ± 51
192 ± 28
Urine output, mL/kg/h
Baseline
0.4 ± 0.2
0.5 ± 0.2
HS
0.3 ± 0.2
0.2 ± 0.2
1 h after HS
2.2 ± 2.3
1.1 ± 1.1
Before sepsis
2.5 ± 2.2
2.1 ± 1.3
4 h after sepsis
0.5 ± 0.3
0.5 ± 0.3
8 h after sepsis
0.6 ± 0.3
0.6 ± 0.4
12 h after sepsis
0.6 ± 0.6
0.6 ± 0.7
Granulocytes cortex, n/mm2
30.9 ± 18.9∗
65.7 ± 19.9
Granulocytes medulla, n/mm2
36.9 ± 19.2∗
92.6 ± 22.6
Cortical injury score
1.4 ± 0.7
1.8 ± 0.9
Medullary injury score
1.4 ± 0.6
2.0 ± 0.4
∗ P ≤ 0.05 vs. vehicle (t test).
Inflammation
Both IL-6 and TNF-alpha increased after induction of sepsis in both groups to similar levels. The TNF-alpha peak was 2 h after sepsis induction in both groups (56,859 ± 43,582 (antibody) vs. 88,828 ± 54,030 (vehicle) pg/mL), and then declined again without differences between groups at study end (130 ± 102 vs. 231 ± 165 pg/mL, P = 0.636, GLM interaction test). IL-6 peaked after 4 h in both groups (15,493 ± 18,601 (antibody) vs. 14,712 ± 21,266 (vehicle) pg/mL), but the IL6 concentration in the vehicle group increased again in sepsis and was higher compared to the antibody group at the end of the study, resulting in a significant treatment effect (8,322 ± 8,655 (antibody) vs. 18,659 ± 22,432 (vehicle) pg/mL, P = 0.028, GLM interaction test) (Table 3 ).
Table 3 -
Systemic inflammation
Adrecizumab
Vehicle
IL 6, pg/mL
Baseline
10 ± 0
49 ± 95
HS
10 ± 0
54 ± 107
1 h after HS
10 ± 0
31 ± 52
Before sepsis
36 ± 64
10 ± 0
4 h after sepsis
15,493 ± 18,601
14,712 ± 21,266
6 h after sepsis
10,855 ± 11,827
11,817 ± 15,458
12 h after sepsis
8,322 ± 8,655
18,659 ± 22,432
TNFα, pg/mL
Baseline
10 ± 0
10 ± 0
HS
10 ± 0
10 ± 0
1 h after HS
10 ± 0
10 ± 0
Before sepsis
10 ± 0
20 ± 24
2 h after sepsis
56,859 ± 43,582
88,828 ± 54,030
4 h after sepsis
946 ± 661
1,091 ± 446
12 h after sepsis
130 ± 102
231 ± 165
IL 6 indicates interleukin 6; TNFα, tumor-necrosis-factor α.
Plasma adrenomedullin concentration
After induction of sepsis and simultaneous antibody or vehicle application, plasma ADM increased immediately in both groups, but the increase was significantly quicker and more pronounced in the antibody group compared to vehicle treatment (P = 0.003, GLM interaction test). In contrast, as shown in Figure 4 , plasma levels of MR-proADM increased similarly upon sepsis induction independently from treatment with Adrecizumab or vehicle (P = 0.625, GLM interaction test).
Fig. 4: Plasma bio-ADM and MR-proADM concentration.
DISCUSSION
In the present study, we tested the efficacy of the humanized anti-adrenomedullin antibody Adrecizumab in a porcine two-hit model of hemorrhagic and septic shock. The treatment with Adrecizumab significantly increased plasma adrenomedullin levels, prevented the development of septic shock, and reduced renal granulocyte extravasation.
The cumulative amount of required volume replacement therapy as well as the percentage of animals requiring noradrenaline on top of fluid resuscitation to achieve the target MAP was significantly lower in the animals receiving antibody therapy. Based on the sepsis-1 definition (32) (33–36) (19)
In our study, the use of Adrecizumab did not significantly improve pulmonary function. Other studies report on an attenuation of pulmonary hyperpermeability, lung injury, and systemic hyperinflammation after adrenomedullin administration in a murine model of lung injury (37)
Our results of renal histopathology showed significantly reduced granulocytes invasion in cortex and medulla in the animals treated with Adrecizumab after sepsis induction, while differences in cortical and medullary injury did not reach the significance levels. In a recent study, in which effects of Adrecizumab on the vascular barrier function and survival in rodent models of systemic inflammation and sepsis were investigated, Adrecizumab also improved kidney barrier function during murine sepsis, exemplified by significantly reduced extravascular albumin and vascular endothelial growth factor expression (20)
Regarding adrenomedullin concentration, the induction of sepsis in the progression to septic shock led to an increase of plasma bio-adrenomedullin in the vehicle animals (15, 16, 30) (20) (38) (31) de novo synthesis of ADM is not induced by Adrecizumab (38)
An increased adrenomedullin activity can cause partly opposite effects. On the one hand, a variety of studies revealed that higher concentrations of adrenomedullin cause vasodilation and thus lead to a decrease in blood pressure (39–42) (19) (38)
On the other hand from numerous in vitro (43–45) in vivo (9, 46) (20) (20)
Pickkers et al. recently presented a hypothesis about the presumed mechanism by which Adrecizumab exerts its beneficial effects (13, 28) (47–50)
Limitations
As a major limitation of our study it must certainly be mentioned that the hypothesis about the presumed mechanism of action of Adrecizumab improving endothelial barrier cannot be proven by the results of the present experiment alone. Another limitation is the small number of animals. So statistical significance could be reached only for very strong effects. In addition, the short observation period of 12 h after induction of sepsis limits the informative value of our study, especially the kidney function that had not been impaired up to this point.
CONCLUSION
In summary, we found that application of the non-neutralizing humanized monoclonal anti-adrenomedullin antibody Adrecizumab significantly increased plasma adrenomedullin levels in a porcine two-hit model of hemorrhagic and septic shock. The volume requirement for hemodynamic stabilization was significantly lower and significantly less animals developed septic shock while renal granulocyte extravasation was significantly reduced. This is in line with the assumed effect of Adrecizumab in supporting endothelial barrier function by increasing the amount of functional adrenomedullin in the intravascular compartment. Thus, therapy with Adrecizumab may provide benefit in experimental sepsis. Further clinical development of this drug candidate is warranted.
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