This was a secondary analysis of data obtained from 2 previous experimental studies focused on mechanical ventilation and ventilation-induced lung injury, involving a total of 78 pigs [5, 9].

In the first study, 36 female pigs with average weight of 23.3(± 2.3)Kg were ventilated for 48 h with an applied mechanical power ranging between 18 and 120 J/min. The tidal volume applied was equal to the functional residual capacity (strain = 1); the respiratory rate was 30 bpm, and the PEEP varied between 0 and 18cmH2O (0, 4, 7, 11, 14, and 18) [9].

In the second study, 42 female pigs with average weight of 24.2(± 2.0)Kg were randomized into six groups, of which three receiving low mechanical power (15 J/min) and the other three higher mechanical power (30 J/min), and then were ventilated for 48 h. In the six groups, mechanical power was delivered with different combinations of respiratory rate, tidal volume and PEEP. The applied tidal volume ranged between 0.5 and 3.8L, the respiratory rate from 5 to 44 bpm, and PEEP from 5 to 25cmH2O [5]. The experimental trials were previously approved by the local ethics committee (Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit: LAVES; Oldenburg, Niedersachsen, Germany. Approval of study 1: 09/01/17; number: 16/2223; title: “Animal experimental study of the relationship between mechanical ventilation energy in the lungs and lung size”. Approval of study 2: 24/05/18; number: 18/2795; title: “Experimental confirmation of mechanical energy threshold in ventilator-induced lung injury”) and were performed in accordance with the 1975 Helsinki Declaration; the manuscript conformed to the ARRIVE guidelines [10].

Management of the study individuals and time-course of the experimental trials:

In both experiments, propofol, midazolam and sufentanil were used for the induction of anesthesia. After endotracheal intubation, standardized baseline mechanical ventilation settings were applied (Vt 6 mL/kg, PEEP 5cmH2O, respiratory rate to maintain PaCO2 between 35 and 45 mmHg). Animals were instrumented with the following devices: orogastric probe with esophageal pressure monitoring system (Nutrivent, Sidam Srl., Modena, Italy); central venous catheter; Swan-Ganz catheter; central arterial PiCCO® (Pulsion Medical System, Germany) catheter and urinary catheter.

Once the animal was clinically stable and ready for the experiment, baseline measurements were obtained and, subsequently, the settings of mechanical ventilation was modified according to the experimental group allocation and maintained unchanged throughout the experiment. In both trials, measurements were collected at 0.5 h and, subsequently, every 6 h until the end of the experiment. Plasma and urine samples were collected at baseline, 6, 12, 24, and 48 h.

The infusion of a balanced crystalloid solution (Sterofundin®; Braun GmbH, Germany) was initiated in all animals before the baseline measurement, at rate 2 mL/h. Whenever clinical signs of hypovolemia/hypoperfusion were detected (MAP < 60 mmHg or increased arterial lactates), additional boluses of 250 mL of crystalloids were delivered. In the absence of fluid responsiveness, a continuous infusion of norepinephrine or epinephrine was initiated to maintain hemodynamic stability.

At the end of the experiment, the animal was euthanized, an autopsy was performed, and three tissues samples were harvested from each lung (basal, medial, apical), together with a tissue sample from the liver, kidneys, bowel and muscle. Each specimen (approximately 2 g) was weighted before and after being heated and dried in an oven at 50 °C for 24 h to obtain the wet-to-dry ratio.

The following variables were calculated at each time-point of the study:

- Respiratory system mechanical power (MPRS) [4]:

$$}}_}} \left(}/}\right)=0.098*}*\left(}}^*\left(0.5*}}_}}+}* \frac:}}:}}*}}_}}\right)+}*}\right)$$

(1)

RR: respiratory rate; Vt: tidal volume; ERS: respiratory system elastance; I:E: inspiratory/expiratory ratio; Raw: airway resistances; PEEP: positive end-expiratory pressure.

- Relative components of mechanical power: the classic equation of mechanical power was partitioned to compute the amount of each of the three components of mechanical power:

Elastic component of mechanical power [4]:

$$}}_}-\mathrm} \left(}/}\right)=0.098*}*}}^*0.5*}}_}}$$

(2)

Resistive component of mechanical power [4]:

$$}}_}-\mathrm} (}/})=0.098*}}^*}}^*\frac:}}:}}*}}_}}$$

(3)

PEEP component of mechanical power [4]:

$$}}_}-\mathrm} \left(}/}\right)=0.098*}*}*}$$

(4)

Mean perfusion pressure (MPP):

$$\mathrm(})=\mathrm-}$$

(5)

MAP mean arterial pressure, CVP central venous pressure;

The animals were grouped into four categories according to the value of plasma creatinine assessed at the end of the experiment, based on the RIFLE criteria for kidney injury [11]. The experimental groups are as follows: 1) No AKI: no increase in creatinine compared to baseline; 2) RIFLE 1-Risk: increase of creatinine of > 50%; 3) RIFLE 2-Injury: a two-fold increase in creatinine; 4) RIFLE 3-Failure: a three-fold increase in creatinine.

Data are reported as median (± 95% confidence interval). Continuous variables were compared using one-way ANOVA. The time-course description of the variables was evaluated via a linear mixed-effects model, with the severity of AKI, time, and their interaction as fixed effects, and the single individual as random effect. To reflect the weight of each variable according to the time of exposure, the time-weighted average of each study period was computed as the area under the curve of the variable.

The association between renal function and the hemodynamic variables was explored with a regression model, where the time-weighted average hemodynamic value recorded during the experimental phase was plotted against the increase in serum creatinine. The strength of the association was also evaluated with a Receiver Operating Characteristic model.

Two-tailed p-value < 0.05 was considered statistically significant. All analyses were performed with SPSS 25 (SPSS inc., Chicago, IL).