Unraveling the Contribution of Fluid Therapy to the Development of Augmented Renal Clearance in a Piglet Model

Augmented renal clearance (ARC) observed in the critically ill pediatric population has received an increased attention over the last years due to its major impact on the disposition and pharmacokinetics of mainly renally excreted drugs. Apart from an important inflammatory trigger, fluid administration has been suggested to contribute to the development of ARC. Therefore, the primary objective of this study was to evaluate the effect of continuous intravenous fluid administration on renal function using a conventional piglet animal model and to quantify the impact of fluid administration on the pharmacokinetics of renally excreted drugs. At baseline, twenty-four piglets (12 treatment/12 control; 7 weeks old, all ♂) received the marker drugs iohexol (64.7 mg/kg body weight (BW)) and para-aminohippuric acid (10 mg/kg BW) to quantify glomerular filtration rate and effective renal plasma flow, respectively. In addition, the hydrophilic antibiotic amikacin (7.5 mg/kg BW) was administered. Following this baseline measurement, the treatment group received fluid therapy as a constant rate infusion of 0.9% saline at 6 mL/kg/h over 36 h. After 24 h of fluid administration, the marker drugs and amikacin were administered again. When comparing both groups, a significant effect of fluid administration on the total body clearances of iohexol (p = 0.032) and amikacin (p = 0.0014) was observed. Clearances of iohexol and amikacin increased with on average 15 and 14%, although large interindividual variability was observed. This led to decreased systemic exposure to amikacin, which was manifested as decrease in area under the plasma concentration-time curve from time 0 h to infinity from 34,807 to 30,804 ng.h/mL. These results suggest that fluid therapy is a key factor involved in the development of ARC and should be taken into account when administering mainly renally excreted drugs. However, further research is necessary to confirm these results in children.

Sample preparation consisted of sequential addition of 50 µL of 1% FA in H2O and 50 µL of IS WS to 100 µL of plasma. After vortex mixing (15 s), 50 µL of 10% trichloroacetic acid (Merck, Darmstadt, Germany) in H2O was added to each sample. Subsequently, samples were vortex mixed for 15 s and centrifuged for 10 min (16,200 × g, 4°C). The obtained supernatant was filtered through a Millex ® nylon syringe filter (0.22 µm) and transferred into an autosampler vial. A 10 μL aliquot of the final solution was injected onto the UHPLC-MS/MS instrument.
Chromatographic separation was achieved on a BEH C18-column (50 mm x 2.1 mm internal diameter, 1.7 µm) in combination with a guard column of the same type, both from Waters (Zellik, Belgium). Mobile phase A consisted of 5 mM pentafluoro propionic acid (PFPA, Sigma-Aldrich, Bornem, Belgium) in H2O:acetonitrile (50:50,V:V). As mobile phase B, 5 mM PFPA in H2O was used. The following gradient elution program was run: start condition (10% A, 90% B), 0.0-3.0 min (linear gradient to 60% B), 3.0-5.0 min (linear gradient to 10% B), 5.0-6.0 min (90% A, 10% B), 6.0-6.1 min (linear gradient to 90% B) and 6.1-10.0 (10% A, 90% B). Flow rate was set at 300 μL/min. Mass spectrometric detection was performed on a Waters Quattro Premier equipped with a heated electrospray ionization (h-ESI) probe operating in the positive ionization mode. The following transitions (m/z) were used for identification and quantification, respectively, for amikacin: 586. Matrix-matched calibration curves were prepared over a concentration range of 0.5-20 µg/mL. The calibration model was quadratic with 1/x 2 weighting. When sample concentrations above the validated concentration range were observed/expected, analysis was performed on a reduced sample volume (50 µL) supplemented with 50 µL blank porcine plasma.
In Table S1 an overview of the validation results is presented. The LLOQ was 0.5 µg/mL. The LOD, defined as the lowest concentration which could be recognized by the detector with a signal-to-noise (S/N) ratio of ≥3, was 22.98 ng/mL. No carry-over was observed in the solvent sample injected after the highest calibrator. Table S1: Validation results of the evaluation of calibration (correlation coefficient (r) and goodnessof-fit coefficient (gof)), lower limit of quantification (LLOQ), limit of detection (LOD), and withinday and between-day accuracy and precision for quantification of amikacin in porcine plasma. Acceptance criteria for linearity: r > 0.99 and gof < 10%

Bio-analysis of para-aminohippuric acid in urine
For the analysis of PAH in urine, the same UHPLC method was used as described by Dhondt et al (2019) (Dhondt et al., 2019). Sample preparation consisted of the addition of 12.5 µL of 1 M hydrochloric acid and 25 µL of H2O to 50 µL of urine. After vortex-mixing, the samples were equilibrated 10 min at room temperature. Next, 1,912.5 µL of H2O was added to the samples. After vortex-mixing, 250 µL of the sample was added together with 750 µL of H2O and 12.5 µL of IS solution (5 µg/mL 13 C6-PAH) in a vial. An aliquot of 3 µL of the final solution was injected on the column. Samples expected to have concentration levels higher than the highest calibrator were diluted 1/10 with H2O. Subsequently, 50 µL of this solution underwent the same sample treatment as the non-diluted samples. LLOQ and LOD were 0.25 µg/mL and 7.6 ng/mL, respectively. The method was validated based on European and international guidelines and recommendations (Knecht and Stork, 1974;European Commission, 2002; Committee for Medicinal Products for Veterinary Use, 2015). The validation encompassed an evaluation of the following characteristics: linearity using matrix-matched calibrator samples (correlation coefficient (r) and goodness-of-fit coefficient (gof)), within-run and between-run precision and accuracy, lower limit of quantification (LLOQ), limit of detection (LOD) and carry-over. Validation results are presented in Table S2. Table S2: Validation results of the evaluation of linearity (correlation coefficient (r) and goodness-offit coefficient (gof)), lower limit of quantification (LLOQ), limit of detection (LOD), and within-day and between-day accuracy and precision for quantification of para-aminohippuric acid in porcine urine. Acceptance criteria for linearity: r > 0.99 and gof < 10%

Normal water intake/ ad libitum access to water
From the urine output observed in the studied pigs under ad libitum access to water, it is possible to estimate the amount of water that the piglets have drunk by using the porcine water balances (inputoutput) reported by Mroz et al. (cited by Patience et al.) (Mroz et al., 1995;Patience, 2012). Using the water balances reported by Mroz et al., an urine output of 3.99 mL/kg/h corresponds with an (oral) water intake of 6.23 mL/kg/h. The latter value approximates the rate of fluid administration (6 mL/kg/h) used in this study. Consequently, this rate overestimated the normal water intake roughly 1.7 (6.23/3.65) times.