#2548 In vitro evaluation of proenkephalin kinetics in hemofiltration and hemodialysis
Authors: Lorenzin Anna , Perin Natascha , de Cal Massimo , Morisi Niccolò , Brendolan Alessandra , Lentini Paolo Luca Maria , Zanella Monica , Ronco Claudio 
Published: May 2024

Abstract

Background and Aims
Proenkephalin A 119-159 (penKid) is a stable surrogate marker for enkephalins (endogenous opioids regulating kidney function).
PenKid plasma concentration correlates to GFR and increases in AKI, for this reason it can be used for prediction and diagnosis of AKI (even subclinical forms) and need of RRT. Furthermore, it seems to be also a reliable biomarker to predict the successful liberation from RRT and may therefore aid in the successful weaning of patients from RRT.
PenKid has the perk of maintaining stable its concentration during RRT, suggesting that is barely filtered, in contrast to other biomarkers.
However, with its small size of 4.5 kDa, penKid can be assumed to be freely filtered through the glomerulus and dialysis membranes.
To address the issue about stable penKid plasma concentrations in RRT (no extracorporeal removal or increased production in response to impaired kidney function), we planned ex vivo experiments simulating different conditions of RRT.
The aim of this study is to determine the sieving coefficient and the diffusive clearance of the penKid molecule in conditions of in vitro continuous veno-venous hemofiltration (CVVH) and continuous veno-venous hemodialysis (CVVHD), respectively.

Method
The lyophilized peptide was solved in 20 mM Dipotassium phosphate, 6 mM Disodium EDTA [pH 8], avoiding concentrations higher than 1 mg/mL. The compound was diluted in 50 mL of plasma to ensure homogeneous distribution. For each experimental modality, a batch of blood of 1000 mL with adjusted hematocrit at 30% and albumin concentration of 3 g% was utilized. Blood was spiked with lyophilized synthetic penKid to achieve two target concentrations of 150 and 500 pmol/L in CVVH and CVVHD modalities, respectively. Blood was adjusted for a volume of 1000 mL, maintained at 37° and continuously stirred during the experiments. CVVH was performed with blood flow (QB) = 150 mL/min and ultrafiltration rate (QUF) = 20 mL/min to reproduce a filtration fraction of 20%. The ultrafiltrate was reinfused in the venous line to maintain initial volume. This allowed to define convective clearance and solute sieving coefficient in pure convective mode. CVVHD was performed with QB = 150 mL/min and dialysate flow (QD) = 35 mL/min (Net QUF = 0 mL/min). This allowed the calculation of diffusive clearance. A polysulfone dialyzer (AV1000 FMC, Bad Homburg, Germany) was utilized. Samples were taken at 10, 30, 60 minutes from the initiation of the circulation, in both modalities.

Results
Initial penKid concentrations in blood corresponded to the desired targets in all the experiments.
Sieving coefficients displayed values between 0.69 and 1.5 and they appear to remain stable over time (Fig. 1).
Diffusive clearance displayed values between 35 and 15.7 mL/min. Values appear to present a slight decrease over time (Fig. 2).
The observed variability can be attributed to several factors: difference in plasma protein interference in various moments of the experiments, difference in concentration of penKid in plasma water versus plasma or blood, variability in the sample measurements.

Conclusion
Significant removal of penKid can be obtained by extracorporeal therapies both in convection (CVVH) and diffusion (CVVHD). Further investigation is needed to assess if these findings are confirmed during in vivo treatments in which penKid kinetics is affected by generation, elimination and changes in distribution volume.