## 3 Discussion
Moreover, [PERSON] and coworkers [23] specifically studied the behavior of the investigated adsorbent material regarding protein-bound hepatic toxins, such as bilirubin, observing the ability to irreversibly adsorb bilirubin without affecting albumin, which remained stable (changes less than 5%). Apart from these experiences, we are not aware of any other study focused on albumin behavior with the DVB-PVP sorbent or any comparative studies among different extracorporeal techniques. Certainly, this should be investigated in future studies. Therefore, we wondered whether the removal of IS and PCS by DVB-PVP HD treatment was linked to the loss of albumin. Based on the observed data, we cannot exclude that the reduction in PBUTs is also due to the loss of albumin, although all the statistically significant reductions in IS and PCS plasma levels in post-HD samples of symbiotic/placebo DVB-PVP HD treated groups were greater than 8%. However, considering the DVB-PVP resin adsorption efficacy recorded in the in vitro study, and given the study limitations, small sample size and only three DVB-PVP HD sessions, we are aware that longer observation periods with a major number of patients are necessary to better investigate the impact of a DVB-PVP sorbent on PBUTs removal in HD patients. The increase in %PB for both IS and PCS after dialysis treatment confirmed literature data [48]. However, [PERSON] and coworkers cannot explain the changes in %PB during hemodialysis by changes in pH. They hypothesized that the increased percentage of toxins PB during HD could be attributed to the slow restoration of the balance between their protein-bound and the free forms during the dialysis session once the pre-dialysis free fraction has been removed. They confirmed their hypothesis, calculating the difference in reduction ratio (RR) between the free and total concentration of highly-bound IS and PCS, which might imply that equilibrium could not be restored during the dialysis session. Indeed, we found that most of the differences between RRs of the total and free IS and PCS concentrations were statistically significant (Table S2), with the RRs of the free form much higher, which means that only the free fraction can be removed from dialysis and will cause an unbalance with the bound fraction, which is released slowly. Although we found a positive correlation between %PB of IS and PCS and pH, both in vitro and in vivo with DVB-PVP resin, we could just suggest a probable influence of pH in restoring the balance of the free and protein-bound form of uremic toxins in HD patients, being aware of the need for further experiments for confirmation. This information might be useful in the improvement in the PBUTs removal strategies and in the development of innovative ones aiming at the optimization of dialysis. In conclusion, this study demonstrates DVB-PVP resin efficacy in removing IS and PCS in vitro. Moreover, it represents a proof of concept of the potential validity of a strategy that combines the gut microbiota modulation and an innovative absorption-based approach in HD patients, aimed at reducing plasma levels of IS and PCS. Extended treatment cycles with a larger number of patients are needed to confirm the efficacy and the applicability of the combined strategy symbiotic/DVB-PVP dialysis in uremic patients, with clinical outcomes that need to be studied.