Vectisol Formulation Enhances Solubility of Resveratrol and Brings Its Benefits to Kidney Transplantation in a Preclinical Porcine Model

Abstract

Current organ shortages have led centers to extend the acceptance criteria for organs, increasing the risk for adverse outcomes. Current preservation protocols have not been adapted so as to efficiently protect these organs. Herein, we target oxidative stress, the key mechanism of ischemia reperfusion injury. Vectisol^® is a novel antioxidant strategy based on the encapsulation of resveratrol into a cyclodextrin, increasing its bioavailability. We tested this compound as an additive to the most popular static preservation solutions and machine perfusion (LifePort) in a preclinical pig model of kidney autotransplantation. In regard to static preservation, supplementation improved glomerular filtration and proximal tubular function early recovery. Extended follow-up confirmed the higher level of protection, slowing chronic loss of function (creatininemia and proteinuria) and the onset of histological lesions. Regarding machine perfusion, the use of Vectisol^® decreased oxidative stress and apoptosis at the onset of reperfusion (30 min post declamping). Improved quality was confirmed with decreased early levels of circulating SOD (Superoxide Dismutase) and ASAT (asparagine amino transferase). Supplementation slowed the onset of chronic loss of function, as well as interstitial fibrosis and tubular atrophy. The simple addition of Vectisol^® to the preservation solution significantly improved the performance of organ preservation, with long-term effects on the outcome. This strategy is thus a key player for future multi-drug therapy aimed at ischemia reperfusion in transplantation.

Keywords: ischemia reperfusion injury; organ preservation; oxidative stress; resveratrol; transplantation.

Figure 1

Vectisol^® chemical composition, rationale, and in vitro effectiveness. (A) Molecular structure of β-cyclodextrin with seven glucose subunits. (B) Schematic representation of the Vectisol^® inclusion complex. (C) Cells were subjected to conditions mimicking organ preservation, using a 24 h hypothermia/hypoxia incubation with University of Wisconsin solution, during which Vectisol^® was added in the Vectisol group, followed by a return to normal culture conditions, at which point the cell survival was assessed. Shown are each data point, as well as mean ±SD, statistics (Wilcoxon Test): †: p < 0.05 to Ctr.

Figure 2

Evaluation of Vectisol^® benefits during static preservation: early follow-up. Vectisol was used to supplement preservation solutions in a porcine model of kidney autotransplantation, where solutions used were: (A) Celsior^®; (B) University of Wisconsin^® (UW); (C) Custodiol^®; and (D) Solution pour la Conservation des Organes en Transplantation (SCOT15^®). The benefits were evaluated over the first two weeks post-reperfusion. Shown are: Top: plasma creatinine, Bottom: sodium excretion. Graphical evolution over the first two weeks is displayed, and AUC (Area Under the Curve) comparison was used to determine the statistical difference. Shown are each data point (empty dots), as well as mean ± SD (filled diamonds), statistics (Wilcoxon Test): †: p < 0.05 to Ctr.

Figure 3

Evaluation of Vectisol^® benefits during static preservation: late follow-up. Vectisol was used to supplement preservation solutions in a porcine model of kidney autotransplantation. Solutions used were: (A) Celsior^®; (B) University of Wisconsin^® (UW); (C) Custodiol^®; and (D) Solution pour la Conservation des Organes en Transplantation (SCOT15^®). Pigs were euthanized after 1 month of follow-up. Shown are: Top: plasma creatinine, Bottom: urinary protein. Shown are each data point (empty dots) as well as mean ± SD (filled diamonds), statistics (Wilcoxon Test): †: p < 0.05 to Ctr.

Figure 4

Evaluation of Vectisol^® benefits during machine preservation 30 min after reperfusion. Cortical biopsies were collected 30 min after reperfusion and snap-frozen in an OCT (Optimal Cutting Temperature) compound. Samples were processed for TUNEL (A) or CellRox (B) staining, and signals were quantified as exposed in the methods. Representative images for each group are shown, and quantification of signals for all animals is presented at the bottom. Shown are each data point (empty dots) as well as mean ± SD (filled diamonds), statistics (Dunn’s Test followed by Kruskal Wallis post-hoc): †: p < 0.05 to BioCtR, †¹: p < 0.05 to BioCtr one-sided test; black dots are outliers.

Figure 5

Evaluation of Vectisol^® benefits during machine preservation: early follow-up. The benefits of supplementation were evaluated during the first two weeks of transplantation. Several pathways were investigated: (A) Oxidative stress, through plasma superoxide dismutase (SOD); (B) cell loss, with asparagine amino transferase (ASAT); (C) glomerular filtration, using serum creatinine; (D) tubular function, through sodium excretion, and (E) kidney necrosis, through Neutrophil gelatinase-associated lipocalin (NGAL). Graphical evolution over the first two weeks is displayed, and AUC comparison was used to determine statistical differences when applicable. Shown are each data point (empty dots), as well as mean ± SD (filled diamonds), statistics (Dunn’s Test followed by Kruskal Wallis post-hoc): †: p < 0.05 to BioCtR, ‡: p < 0.05 to BioCyd; black dots are outliers.

Figure 6

Evaluation of Vectisol^® benefits during static preservation: late follow-up. The benefits of supplementation were evaluated at the end of the three months’ follow-up. Biological evaluation was performed through (A) Plasma Creatinine and (B) Urinary Proteins. Histological evaluations of (C) Tubular Atrophy and (D) Intersitial Fibrosis was performed by a blinded pathologist following a semi-quantitative grading of the percentages of lesions by field: scores: 0 = no alteration, 1 = lesions < 25%, 2 = lesions between 25–50%, 3 = lesions between 51–75%, 4 = lesions between 76–100%. Shown are each data point (empty dots), as well as mean ± SD (filled diamonds), statistics (Dunn’s Test followed by Kruskal Wallis post-hoc): †: p < 0.05 to BioCtR, ‡: p < 0.05 to BioCyd.

Figure 7

Representative images of histology at the end of the follow-up. Animals were euthanized after three months’ follow-up and cortical samples were formalin fixed and processed for PAS (Periodic Acid Schiff) staining. Each column represents a group, displaying several areas of the sample at the indicated magnifications.