Influence of Vitamin C on Antioxidant Capacity of In Vitro Perfused Porcine Kidneys

Abstract

Systemic and localized ischemia and reperfusion injury remain clinically relevant issues after organ transplantation and contribute to organ dysfunctions, among which acute kidney injury is one of the most common. An in vitro test-circuit for normothermic perfusion of porcine kidneys after warm ischemia was used to investigate the antioxidant properties of vitamin C during reperfusion. Vitamin C is known to enhance microcirculation, reduce endothelial permeability, prevent apoptosis, and reduce inflammatory reactions. Based on current evidence about the pleiotropic effects of vitamin C, we hypothesize that the antioxidant properties of vitamin C might provide organ-protection and improve the kidney graft function in this model of ischemia and reperfusion.

Methods: 10 porcine kidneys from 5 Landrace pigs were perfused in vitro for 6 h. For each experiment, both kidneys of one animal were perfused simultaneously with a 1:1 mixture of autologous blood and modified Ringer's solution at 38 °C and 75 mmHg continuous perfusion pressure. One kidney was treated with a 500 mg bolus injection of vitamin C into the perfusate, followed by continuous infusion of 60 mg/h vitamin C. In the control test circuit, an equal volume of Ringer's solution was administered as a placebo. Perfusate samples were withdrawn at distinct points in time during 6 h of perfusion for blood gas analyses as well as measurement of serum chemistry, oxidative stress and antioxidant capacity. Hemodynamic parameters and urine excretion were monitored continuously. Histological samples were analyzed to detect tubular- and glomerular-injury.

Results: vitamin C administration to the perfusate significantly reduced oxidative stress (49.8 ± 16.2 vs. 118.6 ± 23.1 mV; p = 0.002) after 6 h perfusion, and increased the antioxidant capacity, leading to red blood cell protection and increased hemoglobin concentrations (5.1 ± 0.2 vs. 3.9 ± 0.6 g/dL; p = 0.02) in contrast to placebo treatment. Kidney function was not different between the groups (creatinine clearance vit C: 2.5 ± 2.1 vs. placebo: 0.5 ± 0.2 mL/min/100 g; p = 0.9). Hypernatremia (187.8 ± 4.7 vs. 176.4 ± 5.7 mmol/L; p = 0.03), and a lower, but not significant decreased fractional sodium excretion (7.9 ± 2 vs. 27.7 ± 15.3%; p = 0.2) were observed in the vitamin C group. Histological analysis did not show differences in tubular- and glomerular injury between the groups.

Conclusion: Vitamin C treatment increased the antioxidant capacity of in vitro perfused kidney grafts, reduced oxidative stress, preserved red blood cells as oxygen carrier in the perfusate, but did not improve clinically relevant parameters like kidney function or attenuate kidney damage. Nevertheless, due to its antioxidative properties vitamin C might be a beneficial supplement to clinical kidney graft perfusion protocols.

Keywords: acute kidney injury; animal models; antioxidant; ascorbic acid; kidney transplantation; organ dysfunction; oxidative stress; porcine kidney perfusion model in vitro techniques; primary graft dysfunction; reperfusion injury; vitamins.

Figure 1

Perfusion parameters displayed as renal blood flow (A), oxygen consumption (B), arterial hemoglobin (HB, C), and the red blood cell count (RBCs, D). */** p < 0.05/0.01 effect of time for the control group; ### p < 0.001 vs. Control, ## p < 0.01 vs. Control, # p < 0.05 vs. Control.

Figure 2

Creatinine clearance rate (A), and concentration of urine protein (B).

Figure 3

The arterial pH value (A) and the lactate Concentration (B). ** p < 0.01 effect of time for both groups.

Figure 4

The arterial chloride- (A), calcium- (B) and glucose-concentrations (C). ** p < 0.01; *** p < 0.001 effect of time for both groups.

Figure 5

Arterial sodium- (A) and potassium-concentration (B). Fractional sodium- (C) and potassium-excretion (D). ** p < 0.01 effect of time for both groups; # p < 0.05 vs. Control.

Figure 6

Interleukin 10 (IL10; A), IL6 (B), and Tumor necrosis factor-alpha (TNF-α; C). ** p < 0.01 effect of time for both groups.

Figure 7

Oxidative stress was assessed as oxidation reduction potential (A), and antioxidant capacity (B). ** p < 0.01 effect of time for the control group; ## p < 0.01 vs. control, ### p < 0.001 vs. control.