Tannic Acid Improves Renal Function Recovery after Renal Warm Ischemia-Reperfusion in a Rat Model

Abstract

Background and purpose: Ischemia-reperfusion injury is encountered in numerous processes such as cardiovascular diseases or kidney transplantation; however, the latter involves cold ischemia, different from the warm ischemia found in vascular surgery by arterial clamping. The nature and the intensity of the processes induced by ischemia types are different, hence the therapeutic strategy should be adapted. Herein, we investigated the protective role of tannic acid, a natural polyphenol in a rat model reproducing both renal warm ischemia and kidney allotransplantation. The follow-up was done after 1 week.

Experimental approach: To characterize the effect of tannic acid, an in vitro model of endothelial cells subjected to hypoxia-reoxygenation was used.

Key results: Tannic acid statistically improved recovery after warm ischemia but not after cold ischemia. In kidneys biopsies, 3h after warm ischemia-reperfusion, oxidative stress development was limited by tannic acid and the production of reactive oxygen species was inhibited, potentially through Nuclear Factor erythroid-2-Related factor 2 (NRF2) activation. In vitro, tannic acid and its derivatives limited cytotoxicity and the generation of reactive oxygen species. Molecular dynamics simulations showed that tannic acid efficiently interacts with biological membranes, allowing efficient lipid oxidation inhibition. Tannic acid also promoted endothelial cell migration and proliferation during hypoxia.

Conclusions: Tannic acid was able to improve renal recovery after renal warm ischemia with an antioxidant effect putatively extended by the production of its derivatives in the body and promoted cell regeneration during hypoxia. This suggests that the mechanisms induced by warm and cold ischemia are different and require specific therapeutic strategies.

Keywords: cold ischemia; oxidative stress; renal function recovery; tannic acid; warm ischemia.

Figure 1

Chemical formula of tannic acid. Tannic acid has numerous phenol groups supporting a high solubility in aqueous solutions and a great antioxidative capacity. The phenolic groups (ArOH) give electrons to different radicals (LOO°) formed during ischemia–reperfusion and the radical tannic acid produced (ArO°) remains stable and not reactive linked to tautomeric forms produced from the benzenic group.

Figure 2

Plasma kinetic of gallic acid after intraperitoneal injection of tannic acid at 50 mg/kg in rat. Gallic acid was determined in plasma collected from 30 min to 24 h, by high pressure liquid chromatography method coupled with spectrophotometry, n = 3–4 at each times. Values are mean ± SD.

Figure 3

Tannic acid injection improves renal function recovery after warm kidney ischemia– reperfusion. Rats were subjected to sham or bilateral renal ischemia for 60 min by renal pedicle clamping with or without prior injection of tannic acid. Renal function was evaluated by creatinine plasma levels concentrations. n = 8, values are mean ± SD, * p < 0.05.

Figure 4

Tannic acid injection was ineffective to improve renal function recovery after cold ischemia– reperfusion in kidney graft model with 6 h of cold preservation. Rats were subjected to sham or kidney transplantation with bilateral nephrectomy with or without prior injection of tannic acid. Renal function was evaluated by creatinine plasma levels concentrations. n = 7–8 in transplanted groups and n = 6 in sham groups, values are mean ± SD, * p < 0.05.

Figure 5

Tannic acid injection protects from oxidative stress development. In kidneys biopsies obtained 3 h after reperfusion following warm ischemia, tannic acid limits P67phox (A) and xanthine oxidase (B) expressions, and reduces the decrease in total glutathion (C) induced by ischemia–reperfusion injury, without effect on superoxide dismutase activity (D), n = 8, values are mean ± SD, * p < 0.05.

Figure 6

Tannic acid prevents oxidative stress in ischemia–reperfusion kidneys. Reactive oxygen species production induced by ischemia–reperfusion injury was evaluated by cell ROX staining. (A) Representative pictures (40X) of staining in kidney biopsies after 3 h of reperfusion following warm ischemia with or without tannic acid injection; (B) quantitative evaluation of staining. n = 4, values are mean ± SD, * p < 0.05.

Figure 7

Tannic acid induced Nuclear Factor erythroid-2-Related factor 2 (NRF2) expression in kidneys. Protein expression of NRF2 determined by immunostaining (40X; A–B) or by western blot (C). Quantification of fluorescence intensity for NRF2 immunostaining (B). n = 8, values are mean ± SD, * p < 0.05.

Figure 8

Tannic acid and derivatives protect endothelial cells from hypoxia–reoxygenation injury. (A) Cytotoxicity induced by the hypoxia–reoxygenation sequence was prevented by tannic acid (AT) and its main metabolites gallic acid (GA) and penta-O-galloyl-beta-D-glucose (PGG) with a dose-dependent response. Molecules were added in the culture medium during the hypoxia–reoxygenation sequence. n = 3, n = 2. (B) XTT analysis showing that cytotoxicity induced by the hypoxia–reoxygenation sequence was prevented by tannic acid (12.5 mg/mL, 7.3 µmol/L). Molecules were added in the culture medium during the hypoxia–reoxygenation sequence. n = 3, n = 2, values are mean ± SD, * p < 0.05 to the PBS condition (vehicle).

Figure 9

Tannic acid and gallic acid prevent oxidative stress induced by hypoxia–reoxygenation sequence. Reactive oxygen species production was determined by Cell ROX staining. (A) Representative pictures (40X) of staining in human aortic endothelial cells (HAEC) subjected to the hypoxia–reoxygenation sequence with or without tannic acid (7.3 µmol/L) or gallic acid (7.3 µmol/L); (B) quantitative evaluation of the staining. n = 4, values are mean ± SD, * p < 0.05.

Figure 10

Tannic acid improves endothelial cell migration during hypoxia. Cell migration assay was determined by the number of cells migrating in an area after warm hypoxia, or 24 h of reoxygenation with or without tannic acid (7.3 µmol/L). n = 3, values are mean ± SD, * p < 0.05.

Figure 11

The starting conformation (A) and the different representative structures of tannic acid in the solution (B–F). The PGG is in blue, the five different gallic acid tails are in different colours. Oxygen atoms are in red, and hydrogen atoms in white.

Figure 12

The six different representative structures of tannic acid in the membrane, according to the clustering analysis. The PGG is in blue, the five different gallic acid tails are in different colours. Oxygen atoms are in red, and hydrogen atoms in white. The membrane is depicted as spheres with the phosphatidylcholine headgroups in dark orange with hydrogens and oxygens; the tail carbon atoms are pale orange spheres.

Figure 13

The z-coordinate of the center of mass of different parts of the tannic acid, and positions of the different parts of the membrane.

Figure 14

The z-coordinate of the center of mass of the protonated (red) and deprotonated (blue) gallic acid.