Nephro-toxic effects of intraperitoneally injected EGCG in diabetic mice: involvement of oxidative stress, inflammation and apoptosis

Abstract

Epigallocatechin gallate (EGCG) has been studied for its beneficial effects. However, some case reports have associated EGCG supplementation with hepato-toxicity. In the present study, we investigated the possible nephro-toxic effects of EGCG in diabetic mice. Streptozotocin (150 mg/kg, i.p.) was injected in mice for diabetes induction. EGCG (100 mg/kg/day, i.p.) was then given for 4 days. The administration of EGCG to diabetic mice caused 60% mortality with no death recorded in other groups. Blood samples were collected for estimation of serum cystatin C, neutrophil gelatinase-associated lipocalin and blood urea nitrogen. Animals were then sacrificed and kidneys were rapidly excised for estimation of oxidative stress markers (NADPH oxidase, reduced glutathione, total antioxidant capacity, nuclear factor erythroid 2-related factor 2, heat shock protein 90, hemeoxygenase-1), as well as inflammatory markers (nuclear factor kappa-B and tumor necrosis factor-α). Administration of EGCG to diabetic mice showed significant elevation in serum cystatin C and neutrophil gelatinase-associated lipocalin, marked increase in oxidative stress and inflammatory states in addition to marked over expression of active caspase-3. Histopathological examination confirmed EGCG induced renal damage in diabetic mice. In conclusion, despite of its well known favorable effects, EGCG could paradoxically exhibit nephro-toxic effect in the presence of diabetes.

Figure 1

Effect of EGCG on STZ-induced changes in (A) NADPH oxidase, (B) Nrf2, (C) HO-1, (D) TAC (E) HSP 90 and (F) GSH. Each value represents the mean of 6–8 experiments ± S.E.M. ^*P < 0.05 vs. normal, ^@P < 0.05 vs. EGCG, ^#P < 0.05 vs. STZ.

Figure 2

Effect of EGCG on STZ-induced changes in (A) NfκB and (B) TNF-α. Each value represents the mean of 6–8 experiments ± S.E.M. ^*P < 0.05 vs. normal, ^@P < 0.05 vs. EGCG, ^#P < 0.05 vs. STZ.

Figure 3. Effect of EGCG on STZ- induced changes in immunohistochemical expression of active caspase-3 expression.

(A) (saline) showing normal tubules and glomeruli with –ve immunostaining for active caspase (x400), (B) (EGCG) showing normal tubules and glomeruli with –ve immunostaining for active caspase (x400), (C) (STZ) showing degenerated tubules with focal immunostaining of the epithelium for active caspase (x400) and (D) (STZ and EGCG) showing degenerated tubules with vacuolated epithelium showing wide immunostaining for active caspase (x400). (E) Each value represents the mean of 3 experiments ± S.E.M. ^*P < 0.05 vs. normal, ^@P < 0.05 vs. EGCG, ^#P < 0.05 vs. STZ.

Figure 4. Effect of EGCG on STZ- induced changes in histopathological examination.

(A) Kidney of mouse from control group showing no histopathological changes (H & E X 400) (B) kidney of mouse from EGCG group showing no histopathological changes (H & E X 400). (C) Kidney of mouse from diabetic group showing moderate congestion of renal blood vessel (H & E X 400) (D). Kidney of mouse from diabetic group receiving EGCG showing severe congestion of renal blood vessel and vacuolation of renal tubular epithelium (H & E X 400). (E) Kidney of mouse from diabetic group receiving EGCG showing necrosis of epithelial lining renal tubules (H & E X 400).