Fig 1.
Establishment of CIN in rats and renal protective effect of EGCG on CIN.
The rats sequentially received an intravenous infusion of indomethacin, L-NAME, and iopromide to establish CIN. Serum Cr (A) and BUN (B) concentrations were measured 24h, 48h, and 72h after the intravenous injections. Varying dosages of EGCG (5, 10, 20mg/kg body wt) were administered intravenously 15min before the establishment of CIN. Serum Cr (C), BUN (D) and CrCL (E) were measured 24h after the establishment of CIN. (n = 5, * P<0.05 vs. vehicle; # P<0.05 vs. CM).
Fig 2.
Renal protective effect of EGCG on medullary damage and apoptosis in CIN kidney.
EGCG (10mg/kg body wt) was given intravenously 15min before (pre-EGCG+CM) or after the establishment of CIN (post-EGCG+CM). Serum Cr (A) and BUN (B) were measured 24h after the establishment of CIN. (C) Representative images of H&E staining under ×400 magnification in the outer medulla. Arrows showed examples of protein casts and tubular vacuolar degeneration/necrosis. Erythrocytes and infiltration of polymorphonuclear cells could be easily observed in the interstitium. (D) Representative images of TUNEL assay under ×400 magnification in the outer medulla. (E) The histopathologic scores of medullary damage. (F) Quantification of TUNEL-positive nuclei per ×200 field. (n = 5, * P<0.05 vs. vehicle; # P<0.05 vs. CM).
Fig 3.
Role of HO-1 in the antioxidant and renal protective effect of EGCG in CIN.
EGCG (10mg/kg body wt) was intravenously infused 15min before the establishment of CIN. The HO-1 inhibitor ZnPP (30mg/kg body wt) was injected intraperitoneally 7h before EGCG pretreatment. The rats were sacrificed at 24h after the establishment of CIN. Renal MDA level (A) and SOD activity (B) were measured. Renal HO-1 protein detected by immunoblotting was expressed as the ratio of HO-1 and GAPDH (C). Serum Cr (D), and BUN (E) were determined to evalute the renal function. Nrf2 expression in the nuclear fraction of renal tissue detected by immunoblotting was expressed as the ratio of Nrf2 and histone H3 (F). (n = 5, * P<0.05 vs. vehicle; # P<0.05 vs. CM; & P<0.05 vs. EGCG).
Fig 4.
Role of HO-1 in the anti-inflammatory effect of EGCG in CIN.
EGCG (10mg/kg body wt) was intravenously infused 15min before the establishment of CIN. The HO-1 inhibitor ZnPP (30mg/kg body wt) was injected intraperitoneally 7h before EGCG pretreatment. The rats were sacrificed at 24h after the establishment of CIN. Renal MPO activity (A) and IL-1βlevel (B) were measured. Renal NLRP3 protein expression was detected by immunofluorescence microscopy (red fluorescence, ×400) (C), and semi-quantified by immunoblotting, expressed as the ratio of NLRP3 and GAPDH (D). (n = 5, * P<0.05 vs. vehicle; # P<0.05 vs. CM; & P<0.05 vs. EGCG).
Fig 5.
Schematic diagram of possible mechanism of EGCG in protecting against CIN.
HO-1 expression is markedly enhanced by EGCG via activation of the Nrf2/HO-1 pathway. HO-1 is essential to the protective effect of EGCG on CIN in anti-oxidation and anti-inflammation. Dash-line arrow stands for unclear or multiple-step actions. Straight-line arrow stands for direct actions.