Bovine lactoferrin ameliorates ferric nitrilotriacetate-induced renal oxidative damage in rats

Abstract

Milk provides a well-balanced source of amino acids and other ingredients. One of the functional ingredients in milk is lactoferrin (LF). LF presents a wide variety of bioactivities and functions as a radical scavenger in models using iron-ascorbate complexes and asbestos. Human clinical trials of oral LF administration for the prevention of colon polyps have been successful and demonstrated that dietary compounds exhibit direct interactions. However, antioxidative properties of LF in distant organs require further investigation. To study the antioxidant property of LF, we employed bovine lactoferrin (bLF) using the rat model of ferric nitrilotriacetate (Fe-NTA)-induced renal tubular oxidative injury. We fed rats with bLF (0.05%, w/w) in basal chow for 4 weeks and sacrificed them after Fe-NTA treatment. After intraperitoneal administration of 9.0 mg iron/kg Fe-NTA for 4 and 24 h, bLF pretreatment suppressed elevation of serum creatinine and blood urea nitrogen levels. In addition, we observed protective effects against renal oxidative tubular damage and maintenance of antioxidant enzyme activities in the bLF-pretreated group. We thus demonstrated the antioxidative effect of bLF against Fe-NTA-induced renal oxidative injury. These results suggest that LF intake is useful for the prevention of renal tubular oxidative damage mediated by iron.

Keywords: chemoprevention; ferric nitrilotriacetate; glutathione metabolism; lactoferrin; oxidative renal damage.

Fig. 1

The effect of bovine lactoferrin (bLF) on normal growth. Body weight curve. All rats were active and healthy following administration of different concentrations of bLF (1, 0.5, 0.1, 0.05 and 0.01% w/w) (n = 3).

Fig. 2

Serum markers for renal dysfunction at 4 and 24 h after Fe-NTA administration. (A) Serum creatinine: Fe-NTA, 4 h. (B) Serum creatinine: Fe-NTA, 24 h, (C) Serum BUN: Fe-NTA, 4 h. (D) Serum BUN: Fe-NTA 24 h. Fe-NTA treatment induced elevation of serum creatinine and BUN, which are markers of renal failure. Pretreatment with bLF suppressed elevation of serum creatinine and BUN. (ANOVA, p<0.0001 for A–D; ^#p<0.05 vs untreated; *p<0.05 vs Fe-NTA alone; **p<0.01 vs Fe-NTA alone).

Fig. 3

Determination of renal reduced glutathione (GSH) at 4 h and activities of renal antioxidative enzymes at 4 and 24 h after Fe-NTA administration. (A) GSH: Fe-NTA, 4 h. (B) GSH reductase: Fe-NTA, 4 and 24 h. (C) GSH peroxidase: Fe-NTA, 4 and 24 h. (A) Bovine lactoferrin (bLF, 0.05%, w/w) pretreatment demonstrated slight elevation of renal GSH level (p = 0.25, untreated vs bLF alone). GSH depletion was detected in Fe-NTA-administered rats. We observed that bLF pretreatment attenuated Fe-NTA-induced renal oxidative damage. (B) and (C) Protective effect of bLF (0.05%, w/w) pretreatment on Fe-NTA-induced renal oxidative damage was observed based on both parameters (ANOVA, p<0.0001, for A–C; ^#p<0.05 vs untreated or bLF alone; *p<0.05 and **p<0.01 vs Fe-NTA alone).

Fig. 4

Histological and immunohistochemical analyses of kidney at 4 and 24 h after Fe-NTA administration. Hematoxylin and eosin staining (HE). (A) Untreated, (B) Fe-NTA alone, 4 h, (C) or in the presence of bLF + Fe-NTA, 4 h. Immunohistochemical staining of 4-hydroxy-2-nonenal-modified proteins (HNE). (D) Untreated, (E) Fe-NTA alone, 4 h, (F) bLF + Fe-NTA, 4 h. HE staining. (G) Fe-NTA alone, 24 h, and (H) bLF + Fe-NTA, 24 h. Representative images are shown. Scattered necrotic tubules were detected (B). Only a few necrotic tubules and some degenerative tubules were observed (C). HNE immunostaining revealed accumulation of oxidatively modified proteins. No positive tubules in the HNE immunostaining were observed (D), whereas many positive tubules were detected (E). Immunopositivity was markedly decreased (F). After Fe-NTA administration for 24 h, oxidative injury destroyed massive proximal tubules (G). Pretreatment with bLF protected against oxidative injury (H). Few infiltrating inflammatory cells were observed in histological samples (bar, 50 µm).