Development of Dietary Thiol Antioxidant via Reductive Modification of Whey Protein and Its Application in the Treatment of Ischemic Kidney Injury

Abstract

Thiol antioxidants play important roles in cell and body defense against oxidative stress. In body fluid, albumin is the richest source of thiol antioxidants. One recent study showed that the reductive modification of thiol residues in albumin potentiated its antioxidative activity. Given that whey protein (WP) contains albumin and other thiol-active proteins, this property of WP could be exploited to develop novel thiol antioxidants. The aim of this study was to address this possibility. WP was reductively modified with dithiothreitol (DTT). The modified protein exhibited significantly elevated free sulfhydryl groups (-SH) and thiol antioxidative activity. It detoxified H2O2 and prevented H2O2-initiated protein oxidation and cell death in a -SH group-dependent way in vitro. In addition, it reacted with GSH/GSSG and altered the GSH/GSSG ratio via thiol–disulfide exchange. In vivo, oral administration of the reductively modified WP prevented oxidative stress and renal damage in a mouse model of renal injury caused by ischemia reperfusion. It significantly improved renal function, oxidation, inflammation, and cell injury. These protective effects were not observed in the WP control and were lost after blocking the -SH groups with maleimide. Furthermore, albumin, one of the ingredients of WP, also exhibited similar protective effects when reductively modified. In conclusion, the reductive modification of thiol residues in WP transformed it into a potent thiol antioxidant that protected kidneys from ischemia reperfusion injury. Given that oxidative stress underlies many life-threatening diseases, the reductively modified dietary protein could be used for the prevention and treatment of many oxidative-stress-related conditions, such as cardiovascular diseases, cancer, and aging.

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“…Western blot analysis was conducted as previously described [ 19 , 21 ]. Cell and tissue lysates were prepared using SDS and RIPA lysis buffers, respectively, supplemented with a protease inhibitor cocktail.…”

“…Cytotoxicity was quantified using a LDH cytotoxicity detection kit (LDH Cytotoxicity Detection Kit, Takara Bio Inc.) according to the manufacturer’s protocol [ 19 , 21 ]. Cells were seeded in 96-well plates and grown to 80–90% confluence in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) supplemented with fetal bovine serum (FBS).…”

“…Protein sulfenylation was assessed as previously described [ 19 , 21 , 22 ]. Serum samples were diluted 20- to 50-fold, while cell and tissue lysates were prepared at 1–5 mg/ml.…”

“…Cell viability was assessed using a calcein AM/PI live/dead assay kit (Dojindo) according to the manufacturer’s instructions [ 19 , 21 ]. Cells were incubated with a calcein AM and PI mixture for 10–20 min at 37 °C.…”

“…Serum BUN levels were determined using commercial colorimetric assay kits according to the manufacturers' instructions [ 21 ]. Serum samples were diluted as needed and incubated with the assay reagents for 30 min at room temperature.…”