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Diabetic nephropathy (DN) is a serious complication of diabetes mellitus with long repercussions and a high mortality rate. In many developed countries, approximately 40% of DN patients further progress to end-stage renal disease (ESRD). Currently, there is no cure for DN and renal preservation acts as a key approach to prevent ESRD. The pathophysiology of DN is characterized by an increase in hyperglycemia-induced reactive oxygen species (ROS) levels that inflict damage on proximal tubule cells (PTCs), which represent the most abundant cell type in the kidney, leading to inflammation, fibrosis and ESRD.
Lactoferrin (Lf) is a protein found in milk and possesses anti-inflammatory, iron-binding, immunomodulatory, etc. properties and acts as an antioxidant. It is commonly taken as a dietary supplement and it’s purported to enhance iron absorption. Here, we hypothesize that Lf supplementation can reduce renal ROS and exert protection in DN.
We utilized the AnOxPePred deep-learning algorithm to predict the free radical scavenging (FRS) and metal-chelating activities (MCA) of human and bovine Lf (bLf). The antioxidative properties of bLf were validated by 1,1-diphenyl-2-picryl hydrazyl (DPPH) and Ferric Reducing Antioxidant Power (FRAP) assay, respectively. To assess the effect of bLf, human kidney PTCs (HK-2) were cultured in low glucose (LG, 5.5 mM) or high glucose (HG, 55 mM) media and treated with varying bLf concentrations for 72 h. The cell viability by assessed using the 3-(4,5-dimethyl thiazol-2-yl-)-2,5-diphenyl tetrazolium bromide (MTT) assay.
The AnOxPePred algorithm predicted the FRS and MCA in both human and bLf based on their full peptide sequence. Our findings, presented in Figures 1 and 2, revealed that both human and bovine Lf exhibited similar trends in FRS and MCA scores. Notably, the regions responsible for the potential antioxidant effects were identified within the transferrin-like 1 and 2 domains.
We performed further analysis of bLf’s FRS activity in varying concentrations ranging from 78.125 – 10000 µg/mL, using the DPPH assay. Our results showed that 5000 µg/mL bLf has approximately 48% FRS activity (Figure 3). Furthermore, MCA was tested through the FRAP assay. At 5000 µg/mL, bLf exhibited antioxidant activity, measuring 88.2 nmol/mL of ferrous equivalents (Figure 4). Remarkably, the top-scoring bLf peptide sequence demonstrated an FRS and MCA score of 0.60836 and 0.32879, respectively. These findings collectively support a concentration-dependent increase in the antioxidative properties of Lf, confirming our initial prediction for the presence of FRS and MCA within Lf.
We conducted an MTT assay to investigate the impact of bLf on HK-2 cell viability under HG. The results demonstrated a significant enhancement in cell viability after 72 h. At a concentration of 750 and 1250 μg/mL, cell viability increased by approximately 24% (p < 0.01) and 35% (p < 0.05), respectively (Figure 5).
Overall, our data demonstrated Lf possesses antioxidant properties, which protect cells from ROS-induced damage in DN, highlighting its potential therapeutic relevance.