Low-molecular-weight chitosan scavenges methylglyoxal and N ε-(carboxyethyl)lysine, the major factors contributing to the pathogenesis of nephropathy

Chou et al. SpringerPlus (2015) 4:312 DOI 10.1186/s40064-015-1106-4 Open Access RESEARCH Low‑molecular‑weight chitosan scavenges methylglyoxal and Nε‑(carboxyethyl) lysine, the major factors contributing to the pathogenesis of nephropathy Chu‑Kuang Chou1,3, Shih‑Ming Chen2, Yi‑Chieh Li2, Tzu‑Chuan Huang2 and Jen‑Ai Lee2* Abstract Methylglyoxal (MG) can cause protein glycation, resulting in cell damage and dysfunction. Accumulation of MG and its downstream metabolite Nε-(carboxyethyl)lysine (CEL) has been identified in several variations of nephropathy, including diabetic, hypertensive, and gentamicin-induced nephropathies. In this study, we investigated the effects of low-molecular-weight chitosan (lmw-chitosan) on MG-induced carbonyl stress in aristolochic acid-induced nephropa‑ thy. We used a buffer to investigate whether MG could be scavenged by lmw-chitosan in vitro. In addition, we also used a mouse model of aristolochic acid-induced nephropathy, which exhibits 12-fold greater accumulation of MG in the kidneys than that found in control animals, to examine whether lmw-chitosan could decrease MG levels in vivo. Examination of the binding of lmw-chitosan with MG in vitro demonstrated that the concentration of lmw-chitosan necessary to achieve 50% inhibition was 4.60 µg mL−1. Treatment with lmw-chitosan (500 mg kg−1 day−1 orally) for 14 days significantly decreased renal MG accumulation from 212.86 ± 24.34 to 86.15 ± 33.79 µg g−1 protein (p < 0.05) and CEL levels from 4.60 ± 0.27 to 2.84 ± 0.28 µmol µg−1 protein (p < 0.05) in the aristolochic acid-induced nephrop‑ athy model. These data suggest that lmw-chitosan might represent a novel treatment modality for MG-related diseases such as nephropathy. Keywords: Nephropathy, Methylglyoxal, Nε-(carboxyethyl)lysine, Low-molecular-weight chitosan Background Nephropathy can lead to the acquisition of end-stage renal disease, which is associated with major health problems. Causes of nephropathy include medications or disease, as well as the toxicity associated with compounds such as the organic by-product methylglyoxal (MG). Aristolochic acid (AAN)- and gentamicin-induced nephropathies are both related to MG accumulation in the kidney (Li et al. 2012, 2013). The pathogenesis of diabetic nephropathy and its associated complications are primarily related to MG and its downstream metabolites, which cause injury to the kidneys (Rabbani and *Correspondence: 2 School of Pharmacy, College of Pharmacy, Taipei Medical University, No. 250 Wuxing St., Taipei 11031, Taiwan, ROC Full list of author information is available at the end of the article Thornalley 2011). Additionally, increased levels of MG and advanced glycation end products (AGEs) have been identified in the kidneys of spontaneously hypertensive rats (Wang et al. 2004). MG can also cause oxidative stress. MG is generated during the process of tissue injury repair, which places additional energy demands on cells. MG, formed as a byproduct of glycolysis and lipid and amino acid metabolism, modifies proteins and interferes with protein function (Rabbani and Thornalley 2012). Furthermore, Nε-(carboxyethyl)lysine (CEL), the downstream AGE of MG, is also known to enhance inflammation (Rabbani and Thornalley 2011). To date, only metformin has been approved as an effective MGreducing agent by the US Food and Drug Administration. However, this drug is not suggested for use in patients with renal insufficiency due to its toxicity. Therefore, an © 2015 Chou et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Chou et al. SpringerPlus (2015) 4:312 effective and potent anti-MG agent is needed for better prevention and treatment of MG-induced renal injury. Chitosan is a natural polysaccharide with a high molecular weight (500–1,000 kDa) and has been widely used in the pharmaceutical, food, and cosmetic industries (Zhang et al. 2010). Upon hydrolyzation into low-molecular-weight chitosan (lmw-chitosan), it becomes highly water-soluble and therefore has more biological applications. For example, lmw-chitosan can be utilized as an antibacterial agent, antifungal agent, antidiabetic drug, and as a lipid-lowering medication (Lee et al. 2003; Seyfarth et al. 2008; Anraku et al. 2010; Chen et al. 2012). In addition, lmw-chitosan has been shown to have protective effects on the kidneys in studies on diabetes (Yoon et al. 2008). However, the molecular mechanisms underlying these effects have not been elucidated. Lmw-chitosan can also be used as an agent for kidneyspecific targeting and drug delivery. For example, conjugation of lmw-chitosan nanoparticles with drugs that benefit the kidney such as catechol or tripterygium glycoside have been investigated by multiple laboratories (Qiao et al. 2014; Chen et al. 2013). Conversely, the demonstration that the levels of MG are increased 12-fold in the kidneys of AAN mice suggests that this might represent a good model for investigating the effect of lmwchitosan on decreasing physiological MG accumulation in vivo. While MG and CEL are important in disease pathogenesis, the relationships between lmw-chitosan and MG or CEL have not been determined. Therefore, in this study we aimed to elucidate the effects and underlying mechanisms of lmw-chitosan on MG and CEL accumulation in vitro and in vivo. Page 2 of 7 Results Inhibitory activity of lmw‑chitosan in vitro and in vivo The data of MG chelation and inhibition by lmw-chitosan in vitro are shown in Figure 1a. The concentration of lmw-chitosan required for 50% inhibition was determined to be 4.60 µg mL−1. MG levels in the in vivo study were ascertained by HPLC and then normalized with protein assay. MG levels in the kidneys of Group A mice were significantly higher than those in Group C mice (212.86 ± 24.34 vs. 18.23 ± 8.05 μg g−1 protein, respectively, p < 0.05). Treatment with lmw-chitosan decreased MG levels to 86.15 ± 33.79 μg g−1 protein (p < 0.05 vs. Group AM; Figure 1b). Additionally, the lmw-chitosan only group (Group M) exhibited an MG level of 6.26 ± 1.79 μg g−1 protein. Immunohistological staining and quantitative detection of CEL Immunochemistry was performed to visualize CEL expression in the kidneys (Figure 2A). We observed that CEL staining was present in several monocytes of the desquamation tubule region and that lmw-chitosan treatment significantly decreased the level of CEL staining (p < 0.05). The quantitative data of CEL expression in mouse kidneys are shown in Figure 2B. The levels of CEL in Group A mice were significantly higher than those in Group C mice (4.6 ± 0.27 vs. 2.24 ± 0.08 μmol µg−1 protein, respectively). After lmw-chito (...truncated)