Chitosan is widely used in the biomedical field due its chemical and pharmacological properties. However, intake of chitosan results in renal tissue accumulation of chitosan and promotes an increase in calcium excretion. On the other hand, the effect of chitosan on the formation of calcium oxalate crystals (CaOx) has not been described. In this work, we evaluated the antioxidant capacity of chitosan and its interference in the formation of CaOx crystals in vitro. Here, the chitosan obtained commercially had its identity confirmed by nuclear magnetic resonance and infrared spectroscopy. In several tests, this chitosan showed low or no antioxidant activity. However, it also showed excellent copper-chelating activity. In vitro, chitosan acted as an inducer mainly of monohydrate CaOx crystal formation, which is more prevalent in patients with urolithiasis. We also observed that chitosan modifies the morphology and size of these crystals, as well as changes the surface charge of the crystals, making them even more positive, which can facilitate the interaction of these crystals with renal cells. Chitosan greatly influences the formation of crystals in vitro, and in vivo analyses should be conducted to assess the risk of using chitosan.
Chitin and its derivative, chitosan, are biopolymers obtained from a large number of terrestrial and marine sources. They have become increasingly important since the study of fishing tailings of crustaceans began in the 1970s. Since then, various studies have been conducted on their properties, which provided a basis for the use of these two polymers in industrial and biotechnological applications [1]. Another factor that partly explains the industrial applicability of chitin/chitosan is the fact that chitin is the second most abundant natural polymer around the world. Chitin is composed of units of d-N-acetyl glucosamine linked together by β-(1–4). Chitosan is obtained from chitin, which undergoes different processes that remove the acetyl groups. The process of deacetylation of chitin to form chitosan is not complete, so by controlling the reaction conditions, it is possible to obtain chitosan with different levels of acetylation. Therefore, chitosan is defined as a linear polysaccharide formed by the random distribution of two monosaccharides, d-glucosamine and d-N-acetyl-glucosamine, which are joined together by a β-(1–4) bond. The amine groups are positively charged under physiological conditions [2].
Currently, many of the polymers used in various areas are synthetic materials, but their biocompatibility and biodegradability are very limited. On the other hand, chitosan is well known for its biocompatibility by presenting unique characteristics, such as a pH-dependent behavior, namely regarding its molecular conformation and solubility in the environment in which it is found, namely mucoadhesivity and the ease of overcoming the epithelial junctions. Furthermore, chitosan also has many pharmacological properties, making it a strong candidate to replace synthetic polymers [3,4]. In this context, in recent years, chitosan has been widely used for nanoparticle production; by the year 2012, there were approximately 10,000 published articles that had a nanotechnology and chitosan theme, accumulating more than 119,000 citations [5], which shows the considerable interest from the scientific community in the development of compounds made from chitosan. In addition, much of this interest is generated by the fact that chitosan transfers many of its properties to the nanoparticles formed from it. This leads to a question: Can the toxicity of chitosan also be transferred to nanocomposites? Existing data currently suggest that the polysaccharide chitosan is a compound with low toxicity [6,7] and that the chitosan nanoparticle toxicity would be more related to the nanoparticle size and the presence of other compounds than to the presence of chitosan [8].
Most of the chitosan-based nanocomposites, when administered to animals or patients, release chitosan into the blood stream [9]. The chitosan reaches several organs and tends to accumulate in the kidneys 8 h after oral administration [10,11], specifically in the proximal tubule cells [12]. It was also shown that the presence of chitosan in the body increases the amount of calcium excreted in urine [13]. Nevertheless, no studies have reported the toxicity of chitosan in renal tissue nor is there evidence that this caused the calcium oxalate crystal formation and consequent urolithiasis.
Urolithiasis is the formation of kidney stones, which can lead to tissue damage. This condition is a common cause of hospitalization. It is estimated that about 122 of 100,000 hospital admissions can be assigned to it. Moreover, once a patient is diagnosed with kidney stones, it is likely that the patient will develop new stones. Some studies have shown that 50%–70% of patients with a history of urolithiasis will have another crisis over the next 10 years [14].
The process of crystal formation occurs from urinary supersaturation, which provides nucleation of crystals. This event occurs when the crystal is used as a niche for similar crystals and/or macromolecules joined together, creating bigger and bigger particles. Once nucleated, the deposition of crystals in the other niche is easy and does not require such high levels of saturation as earlier in the process. The core can grow and add other crystals or organic matrix, forming the calculus, or be eliminated in the form of crystalluria [15]. After the crystallization, aggregation can occur, which describes the connection process of the crystals, resulting in the formation of agglomerate, which can precipitate [16].
Oxidative stress is another factor related to the formation of kidney stones [17]. Studies with mice that were induced to form kidney stones of calcium oxalate (CaOx) using different agents showed a decrease in plasma activities of antioxidant enzymes superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), glucose-6-phosphate dehydrogenase and glutathione S-transferase, as well as decreased plasma levels of free radical scavengers: vitamin E, vitamin C, protein thiol and reduced glutathione (GSH). It was also demonstrated that there was an increase in plasma lipid peroxidation marker molecules [18]. Data with patients who form CaOx calculus also show similar changes, indicating a positive correlation between the decrease of antioxidant defenses and the presence of oxalate/CaOx and CaOx crystal formation [19]. On the other hand, there are studies showing that antioxidant molecules can inhibit the formation of oxalate crystals in renal tissue [20,21].
As previously stated, chitosan is accumulated in renal tissue. Furthermore, it was demonstrated that the presence of chitosan in the body increases the amount of calcium excreted in urine [13], which is a primary factor in the formation of kidney stones. Given the above, this study investigated whether chitosan is able to induce the formation of CaOx crystals in vitro and whether it has antioxidant activity.