Antilithiatic effects of crocin on ethylene glycol-induced lithiasis in rats

Urinary tract stones (urolithiasis) are the third prevalent disorder affecting the urinary system. This disorder arises as a result of a cascade of events initiated by supersaturation, crystal nucleation, growth, aggregation, retention and migration to the renal papillary surfaces [1, 2]. Almost 80 % of all kidney stones are predominately composed of CaOx [3]. High recurrence rate of this disorder is worrying and long-term treatment strategies have been inadequate [4, 5].

For years, medicinal plants have become an important source of new chemical substances with potential therapeutic effects.

Saffron, the dried stigmas of Crocus sativus L., (family, Iridaceae), is native to the Mediterranean environment and widely cultivated in Iran [6]. Traditionally, this plant has been used as a colorant and seasoning in food. Moreover, in folklore medicine, saffron has been used in numerous illnesses including cough, asthma, menstruation problems, insomnia, pain, colic, chronic uterine hemorrhage, painful urination, kidney stone, cardiovascular disorders and tumors [7, 8, 9]. Avicenna, a Persian physician and the most famous philosopher–scientist of the Islamic world, described the details of a remedy, consisting of saffron plus honey, prescribed to facilitate the passage of kidney stones [10]. Saffron extracts and its active constituents, safranal and crocin, have shown anti-cancer [11, 12], antioxidant [13], hypolipidemic and insulin resistance improvement [14], protection against cisplatin-induced acute renal failure [15], and anti-convulsant [16], anti-depressant [17, 18], anti-anxiety [19], anti-nociceptive and anti-inflammatory properties [20, 21, 22]. In this study we investigated the possible effect of crocin, a main water-soluble carotenoid of C. sativus as a preventive or curative agent against ethylene glycol-induced lithiasis using male Wistar rats.

Male Wistar rats with the weight range of 190–200 g were obtained from the animal house of the school of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran. They were maintained under a 12 h light–dark cycle environment. Animals had access to food and water ad libitum throughout the study. All the procedures were approved by Mashhad University of Medical Sciences and followed the internationally accepted Principles for Laboratory Animal Use and Care [25].

Collection and analysis of tissue for histological assays

After the last urine collection, the kidneys were isolated from killed animals and washed in ice‐cold saline. The right kidneys were fixed in 10 % neutral buffered formalin. The 5 μm-thick sections were stained via a hematoxylin and eosin solution and examined by light microscopy. Calcium oxalate deposits of slides were randomly counted using an Olympus microscope, type BX 40, with a magnification of 40×. As described previously, a sagittal section of each kidney sample was divided into eight equal-sized parts by four virtual lines, to count the number of crystalline deposits, as shown in Fig. 1 [28]. The total number of CaOx deposits in each sample was reported as average of the eight readings. The histological slides were examined by a pathologist who was blind to the experiment.

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The annual incidence and prevalence of urinary tract stones is increasing, while the age of onset of this disorder is decreasing, probably due to change in dietary lifestyle, habits, diets, hypertension and obesity [30, 31]. Despite recent advances in the treatment of kidney stones with modern techniques such as extracorporeal shock wave lithotripsy, high cost, several adverse effects and the recurrence of stones limit their usefulness. The low efficacy of current drugs such as alkali citrate and thiazide diuretics, in addition to their lesser tolerability, necessitates the considerable need for finding new drugs [1, 32, 33]. Herbal medicines have shown protective effects in different in vitro and in vivo models of urolithiasis, perhaps due to multiple constituents acting through different pathways [34, 35, 36, 37, 38]. In the present study, the prophylactic and curative effects of crocin, a main pharmacologically active component of C. sativus, were evaluated against ethylene glycol-induced nephrolithiasis in rats. Only male rats were used in this study because the prominence of stone induction is more in male rats compared to female ones. There is a close similarity between the urinary system of these animals and that of humans [3]. It has been reported that female sex hormones can inhibit renal crystal deposition in EG-treated rats through suppressing urinary oxalate excretion and the expression of osteopontin (OPN) [39]. It has been indicated that chronic administration of EG, which is oxidized to oxalic acid by nonspecific dehydrogenase, causes increased renal retention and excretion of oxalate and the formation of renal calculi composed mainly of calcium oxalate [37, 38]. Consistent with previous reports, in the present study, oxalate increased in animals treated with EG for 30 days. It has been demonstrated that hyperoxaluria is a major risk factor in the pathogenesis of renal calculi [40]. The elevated levels of oxalate provide an environment appropriate for calculi induction through forming calcium oxalate crystals. Although decreased levels of urinary PH has been reported in some studies [41], in the present study the PH of urine did not significantly change over a period of 30 days of EG administration, which is in accordance with the results of Green et al. [42], who concluded that metabolic acidosis does not develop in conventional EG treatments, but may result from renal insufficiency induced by oxalate load.

A decreased level of urinary citrate and magnesium was observed in the ethylene glycol-induced urolithic rats. The low levels of citrate and magnesium are important risk factors for stone formation in the kidney [1]. These protective factors prevent crystallization of salts in urine. Citrate complexes with calcium ions and reduces urinary supersaturation of calcium salts. As a result, crystal growth and aggregation are inhibited. Furthermore, citrate increases the activity of some macromolecules in urine, such as Tamm–Horsfall protein that inhibits calcium oxalate aggregation and may reduce the expression of urinary osteopontin (OPN), an important component of the protein matrix of urinary stones. Magnesium can also form complexes with oxalate and decrease supersaturation. In addition, the excretion of citrate in urine has been reported to be elevated after administration of magnesium [1, 43, 44, 45]. The urinary citrate excretion reached normal levels following prophylactic treatment with 40 mg/kg of crocin and potassium citrate as a positive control. Magnesium levels of urine were restored on the supplementation with prophylactic and curative regimens of crocin in a dose-dependent manner. No significantly increased excretion of uric acid and phosphate was observed in the lithiatic group when compared with intact control animals, which is in agreement with the Shirfule et al. study [46]. It might be speculated that increased levels of such factors need more concentration of EG or longer exposure to it.

The present observation showed that protein excretion increased in the lithogenic treatment with ethylene glycol, which is as a result of proximal tubular dysfunction [47, 48]. Urinary protein loss was brought down significantly by the prophylactic regimen of crocin (20 and 40 mg/kg), but not at a low dose of 10 mg/kg of crocin.

There was limited or no crystal formation in animals receiving 20 and 40 mg/kg of prophylactic treatment of crocin.

It has been indicated that hyperoxaluria induces peroxidative damage to the renal tubular membrane surface (lipid peroxidation), which in turn leads to the production of superoxide and hydroxyl free radicals, oxidative stress-induced cell membrane rupture, cell death (apoptosis) and subsequent development of calcium oxalate urinary stone generation [49, 50]. The involvement of oxidative stress has also been demonstrated in the kidneys of stone-forming patients [51]. Therefore, a reduction of renal oxidative stress could be an effective therapeutic approach [52]. Antioxidants such as vitamin E, catechin and selenium have been reported to prevent hyperoxaluria-induced calcium oxalate crystal deposition [49, 53]. It was observed that oxidative damage as reflected from the increased level of MDA, a marker of oxidative injury, significantly increased in the kidneys of the stone-forming group; however, prophylactic crocin at the dose of 20 and 40 mg/kg attenuated the content of MDA in the kidneys of lithogenic rats. Supplementation with potassium citrate also improved urinary citrate and magnesium and reduced oxalate and crystals number; however, it did not reduce the urinary lipid peroxidation product, MDA.

It is suggested that the nephroprotective effects of crocin in the EG-induced urolithiasis model could be through maintaining balance between stone promoters and inhibitors, reducing deposition of CaOX crystals from the kidney and preventing the EG-induced lipid peroxidation.

Antioxidant and anti-inflammatory properties of this promising agent have been shown in various studies [19, 54, 55]. For example, an aqueous extract of saffron and crocin prevented renal ischemia–reperfusion induced oxidative injury through antioxidant effect in rats [56]. Following cisplatin-induced acute renal failure in mice, crocin elicited a protective effect by suppressing relative oxidative stress [15]. Thushara et al. [57] showed that platelet aggregation induced by oxidative stress was inhibited by crocin through an anti-apoptotic pathway. In a study conducted by Jnaneshwari et al. [58], cyclophosphamide-induced organ toxicity was prevented by crocin through modulating antioxidant status and inflammatory cytokines. Crocin prevented apoptosis following retinal ischemia/reperfusion damage through the PI3K/AKT signaling pathway [59]. In a study by Xu et al. [60], crocin inhibited xylene-induced ear edema in mice and carrageenan-induced paw edema as well as production of lipopolysaccharide-induced prostaglandin E2 (PGE2) in rats. Crocin and crocetin reduced LPS-induced nitric oxide (NO) release, TNF-α, IL-1β, intracellular ROS and NF-κB activation from cultured rat brain microglial cells [61]. Crocin showed antioxidant activity in the liver and kidney of streptozotocin-induced diabetic rats [62]. Recently, crocin has been reported to protect against beryllium chloride toxicity in rats through antioxidant effect and enhancing gene expression of antioxidant enzymes in a study by El-Beshbishy HA and co-workers [63].

Considering that infection is probably associated with the urolithiasis process, antimicrobial activity may also contribute to the antilithiatic effect of crocin [64].

The safety profile of crocin in animals or humans has been reported in some studies. In a study conducted by Wang et al. on rats, a very low toxicity of crocin was reported. A daily dose of crocin (50 mg/kg for 8 days) did not affect hepatic function; however, a higher dosage of 100 mg/kg for 2 weeks caused hepatic damage and black pigmentation, but a lower dose of 100 mg/kg for 40 days did not. Rats receiving 1 % of crocin dyes for 4 months elicited mild hepatic functional disorders and pigmentation, which was completely reversible [65].

A recent study, evaluating biochemical, hematological and pathological criteria in mice and rats treated with crocin (up to 3 g, p.o. and i.p. as well as 15–180 mg/kg, i.p.), showed no damage to any major organs in the body. In a randomized, double-blind, controlled trial on volunteers treated with crocin tablets (20 mg) for a duration of 1 month, no major adverse events were observed during the trial [66].

As, at the doses tested in the present study, crocin showed its effect in the early stages of stone development and did not reverse the established crystal deposits in the curative study, it is helpful to prevent the recurrence of the disease, one of the biggest challenges in urolithic patients. Hence, consumption of this agent in the prophylactic regimens might be a good strategy.

One of the limitations in this study is that Ca²⁺, a facilitating factor for nucleation and precipitation of calcium oxalate, was not evaluated in our study. However, considering that urinary oxalate execration is a more important factor in the induction of CaOx deposition [42, 52], the elevated levels of this factor could be a reason to guarantee the formation of CaOx calculi.

In addition, intraperitoneal administration of crocin was used in this study. Considering that oral administration of drugs is the preferred route of drug delivery [67], further studies are necessary to examine the effect of oral administration of the effective doses of crocin in this study on the biochemical parameters in urine and blood.

It should be noted that orally administered crocin is not absorbed either after a single dose or repeated dose, due to hydrolysis of crocin to crocetin in the intestinal tract [68]. Hence, crocetin might be a better compound for further studies in this topic.

Taken together, it has been suggested that crocin, the main carotenoid constituent of C. sativus with a wide spectrum of therapeutic potentials, could be useful as either alternative or an adjunctive therapy in the management of kidney stones. Antioxidant activity seems to be an important factor in mediating the antilithiatic effects of this compound. However, further studies are required to determine the exact mechanisms by which crocin prevents EG-induced nephrolithiasis.