Urolithiasis (2014) 42:519–526 DOI 10.1007/s00240-014-0695-7

ORIGINAL PAPER

Prophylactic effects of quercetin and hyperoside in a calcium oxalate stone forming rat model Wei Zhu · Yun‑fei Xu · Yuan Feng · Bo Peng · Jian‑ping Che · Min Liu · Jun‑hua Zheng 

Received: 3 March 2014 / Accepted: 20 July 2014 / Published online: 2 August 2014 © Springer-Verlag Berlin Heidelberg 2014

Abstract  Quercetin and hyperoside (QH) are the two main constituents of the total flavone glycosides of Flos Abelmoschus manihot, which has been prescribed for treating chronic kidney disease for decades. This study aimed to investigate the effect of QH on calcium oxalate (CaOx) formation in ethylene glycol (EG)-fed rats. Rats were divided into three groups: an untreated stone-forming group, a QH-treated stone-forming group (20 mg/kg/day) and a potassium citrate-treated stone-forming group (potassium citrate was a worldwide-recognized calculi-prophylactic medicine). Ethylene glycol (0.5 %) was administered to the rats during the last week, and vitamin D3 was force-fed to induce hyperoxaluria and kidney calcium oxalate crystal deposition. 24 h urine samples were collected before and

after inducing crystal deposits. Rats were killed and both kidneys were harvested after 3 weeks. Bisected kidneys were examined under a polarized light microscope for semiquantification of the crystal-formation. The renal tissue superoxide dismutase and catalase levels were measured by Western blot. QH and potassium citrate have the ability to alkalinize urine. The number of crystal deposits decreased significantly in the QH-treated stone-forming group as compared to the other groups. Superoxide dismutase and catalase levels also increased significantly in the QH-treated stone-forming group, as compared with the untreated stoneforming group. QH administration has an inhibitory effect on the deposition of CaOx crystal in EG-fed rats and may be effective for preventing stone-forming disease.

W. Zhu, Y. Xu and Y. Feng are equal first contributors for this work.

Keywords  Nephrolithiasis · Calcium oxalate · Quercetin · Hyperoside · Oxidative stress

W. Zhu · Y. Xu · B. Peng · J. Che · M. Liu (*) · J. Zheng (*)  Department of Urological Surgery, Shanghai Tenth People’s Hospital, Tongji University, Shanghai 200072, China e-mail: [email protected]

Abbreviations QH Quercetin and hyperoside CaOx Calcium oxalate EG Ethylene glycol KCit Potassium citrate SOD Superoxide dismutase NaCl Sodium chloride SDS Sodium dodecyl sulfate PAGE Polyacrylamide gel electrophoresis BSA Bovine serum albumin

J. Zheng e-mail: [email protected] W. Zhu e-mail: [email protected] Y. Xu e-mail: [email protected] B. Peng e-mail: [email protected] J. Che e-mail: [email protected]

Introduction

Y. Feng  Department of Nephrology, Nanjing University Affiliated Drum Tower Hospital, Nanjing 210093, China e-mail: [email protected]

Urolithiasis is a multi-factorial urological disorder common all over the world. Calcium oxalate (CaOx) is the most common cause of stones with a frequency of more than 80 %

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[1]. The prevalence of urolithiasis differs in various parts of the world: 1–5 % in Asia, 5–9 % in Europe, 13 % in North America, and 20 % in Saudi Arabia, and its prevalence continues to increase annually [2]. Although this disorder has been known since ancient times, the mechanism of renal stone formation remains unclear and few substances that effectively prevent urolithiasis are available. Although minimally invasive management techniques, such as percutaneous nephrolithtomy, have been developed in the past two decades, the side effects such as hemorrhage, renal fibrosis are serious; therefore the identification of substances that effectively prevent crystal deposition and the development of urolithiasis are urgently required in the clinic. Quercetin is a potent bioflavonoid commonly found in vegetables and fruit. It is a main component of the total flavone glycosides of Flos Abelmoschus manihot, which is a common plant grown in eastern China and south-east Asia [3]. Moreover, quercetin is a potent antioxidant that directly scavenges free radicals, inhibits xanthine oxidase and lipid peroxidation, and alters antioxidant defense pathways both in vivo and in vitro [4, 5]. Quercetin is an efficient antioxidant that has been implicated as in inhibitor of oxidative damage in renal tubular cells and renal tissues [6]. However, to our knowledge, Park et al. have investigated the effect of quercetin on oxalate-induced urinary stone formation [7]. Hyperoside is a major pharmacologically active constituent of the flavonoid glycosides found in natural plants, and recently has attracted increasing attention due to its diverse antioxidant, anti-inflammatory, diuretic, anti-hyperglycemic, hypo-uricemic, hepatoprotective and anti-fungal properties. However, the anti-urolithiasis effects of this substance have not been confirmed in vivo. Previous studies conducted in our laboratory demonstrated that the combination of quercetin and hyperoside derived from the traditional Chinese herb, Abelmoschl manihot, showed satisfactory anti-proliferative activities in the human renal cancer cell line 786-O [8]. However, to our knowledge, there is no evidence of the prophylactic effects of the two compounds in urolithiasis. Therefore, the present study was undertaken to determine the effect of quercetin and hyperoside (QH) on urinary excretion factors and to determine the anti-oxidative effects on calcium oxalate urolithiasis in a rat hyperoxaluria-induced stone model, compared with those of sodium potassium citrate. This information is important in evaluating the potential of QH for the treatment of renal calculi in the clinic.

Materials and methods Preparation of QH for gastric feeding Polyphenolics were extracted from a standardized hyperoside and quercetin dehydrate supplement (ratio 1:1) in

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capsule form provided by Suzhong Pharmaceutical Co. (Taizhou, Jiangsu, China). The dried samples were ground to a powder for administration by gastric intubation. Dry powder of QH stored at room temperature was dissolved in 500 ml of heated double-distilled water to prepare a stock solution that was stored at room temperature. The feeding solution contained QH at a concentration of 2 mg/ml was prepared immediately before use. Stone‑forming rat model and experimental design All animal experimental protocols were approved by the Animal Research Committee of the Tongji University. Sprague–Dawley rats (n = 18; aged 7 weeks; weight 180–200 g, were acclimated to room temperature for more than 4 weeks and then fed a standard commercial rat chow during the study. Ethylene glycol (EG 0.5 %) was administered to the rats in drinking water during the last week of the 3-week study. Rats were force-fed with 0.5 μmol of vitamin D3 dissolved in 1 ml of salad oil by gastric intubation to induce hyperoxaluria and kidney calcium oxalate crystal deposition. The rats were divided into three groups. All groups were acclimated for 1 week before the start of the experiment. Group 1, EG group (n = 6): animals received normal drinking water for 2 weeks and crystal deposits were induced in week 3. Group 2, potassium citrate (KCit) group (n = 6): the animals were force-fed KCit (100 mg/kg daily) by gastric intubation for 2 weeks before the induction of crystal deposits in week 3 [9, 10]. Group 3, quercetin and hyperoside group (n = 6): the animals were force-fed QH (20 mg/ kg body weight daily in week 3 [11, 12]. The experimental period was 3 weeks. At the end of the experiment, all rats were euthanized with an overdose of anesthesia (diethyl ether) and both kidneys were harvested. One kidney was treated with 10 % formalin, embedded in paraffin, and strained with hematoxylin and eosin solution, while the other kidney was immediately stored at −80 °C for further study. Measurement of urinary variables Twenty-four hour urine samples were collected on day 14 before the start of crystal deposit induction and before euthanization. Rats were kept individually in metabolic cages for 24 h urine collection, which was either analyzed immediately or stored at −80 °C until further analysis. Drinking volume, urine volume, and urine pH were measure manually. Urinary calcium, potassium, sodium, phosphate, creatinine, and magnesium levels were measured using a Model 705 automate analyzer (SRL, Tokyo, Japan). A portion of the urine sample was acidified with 6 mol/L HCl to maintain urine pH at <3 for measuring urine oxalate

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Table 1  General data Before

After

Normal

KCit

QH

EG

KCit+EG

QH+EG

Body weight (g) Urine pH Water intake (ml/24 h)

190 ± 3.2 7.45 ± 0.15 22.6 ± 7.8

185 ± 4.1 8.14 ± 0.18a 24.5 ± 5.7

193 ± 2.7 7.37 ± 0.21b 26.1 ± 3.9

276 ± 5.7 6.87 ± 0.27 31.9 ± 9.7

292 ± 9.6 8.24 ± 0.31c 35.2 ± 3.1

284 ± 7.9 6.19 ± 0.24d,e 40.6 ± 8.4

Water output (ml/24 h)

12.9 ± 1.7

11.2 ± 2.1

14.3 ± 1.6

17.5 ± 5.1

20.7 ± 6.4

22.3 ± 4.2

a

  p < 0.05 KCit vs Normal drinking water

b

  p < 0.05 QH vs KCit

c

  p < 0.05 KCit+EG vs EG

d

  p < 0.05 QH+EG vs EG

e

  p < 0.05 QH+EG vs KCit+EG

levels by capillary electrophoresis (SRL, Tokyo, Japan). Urine citrate was measured with a Citric Acid Enzyme BioAnalysis kit (Megazyme, Wicklow, Ireland). Evaluation of the severity of renal crystal deposition The severity of crystal deposition was assessed by polarized light microscopy as previously described [7]. In brief, the bi-refringent crystals of CaOx were illuminated with a magnification factor of 100 to evaluate the score. A subjective, semi-quantitative scoring system was used with four grades ranging from 0 to 3+ (where 0 = none, 1+ = few, 2+ = several, and 3+ = many crystal deposits) to evaluate the degree of crystal deposition as previously described [13, 14]. The scoring system was applied using the computerassisted image-scoring system Image J version 1.42 [15]. This software allows randomized and blinded selection of digital images spontaneously. Random digital images of one of the eight regions under a polarized light microscope (100×) were acquired using a digital camera (Olympus, California, USA) mounted onto the microscope. Six independent co-workers used this computer-assisted system to score every individual picture in the database. Finally, all of the personal score files were collected and merged for statistical analysis. Evaluation of superoxide dismutase (SOD) and catalase levels The kidney samples were thawed and homogenized in 1 ml of 25 mM Tris–HCl, pH 7.6, 150 mM NaCl, 1 % NP-40, 1 % sodium deoxycholate, and 1 % SDS and centrifuged at 10,000×g for 10 min at 4 °C. The supernatant was recovered and the protein concentration was measured using the Bradford protein assay. Equal amounts of protein were electrophoresed by SDS PAGE and transferred to a PVDF membrane. Blots were blocked with 3 % BSA overnight at 4 °C, probed with appropriate dilutions of SOD1 antibody

(EP1727Y, rabbit monoclonal to SOD1, Abcam Inc., Cambridge, MA, USA) or catalase antibody (EPR1928Y, rabbit monoclonal to catalase, Abcam Inc., Cambridge, MA, USA) and incubated with a 1:1,000 dilution of anti-rabbit IgG conjugated secondary antibody (Cell Signaling Technology, Inc., Danvers, MA, USA) for visualization by enhanced chemiluminescence (Cell Signaling Technology, Inc., Danvers, MA, USA). Blots were quantitatively analyzed using Image J version 1.42 [15]. Statistical analysis All data are presented as the mean ± standard error (SE). The severity of renal crystal deposition was expressed as an ordinal scale using the semi-quantitative scoring method described. The crystal deposit scores were analyzed by the non-parametric Kruskal–Wallis test for inter-group comparisons and by the Wilcoxon rank sum test (Mann–Whitney U) for pair-wise comparisons. A p < 0.05 was considered statistically significant.

Results General and urinary variables in different groups General physiological parameters, including body weight and urine output are listed in Table 1. A similar increase in body weight was observed in all rats at the end of the experiment. There were no significant differences in water intake or urine output among groups. The urine pH increased significantly in the KCit group compared with the EG-fed group. Urine pH levels among the stone-forming rats were markedly higher than the QH-treated rats. There were no statistically significant differences in the urinary variables including urine creatinine, calcium, and phosphate excretion before and after stone formation despite slightly increased urine calcium excretion in

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Table 2  Urine variables changing before and after inducing crystal deposits Before Normal Creatinine (mg/kg/24 h) Calcium (mg/24 h) Phosphate (mg/24 h) Oxalate (mg/24 h) Citrate(mg/24 h)

After KCit

QH

EG

KCit+EG

QH+EG

2.78 ± 0.22 0.61 ± 0.33 9.41 ± 2.73 0.47 ± 0.04

2.83 ± 0.17 0.78 ± 0.29 7.32 ± 1.45 0.81 ± 0.11

2.73 ± 0.13 0.74 ± 0.19 8.83 ± 1.92 0.77 ± 0.16

3.01 ± 0.32 1.52 ± 0.34 11.19 ± 3.86 2.63 ± 0.81a

3.29 ± 0.23 1.73 ± 1.12 14.41 ± 3.19 3.59 ± 1.34

3.32 ± 0.25 1.34 ± 0.89 10.23 ± 2.65 2.12 ± 1.17

65.13 ± 18.79

60.33 ± 16.94

63.42 ± 20.12

51.29 ± 24.19

86.67 ± 39.73b

48.38 ± 22.82

a

  p < 0.05 KCit vs Normal drinking water

b

  p < 0.05 KCit+EG vs EG

stone-forming rats. Urine oxalate excretion increased significantly in untreated stone-forming rats compared with normal control rats. Urine citrate excretion was decreased after EG-induced stone formation and in the QH intervention group; however, this effect was not statistically significant. In contrast, urine citrate excretion increased significantly in the KCit+EG group compared with the untreated stone-forming rats. The data are listed in Table 2. QH reduced the severity of renal crystal deposition As shown in Fig. 1, polarized light microscopy of the hematoxylin- and eosin-stained kidney sections revealed multiple crystal deposits in the renal cortex and medulla of the EG-fed rats as expected. Analyses of the crystal deposit percentage revealed that the scores of the KCit-treated and QH-treated groups (20 mg/day/day) were significantly lower than that of the untreated group (Fig. 2). Overall, QH administration significantly inhibited CaOx crystal deposition in the kidneys of the experimental rats. QH increased SOD and catalase expression levels in vivo The potential mechanism of the prophylactic effect of QH in stone-forming rats was investigated by Western blot analysis of SOD and catalase expression in kidney tissues (Fig. 3a, c). A quantitative analysis of SOD and catalase expression revealed that the SOD level in QH-treated stone-forming rats increased significantly compared with the levels in untreated and KCit-treated stone-forming rats. The catalase level in QH-treated stone-forming rats was significantly higher than that in the untreated stone-forming rats, but not significantly higher than that in the KCittreated stone-forming rats (Fig. 3b, d).

Discussion In spite of the development of minimally invasive therapeutic strategies for the treatment of kidney stones, the high

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recurrence rates and stone residue remain critical problems for urologists. Thus, clinical demand requires an effective prophylactic treatment for nephrolithiasis. Potassium citrate has been widely used as the corner stone of medical management of renal calculus; it is prescribed as a remedy aimed at lengthening the stone-free status of patients [16]. The present study was designed to compare the results obtained with QH to those achieved with potassium citrate. Our results revealed that the severity of renal crystal deposition and the proportion of crystal formation in the 20 mg/ kg/day QH treatment group were significantly lower than those observed in the untreated group, thus demonstrating that QH inhibits CaOx crystal formation. To the best of our knowledge, this is the first evidence of the inhibitory effect of QH on CaOx crystal formation in EG-fed rats. Based on these preliminary results, we aim to perform further investigations on the prophylactic effects of QH on nephrolithiasis in a larger sample size and using different doses as a prelude to investigating the nephrolithiasis–prophylaxis effect of QH in humans. The physical process of stone formation contains a highly complex mechanism. The state of urine saturation, crystal growth, aggregation, retention, as well as inhibitors and promoters of crystal formation are still under discussion. We confirmed that urine pH was increased by KCit under normal and stone-inducing conditions. The primary mechanism for KCit involves the inhibition of stone formation from calcium oxalate and calcium phosphate stone, while QH has the ability to increase urine pH under stoneinducing conditions [17]. Urine citrate has been shown to inhibit crystal formation by causing an increase in urine citrate, resulting in a decrease in stone formation, which is consistent with our results [18]. At present, its seems clear that renal epithelial cell injury plays a vital role in calculus development, and the lithogenic effect of EG must be mainly attributed to the oxidative damage induced by high levels of oxalate generated by ethylene glycol [19]. Thus, it is hypothesized that the renal epithelial cell injury led to the crystal aggregation and retention that contributed to the severity of crystal deposits

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Fig. 2  The percentage of crystal deposits in the renal tubules were significantly lower in QH-treated stone forming rats than in KCittreated and untreated stone forming rats. *p < 0.05 vs. EG; +p < 0.05 vs. KCit+EG

Fig. 1  Represented sections viewed under polarized light of rat kidneys in each groups. Arrows indicate the crystals in the cortex, tubular, and medulla (×100)

in the present study [19]. It is commonly held that oxalateinduced renal tubular epithelial cell damage is caused by the accumulation of reactive oxygen species (ROS). Oxidative stress influences the cell injury and inflammatory processes that promote the aggregation and retention of CaOx crystals [20]. Another recent study reported that renal tubular injury is mediated by free radical formation [21]. Not only are high concentrations of oxalate per se toxic to renal tubular cells, but CaOx crystals themselves also promote cell damage [22]. In a previous animal experiment in which the administration of high oxalate loads led to CaOx crystal formation, elevated urinary levels of cell injury enzyme markers, including N-acetyl-β-glucosidase, indicated renal tubular epithelial cell damage [23]. In this study, SOD levels in QH-treated stone-forming rats increased significantly compared with those in untreated and KCit-treated stone-forming rats. Furthermore, the catalase levels in QH-treated rats were significantly higher than that in the untreated stone-forming rats. These observations suggest that KCit and QH have the ability to decrease renal tubular cell injury from oxidative stress and that QH is more effective. Therefore, we suggest that three key points contribute to the protective effects of QH against nephrolithiasis: first is the regulation of conditions of urinary pH that leads to stone induction; second, the anti-apoptotic effects of QH, which we have demonstrated elsewhere (data not shown [24]) could alleviate the tubular-damage caused by EG; third, the levels of the anti-oxidative enzyme catalase in QH-treated stone-forming rats were significantly higher than those in untreated rats, but not significantly higher than those in KCit-treated stone-forming rats. Thus, since

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Fig. 3  Western blot detection of SOD and catalase levels. a SOD band in rat kidney tissues; b a quantitative analysis of the SOD level revealed a significantly higher level in QH-treated stone forming rats;

c catalase band in rat kidney tissues; d a quantitative analysis of catalase revealed a significantly higher level in QH-treated stone forming rats than in untreated stone forming rats

oxidative stress influences cell injury, our results indicate that the calculus-preventive effects of QH are partially related to anti-oxidative effects. Many phytomedicine plants are used to treat nephrolithiasis worldwide, of which Orthosiphon grandiflorum and Poria cocos Wolf have been shown to inhibit the kidney stone formation process [25, 26]. Orthosiphon grandiflorum (also known as Clerodendranthus spicatus) strongly suppresses the processes of crystal formation, growth and aggregation of CaOx crystals in vitro; furthermore, the renal calcium content has been shown to decrease in Orthosiphon grandiflorum-treated rats [9]. In addition, several studies have shown that this herb significantly decreases the formation of CaOx deposits [27]. All of these reports suggest that herbal medicines may be a useful strategy for preventing renal stones. In our current study we investigated quercetin because it has a greater antioxidant effect than other flavonoids, including catechin [28]. Of the known flavonoids, quercetin is one of the most widely studied and numerous investigators have reported that quercetin has various biological, pharmacological and medicinal

properties. Quercetin was also found to prevent the ethanol-mediated decrease in intracellular antioxidant defense systems, such as SOD, catalase, and glutathione peroxidase. Since hyperoside was initially used in Traditional Chinese medicine to improve blood circulation, it has been used in patients with abnormal coagulation functions, and in cardiovascular ischemic disease without apparent bleeding complications in animals [28]. Our study provides the first evidence of the prophylactic effects of QH in a CaOx stone rat model. Our study confirms that the exact mechanism by which QH prevents calcium oxalate nephrolithiasis involves two key points: first, urine analysis demonstrated that QH increases urine pH under stone-inducing conditions; second, tubular cell injury from oxidative stress was attenuated by QH intervention. In addition, as reported previously, QH is efficient in reducing cell apoptosis in chronic kidney disease, which is characterized by the accumulation of extracellular matrix (ECM) components in the glomeruli (glomerular fibrosis, glomerulosclerosis) and the tubular interstitium (tubulointerstitial fibrosis). However, since numerous reports have described the importance of

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apoptosis in the etiology of chronic kidney diseases [3, 27], we did not investigate the anti-apoptotic effects of QH in nephrolithiasis patients owing to budgetary constraints, although this is likely to form part of our future research. The present study has some limitations. First, the antilithic mechanism of QH remains unclear. Details of the cellular response to QH as well as whether the extract changes EG metabolism of oxalate or inhibits crystallization remain to be elucidated. A complete characterization of the model and the effects of the extract on urinary components is required for a more complete interpretation of our results. Second, the accurate concentration of QH was not evaluated in the current study. According to previous studies, high concentrations of quercetin were found to inhibit cell survival in vitro [7]. Thus, the appropriate proportion of QH combined therapy should be further investigated. In summary, our results revealed that QH administration led to a significant reduction in CaOx formation as compared with the untreated EG-caused renal stone group. The herbal medicine comprising quercetin and hyperoside appears to effectively prevent nephrolithiasis formation [27]. However, at present, we cannot state the optimum level of QH in serum required to prevent renal stone formation in humans. Therefore, clinical trials should be performed to verify the efficacy of QH in CaOx nephrolithiasis patients, and to ascertain the potential of QH as a novel treatment for long-term prophylaxis in recurrent urolithiasis patients. Acknowledgments  This work was partially supported by grants from the National Natural Science Foundation of China (No. 81000311 and No. 81270831). Conflict of interest  The authors declared no potential conflicts of interests with respect to the authorship and/or publication of this article.

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