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Renoprotective potential of Macrothelypteris torresiana via ameliorating oxidative stress and proinflammatory cytokines
Journal of Ethnopharmacology 139 (2012) 207–213
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Renoprotective potential of Macrothelypteris torresiana via ameliorating oxidative stress and proinflammatory cytokines Jinglou Chen a , Yongfang Lei a , Guanghua Wu a , Yonghui Zhang a,∗ , Wei Fu b , Chaomei Xiong a , Jinlan Ruan a,∗ a Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation of Hubei Province, College of Pharmacy, Tongji Medical Center, Huazhong University of Science and Technology, Wuhan, China b Department of Pharmacy, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
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Article history: Received 7 July 2011 Received in revised form 15 October 2011 Accepted 1 November 2011 Available online 10 November 2011 Keywords: Macrothelypteris torresiana Nephrotic syndrome Oxidative stress Vascular endothelial growth factor Nitric oxide
a b s t r a c t Ethnopharmacological relevance: Macrothelypteris torresiana is traditionally used in Chinese folk medicine for the treatment of edema for patients suffering from kidney/bladder problems due to its satisfactory therapeutic effectiveness. Aim of the study: The aim of this study was to investigate the renoprotective nature of the total polyphenols fraction from Macrothelypteris torresiana (PMT). Materials and methods: The biochemical criterions of plasma and kidney tissues were evaluated to study the effects of PMT on puromycin aminonucleoside-induced chronic nephrotic syndrome (NS) in hyperlipidemic mice. Results: In this study, the NS and hyperlipidemia were ameliorated after 9 weeks administration of PMT. Besides, PMT was able to modulate the level of renal oxidative stress and vascular endothelial growth factor–nitric oxide (VEGF–NO) pathway. Conclusions: It represented a potential resource of PMT for the treatment of NS involved in metabolic syndrome. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The nephrotic syndrome (NS) is a chronic renal disease and usu˜ ally involved with hyperlipidemia (Pena-Rico et al., 2006). High levels of total cholesterol (TC) and triglyceride (TG) always contribute to NS (Jeong et al., 2010). Besides, more and more evidences show that reactive oxygen species (ROS) are associated with the progress of NS. High levels of ROS can produce cell injury including peroxidation of membrane lipids, proteins denaturation and DNA
Abbreviations: NS, nephrotic syndrome; TC, total cholesterol; TG, triglyceride; ROS, reactive oxygen species; SOD, superoxide dismutase; CAT, catalase; GPx, glutathione peroxidase; PAN, puromycin aminonucleoside; VEGF, vascular endothelial growth factor; NO, nitric oxide; iNOS, inducible-NO synthase; PMT, total polyphenols fraction from Macrothelypteris torresiana; CMT, crude methanol extract from Macrothelypteris torresiana; BUN, blood urea nitrogen; LDL, low density lipoprotein; HDL, high density lipoprotein; MDA, malondialdehyde; RT-PCR, real time-polymerase chain reaction; CT, comparative cycle threshold; eNOS, endothelial-NOS; ARF, acute renal failure. ∗ Corresponding authors at: Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation of Hubei Province, College of Pharmacy, Huazhong University of Science and Technology, No. 13 Hangkong Road, Wuhan 430030, Hubei Province, China. Fax: +86 27 83692762. E-mail addresses: yfl[email protected] (Y. Lei), [email protected] (Y. Zhang), [email protected] (J. Ruan). 0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.11.002
damage (Khan et al., 2009). Several biological processes affected by the unstable ROS are potentially important in the pathogenesis of renal injury (Parlakpinar et al., 2006). Increased level of ROS will induce mesangial cells contraction, filtration surface change, ultrafiltration coefficient modify and glomerular filtration rate decrease (Pedraza-Chaverrí et al., 2003). ROS will finally lead to the renal histologic changes as well as the functional abnormalities (Ha and Kim, 1999). This is considered as one of the major mechanisms for nephrotoxicity and other deleterious effects (Khan et al., 2009). As a result, enhancing the activities of the antioxidant enzymes is useful to prevent the renal injury induced by ROS (PedrazaChaverrí et al., 2004). Some polyphenols and flavonoids (such as naringenin and quercetin) are reported to have the potential effect of enhancing the activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) (Vitor et al., 2004; Ratheesh et al., 2011), and finally attenuating the nephrotoxicity in streptozotocin-induced diabetic rats (Sato et al., 2005). Puromycin aminonucleoside (PAN) is usually used to induce the experimental chronic NS which involves the imbalance of the generation and elimination of ROS (Pedraza-Chaverrí et al., 2004). The proinflammatory cytokine vascular endothelial growth factor (VEGF) is also closely associated with the pathogenesis of NS (Cooper et al., 1999). The nitric oxide (NO) comes from
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inducible-NO synthase (iNOS) is also implicated in the renal injury (Tain et al., 2008). Over-expression of iNOS and excessive NO are proposed as one of the causes of vascular dysfunction in kidney (Bhatia et al., 2003). Moreover, NO can react with ROS to form peroxynitrite, and finally lead to a decrease in the glomerular filtration rate (Pedraza-Chaverrí et al., 2003). Furthermore, it is demonstrated that the generation of NO could be stimulated by VEGF (Fan et al., 2008). Summarily, the VEGF-NO pathway is also involved in the process of NS. Macrothelypteris torresiana (Gaud.) Ching is a member of Thelypteridaceae and widely distributed in the south of China. The entire plant has been used in Chinese folk medicine for the treatment of edema for patients suffering from kidney/bladder problems due to its satisfactory therapeutic effectiveness (Zhao et al., 2009). It is usually decocted in water for oral dose. A number of novel polyphenols and flavonoids (such as (2S)-5,7,2 ,5 tetrahydroxyflavanone-2 -O--d-6 -O-acetylglucopyranoside and (2S)-5,7,2 ,5 -tetrahydroxyflavanone-2 -O--d-glucopyranoside) were isolated from this polyphenols-rich plant (Fu et al., 2009; Tang et al., 2009). Our previous study (not showed here) indicated that the methanol extract had remarkable radical scavenging activity and could inhibit malondialdehyde formation in vitro. Then, according to the bioassay guided fractionation method (Kasetti et al., 2010), the experimental acute renal injury induced by K2 Cr2 O7 was used for the preliminary screening of the renoprotective active fraction (Barrera et al., 2003), and the total cholesterol lowering effects on mice was used to identify the hypolipidemic active fraction from the methanol extract (Xu et al., 2003). Interestingly, a fraction which had relative high content of polyphenols were identified as the common active fraction and purified by the method of alkali extraction and acid precipitation. This fraction from Macrothelypteris torresiana was defined as the total polyphenols fraction (PMT). In this study, we investigated the effects of PMT on the acute nephrotoxicity induced by K2 Cr2 O7 and the chronic NS induced by puromycin aminonucleoside in hyperlipidemic mice.
2. Methods and materials 2.1. Plant material Macrothelypteris torresiana was collected in the Jiangxi province, China and identified by Prof. Ceming Tan at the Jiujiang Forest Plants Specimen Mansion, China. After washing with distilled water thoroughly and drying in the shade for 3 days, they were powdered and passed through 40 mesh sieves. The powder was soaked in methanol (v/w) for 6 times and extracted by heat reflux for 3 times, each for 1 h. After filtered with Whatman No. l filter paper, the filtrate was concentrated under vacuum by means of rotavapor to get crude methanol extract from Macrothelypteris torresiana (CMT). Then CMT was partitioned successively between petroleum ether/water, chloroform/water, ethyl acetate/water and n-butanol/water. The parts of chloroform, ethyl acetate and nbutanol were mixed and concentrated in vacuo. The concentrated extract was dried in freeze dryer. The resulting crude extract was mixed with 2% NaOH solution and filtered for the removal of the precipitation. Appropriate volume of the concentrated hydrochloric acid was added into the filtrate to control the pH value at 1, and then stood for 24 h at 4 ◦ C. The mixture was filtered to obtain the sediment, and named as the total polyphenols from CMT (PMT). The total polyphenols content in PMT was estimated using Folin-Ciocalteu reagent (Ainsworth and Gillespie, 2007). The yield of PMT was 67.4 ± 2.0 g/kg of the raw medicinal herbs. The total polyphenols contained in PMT was 514.7 ± 18.1 g/kg of the PMT.
2.2. Animals The Kunming mice were obtained from the Experimental Animal Center of Huazhong University of Science and Technology. The animals were maintained under the standard laboratory conditions with water and food ad libitum. This study protocol was along the “Principles of Laboratory Animal Care” (Xu et al., 2003). The study was approved by the Huazhong University of Science and Technology Committee on Animal Care and Use. After acclimatized for a period of 3 days in the new environment, the animals were used for the study. 2.3. Acute nephrotoxicity in mice induced by K2 Cr2 O7 Forty male mice weighing 20 ± 2 g (4–5 week old) were randomly divided into four groups (n = 10): the vehicle control group, the model group and the PMT treated groups (200 mg/kg/d or 100 mg/kg/d). PMT were orally administrated in the morning for 7 days. The vehicle control and the model groups were given tales doses (the same volume) physiological saline. On the morning of the seventh day, the animals of the model and PMT treated groups were given 15 mg/kg K2 Cr2 O7 by a single subcutaneous injection 30 min after the administration. The vehicle control group was subcutaneously injected tales doses physiological saline. Blood samples were collected at 0, 24 and 48 h after the K2 Cr2 O7 injection. Samples were spun at 2000 r.p.m. for 20 min at 4 ◦ C. The serum was prepared and stored at −80 ◦ C until the determination of serum creatinine and blood urea nitrogen (BUN). 2.4. Chronic NS in hyperlipidemic mice induced by PAN Forty male mice weighing 20 ± 2 g (4–5 week old) were randomly divided into four groups (n = 10): the vehicle control group, the model group and the PMT treated groups (200 mg/kg/d or 100 mg/kg/d). The vehicle control group was fed on the normal forage. The others were fed on 9 weeks’ high-fat diet consisting of normal diet 470 g/kg, sucrose 100 g/kg, egg yolk 200 g/kg, lard 200 g/kg, cholesterol 20 g/kg and sodium deoxycholate 10 g/kg (Chen et al., 2009). PMT were given orally once a day in the morning for 9 weeks. The vehicle control and the model groups were orally given tales doses physiological saline. The animals of the model group and the PMT treated groups were subcutaneously injected 50, 40, 40 and 25 mg/kg PAN (Sigma–Aldrich Chemical Co., USA) on the first day of weeks 5–8, respectively. The vehicle control group was subcutaneously injected with tales doses physiological saline. All the mice were kept in the metabolic cages on the day before sacrificed. Urine samples of 12 h were collected to determine the value of urine protein. Then, the animals were deprived of food for 12 h and were accessible to water freely before they were killed. The blood samples were collected. The serum was prepared and stored at −80 ◦ C until the analysis of plasma lipid profiles, blood glucose, renal function and other plasma biochemical criterions. The kidney samples were rapidly removed and excised. One section of each sample was homogenized in 10 volumes (v/w) of ice cold physiological saline. The homogenate was spun at 15,000 r.p.m. for 20 min at 4 ◦ C. The supernatant was then stored at −80 ◦ C for the subsequent assay for the renal level of oxidative stress. Another section of each sample was stored at −80 ◦ C for the analysis of the expression of renal VEGF. 2.5. Assay of plasma lipid profiles and blood glucose level The blood glucose level was estimated by glucose oxidase peroxidase method (Arora et al., 2010). The levels of TC, TG,
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low density lipoprotein (LDL) and high density lipoprotein (HDL) in plasma were spectrophotometrically determined by the cholesterol esterase/peroxidase method, glycerol kinase method, PVS precipitation method and PTA-Mg2+ precipitation method, respectively (Li et al., 2009). All the manipulations were performed according to the directions of the commercially available kits (Nanjing Jiancheng Bioengineering Institute, China). The results were expressed as mmol/L. 2.6. Assay of renal function The renal function was assessed by estimating the serum creatinine, BUN and 12 h proteinuria. The assays were based on the reported methods (Arora et al., 2010). All the manipulations were performed according to the directions of the commercially available kits (Nanjing Jiancheng Bioengineering Institute, China). The results of creatinine, BUN and proteinuria were expressed as mol/L, mmol/L and mg/12 h, respectively. 2.7. Assay of the levels of oxidative stress and lipid peroxidation in kidney tissue The levels of oxidative stress in kidney tissue was evaluated by determined the levels of SOD, CAT and GPx in 10% kidney supernatant. Also, the malondialdehyde (MDA) level was measured as an index for lipid peroxidation of the tissue. The activities of SOD, CAT and GPx were measured according to the reported methods (Pedraza-Chaverrí et al., 2004) using commercially available kits (Nanjing Jiancheng Bioengineering Institute, China). The assay for total SOD was based on its ability to inhibit the oxidation of oxymine by the xanthine–xanthine oxidase system. The hydroxylamine nitrite produced by the oxidation of oxymine had an absorbance peak at 550 nm. CAT activity was measured by employing H2 O2 into H2 O and O2 . The decomposition of H2 O2 was monitored at 240 nm at 4 ◦ C. The activity of GPx was measured indirectly by a coupled reaction with glutathione reductase and determined the absorbance at 340 nm. All the procedures were performed according to the manufacturer’s instructions. The results were expressed as U/mg protein. The MDA level was measured according to the thiobarbituric acid reaction method (Godard et al., 2009). Briefly, 0.1 ml samples were mixed with 1 ml (17.5%) trichloroacetic acid and 1 ml (0.6%) 2thiobarbituric acid. After being incubated for 1 h at 80 ◦ C they were cooled soon using cold water. The mixture was centrifugated at 3000 r.p.m. for 15 min, the absorbance of the supernatant was measured at 535 nm. 4-Ethoxy-propane (10 nmol/ml) was used as the standard. The result was expressed as nmol/mg protein.
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2.9. Real time-polymerase chain reaction (RT-PCR) The total RNA was isolated from the kidney sample using the Trizol Reagent (Invitrogen, USA). The quality and integrity of RNA were confirmed by spectrophotometric analysis (OD260/280) and gel electrophoresis. cDNA was synthesized using the cDNA Reverse Transcription Kit (Dingguo Biotechnology, China) on a TC-512 DNA machine (Barloworld Scientific, USA). The total volume of 20 l consisted of 1 l random primer, 1 l dNTPs, 4 l 5× buffer, 1 l retroviridase, 6 l (1.5 g) total RNA and 7 l DEPC-H2 O. The reaction parameters were 65 ◦ C × 5 min, 37 ◦ C × 60 min, 70 ◦ C × 15 min and 4 ◦ C × 5 min. Then, cDNA was amplified by RT-PCR. The reaction was performed in a total volume of 25 l containing 2.5 l cDNA, 12.5 l SYBR Green Mix (Invitrogen, USA), 2 l primers and 8 l ddH2 O. Quantitative RT-PCR was performed using a Real-time PCR Detection System (Stratagene Mx3000p). The forward primer of VEGF was 5 -GAGCAAAGGTCACGAAAG-3 , the reverse primer of VEGF was 5 -ATTGAGGGTGAGAAGACG-3 (GenBank: AB086118.1). Actin was used as the internal control. The cycling program consisted of initial denaturation for 3 min at 94 ◦ C followed by 40 cycles of 94 ◦ C for 10 s, 55 ◦ C for 20 s, and 72 ◦ C for 20 s. Relative quantification was performed using the comparative cycle threshold (CT) method (Wang et al., 2006). 2.10. Statistical analyses Data are presented as means ± S.D. of ten measurements per group. Results were analyzed statistically by one-way ANOVA followed by Tukey’s multiple comparison using SPSS software for Windows (Student’s version). Differences were considered significant at p < 0.05. 3. Results 3.1. Acute nephrotoxicity in mice induced by K2 Cr2 O7 The effects of oral administration of PMT (200 or 100 mg/kg/d) on the levels of BUN and creatinine in mice 24 and 48 h after K2 Cr2 O7 injection are summarized in Fig. 1. In the model group, the level of BUN was increased 2.31- and 4.05-fold at 24 and 48 h in comparison with the vehicle control group, respectively. Similarly, the level of creatinine in the model group was enhanced 1.25-and 1.48-fold at 24 and 48 h. Nevertheless, the tendency was attenuated by PMT. The levels of BUN and creatinine in both PMT treated groups were significantly lowered at 24 and 48 h when compared to the model group. 3.2. The lipid profiles and glucose level
2.8. Assay of plasma iNOS activity and NO and VEGF levels The activity of VEGF-NO pathway in mice was evaluated by measuring the levels of VEGF and NO, and the activity of iNOS. The level of NO was determined after conversion of nitrate to nitrite by nitrate reductase according to the Griess reaction, the activity of iNOS was measured based on their activities to catalyze the transformation of l-arginine to NO (Milsom et al., 2010; Yaman and Balikci, 2010). The absorbance of NO and iNOS were determined at 550 nm and 530 nm, respectively. All the procedures were performed according to the manufacturer’s instructions of the kits (Nanjing Jiancheng Bioengineering Institute, China). Assay of the VEGF level was determined by specific ELISA kits (R&D Corporation, USA). All the procedures were performed according to the manufacturer’s instructions. The results of NO, iNOS, and VEGF were expressed as mol/L, U/ml and ng/L, respectively.
Fig. 2 represents the lipid profiles and glucose level in the four groups of mice. The levels of TC, TG, LDL and glucose of plasma were significantly elevated in the model group. Treatment with PMT (200 or 100 mg/kg/d) decreased all of them in a concentrationdependent manner. In the meantime, the HDL level was increased after administrated PMT (200 or 100 mg/kg/d) for 9 weeks compared to the model group. 3.3. The renal function As can be seen from Fig. 3, the renal function of the model group was deteriorated when compared to the vehicle control group. The levels of BUN, creatinine and 12 h urinary protein in model group were significantly increased. Oral administration of PMT (200 or 100 mg/kg/d) revealed a notable (p < 0.05) decline in all of them when compared to the model group.
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Fig. 1. Effects of PMT (200 or 100 mg/kg/d) on acute nephrotoxicity in mice induced by K2 Cr2 O7 . Values are given as means ± S.D. from 10 mice in each group. Values with different alphabets (a–f) in each index (BUN or Creatinine) are significantly different. The mean difference is significant at p < 0.05 (groups with different superscript alphabets are statistically significant). (A) The changes of the level of BUN from 0 h to 48 h. (B) The changes of level of creatinine from 0 h to 48 h.
3.4. The levels of oxidative stress and VEGF-NO pathway in vivo The effects of PMT (200 or 100 mg/kg/d) on modulating the activities of antioxidant enzymes and the level of MDA are described in Table 1. The levels of enzymatic antioxidants were significantly diminished in the model group, accompanied with the MDA level was enhanced. Oral treatment of PMT attenuated the alteration. The activities of SOD, GPx and CAT in both PMT treated groups were notably increased while the level of MDA was significantly decreased. Table 1 also details the altered levels of VEGF and NO, and the activity of iNOS of four groups mice. A significant elevation of the NO level was observed in the model group. Similarly, the iNOS activity of the model group was enhanced more than 3-fold when compared with the vehicle control. As expected, PMT significantly decreased the activity of iNOS and the level of NO. In the meantime, the level of VEGF was also increased about 2-fold in the model group when compared with the vehicle control. Treatment with PMT lowered it in a dose-dependent manner. Fig. 2. Effects of PMT (200 or 100 mg/kg/d) on plasma lipid profiles and glucose level. Values are given as means ± S.D. from 10 mice in each group. Values with different alphabets (a–d) in each index (TC, TG, HDL, LDL or glucose) are significantly different. The mean difference is significant at p < 0.05 (groups with different superscript alphabets are statistically significant).
3.5. The renal expression of VEGF The renal expression of VEGF was determined by quantitative RT-PCR assessment. As can be seen from Fig. 4, the expression of kidney VEGF mRNA was significantly enhanced in the model group. The value was 2.74-fold of the vehicle control group. PMT at 200
Fig. 3. Effects of PMT (200 or 100 mg/kg/d) on the renal function of chronic NS in hyperlipidemic mice. Values are given as means ± S.D. from 10 mice in each group. Values with different alphabets (a–d) in each index (BUN, Creatinine or Urinary protein) are significantly different. The mean difference is significant at p < 0.05 (groups with different superscript alphabets are statistically significant). (A) The effect on the level of BUN. (B) The effect on the level of creatinine. (C) The effect on the level of urinary protein.
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Table 1 Effects of PMT (200 or 100 mg/kg/d) on the levels of SOD, GPx, CAT, MDA, VEGF, NO and iNOS in vivo. Vehicle control group SOD (U/mg protein) GPx (U/mg protein) CAT (U/mg protein) MDA (nmol/mg protein) VEGF (pg/ml) NO (mol/L) iNOS (mol/L)
15.70 19.14 0.42 3.32 121.5 69.69 0.02
± ± ± ± ± ± ±
0.62a 1.31a 0.06a 0.22a 7.74a 3.49a 0.007a
Model group 8.94 7.10 0.11 11.79 243.9 159.91 0.07
± ± ± ± ± ± ±
0.62b 0.54b 0.03b 0.56b 13.47b 7.94b 0.018b
High dose group 13.14 10.47 0.34 5.12 153.8 88.93 0.04
± ± ± ± ± ± ±
0.54c 0.47c 0.06a 0.40c 7.72c 4.25c 0.012c
Low dose group 11.32 8.45 0.21 6.71 181.6 92.28 0.04
± ± ± ± ± ± ±
0.42d 0.36d 0.04c 0.39d 6.20d 3.58c 0.018c
Values are given as means ± S.D. from 10 mice in each group. Values with different superscripts (a–d) in a row are significantly different. The mean difference is significant at p < 0.05 (groups with different superscript alphabets are statistically significant).
or 100 mg/kg/d decreased the value to 0.493- or 0.825-fold of the model group, respectively. 4. Discussion NS is usually related to the disorder of metabolism and associated with high morbidity and mortality (Bhatia et al., 2003). In addition, hyperlipidemia has also been demonstrated to be a major risk factor of the progression of nephropathy (Arora et al., 2010). In our study the animals were fed on a high-fat diet for 9 weeks to induce hyperlipidemia. Besides, PAN was injected to induce ˜ nephrotic syndrome (Pena-Rico et al., 2006). After 9 weeks, the lipid levels of model group were increased accompanied with the renal function was deteriorated, however, PMT attenuated the tendency. Administration of PMT for 9 weeks lowered the levels of TC, TG and LDL, while the level of HDL was elevated. As expected, the renal function was improved. The results indicated that PMT could ameliorate the hyperlipidemia and protect the kidney against the impairments induced by PAN in hyperlipidemic mice. Furthermore, the possible mechanism was also investigated. It was reported that oxidative stress played a key role in the progress of NS (Ha and Kim, 1999). Taken into account that PMT could reduce the acute nephrotoxicity induced by K2 Cr2 O7 , which was closely associated with oxidative stress (Barrera et al., 2003), a possible mechanism emerged: the renal protective effect of PMT seemed related to its antioxidant activity. Therefore, the changes of the activities of renal antioxidant enzymes were investigated to evaluate the renal levels of oxidative stress. The level of MDA, which was the decomposed product of ox-LDL and an index for lipid peroxidation, was also studied. The activities of SOD, CAT and GPx in the model group
Fig. 4. Effects of PMT (200 or 100 mg/kg/d) on renal expression of VEGF. Values are given as means ± S.D. from 10 mice in each group. Values with different alphabets (a–d) are significantly different. The mean difference is significant at p < 0.05 (groups with different superscript alphabets are statistically significant).
were decreased and the level of MDA was elevated. Daily treatment of PMT for 9 weeks significantly increased the activities of these antioxidant enzymes. Similarly, the lowered level of MDA was observed. At this stage, a most possible hypothesis could be proposed: PMT could modulate the activities of kidney antioxidant enzymes, alleviate oxidative stress and decrease the levels of lipid profiles in mice, and finally improve the renal function. VEGF is closely related to the pathogenesis of NS (Cooper et al., 1999; Wasilewska and Zoch-Zwierz, 2006). Marked up-regulation of VEGF gene expression has been observed in NS. And VEGF excretion is significantly increased in accordance with the degree of renal injury (Libby et al., 2002; McKnight et al., 2007). In this study, the renal expression of VEGF was significantly enhanced. As expected, the level of VEGF was significantly increased in the model group accompanied with the levels of BUN, creatinine and urinary protein were notably enhanced. All of them were lowered by 9 weeks administration of PMT (both 200 mg/kg and 100 mg/kg). On the other hand, NO is reported to play an important role in the body and involve in the control of renal function (Plotnikov et al., 2009). Especially in the health status, the endothelial NOS (eNOS) is identified as a protective NO-generating enzyme and contributes to the maintenance of the normal renal structure (Sandovici et al., 2004). The NO derived from eNOS usually plays a protective effect, however, the one comes from iNOS might be implicated in the renal injury (Tain et al., 2008). Indeed, over-expression of iNOS and excessive NO participate in the development of NS (Becker et al., 2009; Milsom et al., 2010). Furthermore, increased level of NO can interact with oxygen radicals and finally lead to the deterioration of acute renal failure (ARF) (Traylor and Mayeux, 1997; Plotnikov et al., 2009). In our study, an elevated level of NO was observed in the model group. In the meantime, the activity of iNOS was significantly enhanced. However, administration of PMT attenuated the tendency. Additionally, report shows that the generation of NO can be stimulated by VEGF (Fan et al., 2008). It is suggested that increased level of VEGF would enhance the NO level and finally lead to the renal dysfunction (de Vriese et al., 2001). VEGF is suggested to be a potential regulator of NO generation. It has the capacity of regulating PKC signaling pathway (Wang et al., 2004; Farhangkhoee et al., 2006). Summarily, the VEGF-NO pathway is also involved in the NS. The whole findings indicated that PMT could suppress the enhancement of the renal VEGF expression in NS mice. And the decreased level of VEGF was observed in both PMT treated group. Taking into account that the levels of NO and iNOS were decreased in PMT treated groups, another possible hypothesis could be given: The renal protective effect of PMT was also associated with the activity of regulating the NO signaling pathway following the VEGF stimulation. Some herb extracts had been demonstrated could obviously inhibit oxidative stress and decrease NO level in rats, such as grape seed extract and Nigella sativa (Mattoo and Kovacevic, 2003; Yaman and Balikci, 2010). Our findings in this work were in line with the other reports about flavonoids and polyphenols were able to significantly enhance the activities of GPx, SOD and CAT, and prevented
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the overexpression of iNOS in rats suffered with kidney diseases (Badary et al., 2005; Morales et al., 2006). 5. Conclusions In summary, PMT could modulate the levels of renal oxidative stress and the VEGF-NO pathway, and finally attenuate the K2 Cr2 O7 -induced ARF and the PAN-induced chronic NS in hyperlipidemic mice. These results suggested that PMT had renoprotective potential, and represented a potential resource for the treatment of renal injury. Further investigations on detailed analysis of the mechanisms of PMT using longer times and varied dosages are required. And further toxicological studies also have a considerable impact on future clinical treatments of patients with renal disease to test the value of PMT as potential agents. Acknowledgements The authors thank Prof. Ceming Tan from the Jiujiang Forest Plants Specimen Mansion for the identification of the plant, the financial support from the State Natural Sciences Fund (NO30973864), the Ministry of Education Fund for Doctoral Program of higher subjects (NO20090142110021) and the Key Natural Science Fund of Hubei Province (NO2009CDA067). References Ainsworth, E.A., Gillespie, K.M., 2007. Estimation of total phenolic content and other oxidation substrates in plant tissues using Folin-Ciocalteu reagent. Nature Protocols 2, 875–877. Arora, M.K., Reddy, K., Balakumar, P., 2010. The low dose combination of fenofibrate and rosiglitazone halts the progression of diabetes-induced experimental nephropathy. European Journal of Pharmacology 636, 137–144. Badary, O.A., Abdel-Maksoud, S., Ahmed, W.A., Owieda, G.H., 2005. Naringenin attenuates cisplatin nephrotoxicity in rats. Life Sciences 76, 2125–2135. Barrera, D., Maldonado, P.D., Medina-Campos, O.N., Hernández-Pando, R., IbarraRubio, M.E., Pedraza-Chaverrí, J., 2003. Protective effect of SnCl2 on K2 Cr2 O7 induced nephrotoxicty in rats: the indispensability of HO-1 preinduction and lack of association with some antioxidant enzymes. Life Sciences 73, 3027–3041. Becker, T., Mevius, I., de Vries, D.K., Schaapherder, A.F., zu Vilsendorf, A.M., Klempnauer, J., Frölich, J.C., Tsikas, D., 2009. The l-arginine/NO pathway in end-stage liver disease and during orthotopic liver and kidney transplantation: biological and analytical ramifications. Nitric Oxide 20, 61–67. Bhatia, S., Shukla, R., Venkata-Madhu, S., Kaur-Gambhir, J., Madhava-Prabhu, K., 2003. Antioxidant status, lipid peroxidation and nitric oxide end products in patients of type 2 diabetes mellitus with nephropathy. Clinical Biochemistry 36, 557–562. Chen, X.H., Bai, X., Liu, Y.H., Tian, L.Y., Zhou, J.Q., Zhou, Q., Fang, J.B., Chen, J.C., 2009. Anti-diabetic effects of water extract and crude polysaccharides from tuberous root of Liriope spicata var. prolifera in mice. Journal of Ethnopharmacology 122, 205–209. Cooper, M.E., Vranes, D., Youssef, S., Stacker, S.A., Cox, A.J., Rizkalla, B., Casley, D.J., Bach, L.A., Kelly, D.J., Gilbert, R.E., 1999. Increased renal expression of vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 in experimental diabetes. Diabetes 48, 2229–2239. de Vriese, A.S., Tilton, R.G., Elger, M., Stephan, C.C., Kriz, W., Lameire, N.H., 2001. Antibodies against vascular endothelial growth factor improve early renal dysfunction in experimental diabetes. Journal of the American Society of Nephrology 12, 993–1000. Fan, H., Sulochana, K.N., Chong, Y.S., Ge, R., 2008. Decorin derived antiangiogenic peptide LRR5 inhibits endothelial call migration by interfering with VEGFstimulated NO release. The International Journal of Biochemistry & Cell Biology 40, 2120–2128. Farhangkhoee, H., Khan, Z.A., Kaur, H., Xin, X., Chen, S., Chakrabarti, S., 2006. Vascular endothelial dysfunction in diabetic cardiomyopathy: pathogenesis and potential treatment targets. Pharmacology & Therapeutics 111, 384–399. Fu, W., Fang, W., Ruan, J.L., 2009. Two new flavanone glycosides from Macrothelypteris torresiana (Gaud.) Ching. Chinese Chemical Letters 20, 579–581. Godard, M., Décordé, K., Ventura, E., Soteras, G., Baccou, J.C., Cristol, J.P., Rouanet, J.M., 2009. Polysaccharides from the green alga Ulva rigida improve the antioxidant status and prevent fatty streak lesions in the high cholesterol fed hamster, an animal model of nutritionally-induced atherosclerosis. Food Chemistry 115, 176–180. Ha, H., Kim, K.H., 1999. Pathogenesis of diabetic nephropathy: the role of oxidative stress and protein kinase C. Diabetes Research and Clinical Practice 45, 147– 151. Jeong, S.C., Jeong, Y.T., Yang, B.K., Islam, R., Koyyalamudi, S.R., Pang, G., Cho, K.Y., Song, C.H., 2010. White button mushroom (Agaricus bisporus) lowers blood
glucose and cholesterol levels in diabetic and hypercholesterolemic rats. Nutrition Research 30, 49–56. Kasetti, R.B., Rajasekhar, M.D., Kondeti, V.K., Fatima, S.S., Kumar, E.G., Swapna, S., Ramesh, B., Rao, C.A., 2010. Antihyperglycemic and antihyperlipidemic activities of methanol:water (4:1) fraction isolated from aqueous extract of Syzygium alternifolium seeds in streptozotocin induced diabetic rats. Food and Chemical Toxicology 48, 1078–1084. Khan, S.A., Priyamvada, S., Farooq, N., Khan, S., Khan, M.W., Yusufi, A.N., 2009. Protective effect of green tea extract on gentamicin-induced nephrotoxicity and oxidative damage in rat kidney. Pharmacological Research 59, 254–262. Li, S.Y., Chang, C.Q., Ma, F.Y., Yu, C.L., 2009. Modulating effects of chlorogenic acid on lipids and glucose metabolism and expression of hepatic peroxisome proliferator-activated receptor-␣ in golden hamsters fed on high fat diet. Biomedical and Environmental Sciences 22, 122–129. Libby, P., Ridker, P.M., Maseri, A., 2002. Inflammation and atherosclerosis. Circulation 105, 1135–1143. Mattoo, T.K., Kovacevic, L., 2003. Effect of grape seed extract on puromycinaminonucleoside-induced nephrosis in rats. Pediatric Nephrology 18, 872– 877. McKnight, A.J., Maxwell, A.P., Patterson, C.C., Brady, H.R., Savage, D.A., 2007. Association of VEGF-1499C → T polymorphism with diabetic nephropathy in type 1 diabetes mellitus. Journal of Diabetes and Its Complications 21, 242– 245. Milsom, A.B., Patel, N.S., Mazzon, E., Tripatara, P., Storey, A., Mota-Filipe, H., Sepodes, B., Webb, A.J., Cuzzocrea, S., Hobbs, A.J., Thiemermann, C., Ahluwalia, A., 2010. Role for endothelial nitric oxide synthase in nitrite-induced protection against renal ischemia–reperfusion injury in mice. Nitric Oxide 22, 141–148. Morales, A.I., Vicente-Sánchez, C., Jerkic, M., Santiago, J.M., Sánchez-González, P.D., Pérez-Barriocanal, F., López-Novoa, J.M., 2006. Effect of quercetin on metallothionein, nitric oxide synthases and cyclooxygenase-2 expression on experimental chronic cadmium nephrotoxicity in rats. Toxicology and Applied Pharmacology 210, 128–135. Parlakpinar, H., Tasdemir, S., Polat, A., Bay-Karabulut, A., Vardi, N., Ucar, M., Yanilmaz, M., Kavakli, A., Acet, A., 2006. Protective effect of chelerythrine on gentamicininduced nephrotoxicity. Cell Biochemistry and Function 24, 41–48. Pedraza-Chaverrí, J., Barrera, D., Hernández-Pando, R., Medina-Campos, O.N., Cruz, C., Murguía, F., Juárez-Nicolás, C., Correa-Rotter, R., Torres, N., Tovar, A.R., 2004. Soy protein diet ameliorates renal nitrotyrosine formation and chronic nephropathy induced by puromycin aminonucleoside. Life Sciences 74, 987–999. Pedraza-Chaverrí, J., González-Orozco, A.E., Maldonado, P.D., Barrera, D., MedinaCampos, O.N., Hernández-Pando, R., 2003. Diallyl disulfide ameliorates gentamicin-induced oxidative stress and nephropathy in rats. European Journal of Pharmacology 473, 71–78. ˜ M., Guadalupe Ortiz-Lápez, M., Camacho-Castillo, L., Cárdenas, M., Pena-Rico, Pedraza-Chaverri, J., Menjívar, M., 2006. Steroidogenic impairment due to reduced ovarian transcription of cytochrome P450 side-chain-cleavage (P450scc) and steroidogenic acute regulatory protein (StAR) during experimental nephrotic syndrome. Life Sciences 79, 702–708. Plotnikov, E.Y., Chupyrkina, A.A., Pevzner, I.B., Isaev, N.K., Zorov, D.B., 2009. Myoglobin causes oxidative stress, increase of NO production and dysfunction of kidney’s mitochondria. Biochimica et Biophysica Acta 1792, 796–803. Ratheesh, M., Shyni, G.L., Sindhu, G., Helen, A., 2011. Inhibitory effect of Ruta graveolens L. on oxidative damage, inflammation and aortic pathology in hypercholesteromic rats. Experimental and Toxicologic Pathology 63, 285– 290. Sandovici, M., Henning, R.H., Hut, R.A., Strijkstra, A.M., Epema, A.H., van Goor, H., Deelman, L.E., 2004. Differential regulation of glomerular and interstitial endothelial nitric oxide synthase expression in the kidney of hibernating ground squirrel. Nitric Oxide 11, 194–200. Sato, S., Yamate, J., Hori, Y., Hatai, A., Nozawa, M., Sagai, M., 2005. Protective effect of polyphenol-containing azuki bean (Vigna angularis) seed coats on the renal cortex in streptozotocin-induced diabetic rats. Journal of Nutritional Biochemistry 16, 547–553. Tain, Y.L., Muller, V., Szabo, A.J., Erdely, A., Smith, C., Baylis, C., 2008. Renal cortex neuronal nitric oxide synthase in response to rapamycin in kidney transplantation. Nitric Oxide 18, 80–86. Tang, Y., Fang, W., Ma, Y.T., Cai, Y.L., Ruan, J.L., 2009. A novel flavonoid from the root of Macrothelypteris torresiana (Gaud.) Ching. Chinese Chemical Letters 20, 815–816. Traylor, L.A., Mayeux, P.R., 1997. Superoxide generation by renal proximal tubule nitric oxide synthase. Nitric Oxide 1, 432–438. Vitor, R.F., Mota-Filipe, H., Teixeira, G., Borges, C., Rodrigues, A.I., Teixeira, A., Paulo, A., 2004. Flavonoids of an extract of Pterospartum tridentatum showing endothelial protection against oxidative injury. Journal of Ethnopharmacology 93, 363–370. Wang, Y., Nagase, S., Koyama, A., 2004. Stimulatory effect of IGF-I and VEGF on eNOS message, protein expression, eNOS phosphorylation and nitric oxide production in rat glomeruli, and the involvement of PI3-K signaling pathway. Nitric Oxide 10, 25–35. Wang, Y., Wang, Y., Yang, Q., Yan, J.T., Zhao, C.X., Cianflone, K., Wang, D.W., 2006. Effects of bezafibrate on the expression of endothelial nitric oxide synthase gene and its mechanisms in cultured bovine endothelial cells. Atherosclerosis 187, 265–273.
J. Chen et al. / Journal of Ethnopharmacology 139 (2012) 207–213 Wasilewska, A., Zoch-Zwierz, W., 2006. Glucocorticoid receptor and vascular endothelial growth factor in nephrotic syndrome. Acta Paediatrica 95, 587– 593. Xu, S.Y., Bian, L., Chen, X., 2003. Experimental Methodology of Pharmacology. People’s Medical Publishing House, Beijing, pp. 155–163.
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Yaman, I., Balikci, E., 2010. Protective effects of Nigella sativa against gentamicininduced nephrotoxicity in rat. Experimental and Toxicologic Pathology 62, 183–190. Zhao, N.W., Zhang, J.J., Zhao, H.J., Pan, L.T., 2009. From Guizhou Thelypteridaceae the types and distribution of medicinal plants research. Lishizhen Medicine and Materia Medica Research 20, 1743–1745.
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Renoprotective potential of Macrothelypteris torresiana via ameliorating oxidative stress and proinflammatory cytokines Jinglou Chen a , Yongfang Lei a , Guanghua Wu a , Yonghui Zhang a,∗ , Wei Fu b , Chaomei Xiong a , Jinlan Ruan a,∗ a Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation of Hubei Province, College of Pharmacy, Tongji Medical Center, Huazhong University of Science and Technology, Wuhan, China b Department of Pharmacy, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
a r t i c l e
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Article history: Received 7 July 2011 Received in revised form 15 October 2011 Accepted 1 November 2011 Available online 10 November 2011 Keywords: Macrothelypteris torresiana Nephrotic syndrome Oxidative stress Vascular endothelial growth factor Nitric oxide
a b s t r a c t Ethnopharmacological relevance: Macrothelypteris torresiana is traditionally used in Chinese folk medicine for the treatment of edema for patients suffering from kidney/bladder problems due to its satisfactory therapeutic effectiveness. Aim of the study: The aim of this study was to investigate the renoprotective nature of the total polyphenols fraction from Macrothelypteris torresiana (PMT). Materials and methods: The biochemical criterions of plasma and kidney tissues were evaluated to study the effects of PMT on puromycin aminonucleoside-induced chronic nephrotic syndrome (NS) in hyperlipidemic mice. Results: In this study, the NS and hyperlipidemia were ameliorated after 9 weeks administration of PMT. Besides, PMT was able to modulate the level of renal oxidative stress and vascular endothelial growth factor–nitric oxide (VEGF–NO) pathway. Conclusions: It represented a potential resource of PMT for the treatment of NS involved in metabolic syndrome. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The nephrotic syndrome (NS) is a chronic renal disease and usu˜ ally involved with hyperlipidemia (Pena-Rico et al., 2006). High levels of total cholesterol (TC) and triglyceride (TG) always contribute to NS (Jeong et al., 2010). Besides, more and more evidences show that reactive oxygen species (ROS) are associated with the progress of NS. High levels of ROS can produce cell injury including peroxidation of membrane lipids, proteins denaturation and DNA
Abbreviations: NS, nephrotic syndrome; TC, total cholesterol; TG, triglyceride; ROS, reactive oxygen species; SOD, superoxide dismutase; CAT, catalase; GPx, glutathione peroxidase; PAN, puromycin aminonucleoside; VEGF, vascular endothelial growth factor; NO, nitric oxide; iNOS, inducible-NO synthase; PMT, total polyphenols fraction from Macrothelypteris torresiana; CMT, crude methanol extract from Macrothelypteris torresiana; BUN, blood urea nitrogen; LDL, low density lipoprotein; HDL, high density lipoprotein; MDA, malondialdehyde; RT-PCR, real time-polymerase chain reaction; CT, comparative cycle threshold; eNOS, endothelial-NOS; ARF, acute renal failure. ∗ Corresponding authors at: Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation of Hubei Province, College of Pharmacy, Huazhong University of Science and Technology, No. 13 Hangkong Road, Wuhan 430030, Hubei Province, China. Fax: +86 27 83692762. E-mail addresses: yfl[email protected] (Y. Lei), [email protected] (Y. Zhang), [email protected] (J. Ruan). 0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.11.002
damage (Khan et al., 2009). Several biological processes affected by the unstable ROS are potentially important in the pathogenesis of renal injury (Parlakpinar et al., 2006). Increased level of ROS will induce mesangial cells contraction, filtration surface change, ultrafiltration coefficient modify and glomerular filtration rate decrease (Pedraza-Chaverrí et al., 2003). ROS will finally lead to the renal histologic changes as well as the functional abnormalities (Ha and Kim, 1999). This is considered as one of the major mechanisms for nephrotoxicity and other deleterious effects (Khan et al., 2009). As a result, enhancing the activities of the antioxidant enzymes is useful to prevent the renal injury induced by ROS (PedrazaChaverrí et al., 2004). Some polyphenols and flavonoids (such as naringenin and quercetin) are reported to have the potential effect of enhancing the activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) (Vitor et al., 2004; Ratheesh et al., 2011), and finally attenuating the nephrotoxicity in streptozotocin-induced diabetic rats (Sato et al., 2005). Puromycin aminonucleoside (PAN) is usually used to induce the experimental chronic NS which involves the imbalance of the generation and elimination of ROS (Pedraza-Chaverrí et al., 2004). The proinflammatory cytokine vascular endothelial growth factor (VEGF) is also closely associated with the pathogenesis of NS (Cooper et al., 1999). The nitric oxide (NO) comes from
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inducible-NO synthase (iNOS) is also implicated in the renal injury (Tain et al., 2008). Over-expression of iNOS and excessive NO are proposed as one of the causes of vascular dysfunction in kidney (Bhatia et al., 2003). Moreover, NO can react with ROS to form peroxynitrite, and finally lead to a decrease in the glomerular filtration rate (Pedraza-Chaverrí et al., 2003). Furthermore, it is demonstrated that the generation of NO could be stimulated by VEGF (Fan et al., 2008). Summarily, the VEGF-NO pathway is also involved in the process of NS. Macrothelypteris torresiana (Gaud.) Ching is a member of Thelypteridaceae and widely distributed in the south of China. The entire plant has been used in Chinese folk medicine for the treatment of edema for patients suffering from kidney/bladder problems due to its satisfactory therapeutic effectiveness (Zhao et al., 2009). It is usually decocted in water for oral dose. A number of novel polyphenols and flavonoids (such as (2S)-5,7,2 ,5 tetrahydroxyflavanone-2 -O--d-6 -O-acetylglucopyranoside and (2S)-5,7,2 ,5 -tetrahydroxyflavanone-2 -O--d-glucopyranoside) were isolated from this polyphenols-rich plant (Fu et al., 2009; Tang et al., 2009). Our previous study (not showed here) indicated that the methanol extract had remarkable radical scavenging activity and could inhibit malondialdehyde formation in vitro. Then, according to the bioassay guided fractionation method (Kasetti et al., 2010), the experimental acute renal injury induced by K2 Cr2 O7 was used for the preliminary screening of the renoprotective active fraction (Barrera et al., 2003), and the total cholesterol lowering effects on mice was used to identify the hypolipidemic active fraction from the methanol extract (Xu et al., 2003). Interestingly, a fraction which had relative high content of polyphenols were identified as the common active fraction and purified by the method of alkali extraction and acid precipitation. This fraction from Macrothelypteris torresiana was defined as the total polyphenols fraction (PMT). In this study, we investigated the effects of PMT on the acute nephrotoxicity induced by K2 Cr2 O7 and the chronic NS induced by puromycin aminonucleoside in hyperlipidemic mice.
2. Methods and materials 2.1. Plant material Macrothelypteris torresiana was collected in the Jiangxi province, China and identified by Prof. Ceming Tan at the Jiujiang Forest Plants Specimen Mansion, China. After washing with distilled water thoroughly and drying in the shade for 3 days, they were powdered and passed through 40 mesh sieves. The powder was soaked in methanol (v/w) for 6 times and extracted by heat reflux for 3 times, each for 1 h. After filtered with Whatman No. l filter paper, the filtrate was concentrated under vacuum by means of rotavapor to get crude methanol extract from Macrothelypteris torresiana (CMT). Then CMT was partitioned successively between petroleum ether/water, chloroform/water, ethyl acetate/water and n-butanol/water. The parts of chloroform, ethyl acetate and nbutanol were mixed and concentrated in vacuo. The concentrated extract was dried in freeze dryer. The resulting crude extract was mixed with 2% NaOH solution and filtered for the removal of the precipitation. Appropriate volume of the concentrated hydrochloric acid was added into the filtrate to control the pH value at 1, and then stood for 24 h at 4 ◦ C. The mixture was filtered to obtain the sediment, and named as the total polyphenols from CMT (PMT). The total polyphenols content in PMT was estimated using Folin-Ciocalteu reagent (Ainsworth and Gillespie, 2007). The yield of PMT was 67.4 ± 2.0 g/kg of the raw medicinal herbs. The total polyphenols contained in PMT was 514.7 ± 18.1 g/kg of the PMT.
2.2. Animals The Kunming mice were obtained from the Experimental Animal Center of Huazhong University of Science and Technology. The animals were maintained under the standard laboratory conditions with water and food ad libitum. This study protocol was along the “Principles of Laboratory Animal Care” (Xu et al., 2003). The study was approved by the Huazhong University of Science and Technology Committee on Animal Care and Use. After acclimatized for a period of 3 days in the new environment, the animals were used for the study. 2.3. Acute nephrotoxicity in mice induced by K2 Cr2 O7 Forty male mice weighing 20 ± 2 g (4–5 week old) were randomly divided into four groups (n = 10): the vehicle control group, the model group and the PMT treated groups (200 mg/kg/d or 100 mg/kg/d). PMT were orally administrated in the morning for 7 days. The vehicle control and the model groups were given tales doses (the same volume) physiological saline. On the morning of the seventh day, the animals of the model and PMT treated groups were given 15 mg/kg K2 Cr2 O7 by a single subcutaneous injection 30 min after the administration. The vehicle control group was subcutaneously injected tales doses physiological saline. Blood samples were collected at 0, 24 and 48 h after the K2 Cr2 O7 injection. Samples were spun at 2000 r.p.m. for 20 min at 4 ◦ C. The serum was prepared and stored at −80 ◦ C until the determination of serum creatinine and blood urea nitrogen (BUN). 2.4. Chronic NS in hyperlipidemic mice induced by PAN Forty male mice weighing 20 ± 2 g (4–5 week old) were randomly divided into four groups (n = 10): the vehicle control group, the model group and the PMT treated groups (200 mg/kg/d or 100 mg/kg/d). The vehicle control group was fed on the normal forage. The others were fed on 9 weeks’ high-fat diet consisting of normal diet 470 g/kg, sucrose 100 g/kg, egg yolk 200 g/kg, lard 200 g/kg, cholesterol 20 g/kg and sodium deoxycholate 10 g/kg (Chen et al., 2009). PMT were given orally once a day in the morning for 9 weeks. The vehicle control and the model groups were orally given tales doses physiological saline. The animals of the model group and the PMT treated groups were subcutaneously injected 50, 40, 40 and 25 mg/kg PAN (Sigma–Aldrich Chemical Co., USA) on the first day of weeks 5–8, respectively. The vehicle control group was subcutaneously injected with tales doses physiological saline. All the mice were kept in the metabolic cages on the day before sacrificed. Urine samples of 12 h were collected to determine the value of urine protein. Then, the animals were deprived of food for 12 h and were accessible to water freely before they were killed. The blood samples were collected. The serum was prepared and stored at −80 ◦ C until the analysis of plasma lipid profiles, blood glucose, renal function and other plasma biochemical criterions. The kidney samples were rapidly removed and excised. One section of each sample was homogenized in 10 volumes (v/w) of ice cold physiological saline. The homogenate was spun at 15,000 r.p.m. for 20 min at 4 ◦ C. The supernatant was then stored at −80 ◦ C for the subsequent assay for the renal level of oxidative stress. Another section of each sample was stored at −80 ◦ C for the analysis of the expression of renal VEGF. 2.5. Assay of plasma lipid profiles and blood glucose level The blood glucose level was estimated by glucose oxidase peroxidase method (Arora et al., 2010). The levels of TC, TG,
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low density lipoprotein (LDL) and high density lipoprotein (HDL) in plasma were spectrophotometrically determined by the cholesterol esterase/peroxidase method, glycerol kinase method, PVS precipitation method and PTA-Mg2+ precipitation method, respectively (Li et al., 2009). All the manipulations were performed according to the directions of the commercially available kits (Nanjing Jiancheng Bioengineering Institute, China). The results were expressed as mmol/L. 2.6. Assay of renal function The renal function was assessed by estimating the serum creatinine, BUN and 12 h proteinuria. The assays were based on the reported methods (Arora et al., 2010). All the manipulations were performed according to the directions of the commercially available kits (Nanjing Jiancheng Bioengineering Institute, China). The results of creatinine, BUN and proteinuria were expressed as mol/L, mmol/L and mg/12 h, respectively. 2.7. Assay of the levels of oxidative stress and lipid peroxidation in kidney tissue The levels of oxidative stress in kidney tissue was evaluated by determined the levels of SOD, CAT and GPx in 10% kidney supernatant. Also, the malondialdehyde (MDA) level was measured as an index for lipid peroxidation of the tissue. The activities of SOD, CAT and GPx were measured according to the reported methods (Pedraza-Chaverrí et al., 2004) using commercially available kits (Nanjing Jiancheng Bioengineering Institute, China). The assay for total SOD was based on its ability to inhibit the oxidation of oxymine by the xanthine–xanthine oxidase system. The hydroxylamine nitrite produced by the oxidation of oxymine had an absorbance peak at 550 nm. CAT activity was measured by employing H2 O2 into H2 O and O2 . The decomposition of H2 O2 was monitored at 240 nm at 4 ◦ C. The activity of GPx was measured indirectly by a coupled reaction with glutathione reductase and determined the absorbance at 340 nm. All the procedures were performed according to the manufacturer’s instructions. The results were expressed as U/mg protein. The MDA level was measured according to the thiobarbituric acid reaction method (Godard et al., 2009). Briefly, 0.1 ml samples were mixed with 1 ml (17.5%) trichloroacetic acid and 1 ml (0.6%) 2thiobarbituric acid. After being incubated for 1 h at 80 ◦ C they were cooled soon using cold water. The mixture was centrifugated at 3000 r.p.m. for 15 min, the absorbance of the supernatant was measured at 535 nm. 4-Ethoxy-propane (10 nmol/ml) was used as the standard. The result was expressed as nmol/mg protein.
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2.9. Real time-polymerase chain reaction (RT-PCR) The total RNA was isolated from the kidney sample using the Trizol Reagent (Invitrogen, USA). The quality and integrity of RNA were confirmed by spectrophotometric analysis (OD260/280) and gel electrophoresis. cDNA was synthesized using the cDNA Reverse Transcription Kit (Dingguo Biotechnology, China) on a TC-512 DNA machine (Barloworld Scientific, USA). The total volume of 20 l consisted of 1 l random primer, 1 l dNTPs, 4 l 5× buffer, 1 l retroviridase, 6 l (1.5 g) total RNA and 7 l DEPC-H2 O. The reaction parameters were 65 ◦ C × 5 min, 37 ◦ C × 60 min, 70 ◦ C × 15 min and 4 ◦ C × 5 min. Then, cDNA was amplified by RT-PCR. The reaction was performed in a total volume of 25 l containing 2.5 l cDNA, 12.5 l SYBR Green Mix (Invitrogen, USA), 2 l primers and 8 l ddH2 O. Quantitative RT-PCR was performed using a Real-time PCR Detection System (Stratagene Mx3000p). The forward primer of VEGF was 5 -GAGCAAAGGTCACGAAAG-3 , the reverse primer of VEGF was 5 -ATTGAGGGTGAGAAGACG-3 (GenBank: AB086118.1). Actin was used as the internal control. The cycling program consisted of initial denaturation for 3 min at 94 ◦ C followed by 40 cycles of 94 ◦ C for 10 s, 55 ◦ C for 20 s, and 72 ◦ C for 20 s. Relative quantification was performed using the comparative cycle threshold (CT) method (Wang et al., 2006). 2.10. Statistical analyses Data are presented as means ± S.D. of ten measurements per group. Results were analyzed statistically by one-way ANOVA followed by Tukey’s multiple comparison using SPSS software for Windows (Student’s version). Differences were considered significant at p < 0.05. 3. Results 3.1. Acute nephrotoxicity in mice induced by K2 Cr2 O7 The effects of oral administration of PMT (200 or 100 mg/kg/d) on the levels of BUN and creatinine in mice 24 and 48 h after K2 Cr2 O7 injection are summarized in Fig. 1. In the model group, the level of BUN was increased 2.31- and 4.05-fold at 24 and 48 h in comparison with the vehicle control group, respectively. Similarly, the level of creatinine in the model group was enhanced 1.25-and 1.48-fold at 24 and 48 h. Nevertheless, the tendency was attenuated by PMT. The levels of BUN and creatinine in both PMT treated groups were significantly lowered at 24 and 48 h when compared to the model group. 3.2. The lipid profiles and glucose level
2.8. Assay of plasma iNOS activity and NO and VEGF levels The activity of VEGF-NO pathway in mice was evaluated by measuring the levels of VEGF and NO, and the activity of iNOS. The level of NO was determined after conversion of nitrate to nitrite by nitrate reductase according to the Griess reaction, the activity of iNOS was measured based on their activities to catalyze the transformation of l-arginine to NO (Milsom et al., 2010; Yaman and Balikci, 2010). The absorbance of NO and iNOS were determined at 550 nm and 530 nm, respectively. All the procedures were performed according to the manufacturer’s instructions of the kits (Nanjing Jiancheng Bioengineering Institute, China). Assay of the VEGF level was determined by specific ELISA kits (R&D Corporation, USA). All the procedures were performed according to the manufacturer’s instructions. The results of NO, iNOS, and VEGF were expressed as mol/L, U/ml and ng/L, respectively.
Fig. 2 represents the lipid profiles and glucose level in the four groups of mice. The levels of TC, TG, LDL and glucose of plasma were significantly elevated in the model group. Treatment with PMT (200 or 100 mg/kg/d) decreased all of them in a concentrationdependent manner. In the meantime, the HDL level was increased after administrated PMT (200 or 100 mg/kg/d) for 9 weeks compared to the model group. 3.3. The renal function As can be seen from Fig. 3, the renal function of the model group was deteriorated when compared to the vehicle control group. The levels of BUN, creatinine and 12 h urinary protein in model group were significantly increased. Oral administration of PMT (200 or 100 mg/kg/d) revealed a notable (p < 0.05) decline in all of them when compared to the model group.
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Fig. 1. Effects of PMT (200 or 100 mg/kg/d) on acute nephrotoxicity in mice induced by K2 Cr2 O7 . Values are given as means ± S.D. from 10 mice in each group. Values with different alphabets (a–f) in each index (BUN or Creatinine) are significantly different. The mean difference is significant at p < 0.05 (groups with different superscript alphabets are statistically significant). (A) The changes of the level of BUN from 0 h to 48 h. (B) The changes of level of creatinine from 0 h to 48 h.
3.4. The levels of oxidative stress and VEGF-NO pathway in vivo The effects of PMT (200 or 100 mg/kg/d) on modulating the activities of antioxidant enzymes and the level of MDA are described in Table 1. The levels of enzymatic antioxidants were significantly diminished in the model group, accompanied with the MDA level was enhanced. Oral treatment of PMT attenuated the alteration. The activities of SOD, GPx and CAT in both PMT treated groups were notably increased while the level of MDA was significantly decreased. Table 1 also details the altered levels of VEGF and NO, and the activity of iNOS of four groups mice. A significant elevation of the NO level was observed in the model group. Similarly, the iNOS activity of the model group was enhanced more than 3-fold when compared with the vehicle control. As expected, PMT significantly decreased the activity of iNOS and the level of NO. In the meantime, the level of VEGF was also increased about 2-fold in the model group when compared with the vehicle control. Treatment with PMT lowered it in a dose-dependent manner. Fig. 2. Effects of PMT (200 or 100 mg/kg/d) on plasma lipid profiles and glucose level. Values are given as means ± S.D. from 10 mice in each group. Values with different alphabets (a–d) in each index (TC, TG, HDL, LDL or glucose) are significantly different. The mean difference is significant at p < 0.05 (groups with different superscript alphabets are statistically significant).
3.5. The renal expression of VEGF The renal expression of VEGF was determined by quantitative RT-PCR assessment. As can be seen from Fig. 4, the expression of kidney VEGF mRNA was significantly enhanced in the model group. The value was 2.74-fold of the vehicle control group. PMT at 200
Fig. 3. Effects of PMT (200 or 100 mg/kg/d) on the renal function of chronic NS in hyperlipidemic mice. Values are given as means ± S.D. from 10 mice in each group. Values with different alphabets (a–d) in each index (BUN, Creatinine or Urinary protein) are significantly different. The mean difference is significant at p < 0.05 (groups with different superscript alphabets are statistically significant). (A) The effect on the level of BUN. (B) The effect on the level of creatinine. (C) The effect on the level of urinary protein.
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Table 1 Effects of PMT (200 or 100 mg/kg/d) on the levels of SOD, GPx, CAT, MDA, VEGF, NO and iNOS in vivo. Vehicle control group SOD (U/mg protein) GPx (U/mg protein) CAT (U/mg protein) MDA (nmol/mg protein) VEGF (pg/ml) NO (mol/L) iNOS (mol/L)
15.70 19.14 0.42 3.32 121.5 69.69 0.02
± ± ± ± ± ± ±
0.62a 1.31a 0.06a 0.22a 7.74a 3.49a 0.007a
Model group 8.94 7.10 0.11 11.79 243.9 159.91 0.07
± ± ± ± ± ± ±
0.62b 0.54b 0.03b 0.56b 13.47b 7.94b 0.018b
High dose group 13.14 10.47 0.34 5.12 153.8 88.93 0.04
± ± ± ± ± ± ±
0.54c 0.47c 0.06a 0.40c 7.72c 4.25c 0.012c
Low dose group 11.32 8.45 0.21 6.71 181.6 92.28 0.04
± ± ± ± ± ± ±
0.42d 0.36d 0.04c 0.39d 6.20d 3.58c 0.018c
Values are given as means ± S.D. from 10 mice in each group. Values with different superscripts (a–d) in a row are significantly different. The mean difference is significant at p < 0.05 (groups with different superscript alphabets are statistically significant).
or 100 mg/kg/d decreased the value to 0.493- or 0.825-fold of the model group, respectively. 4. Discussion NS is usually related to the disorder of metabolism and associated with high morbidity and mortality (Bhatia et al., 2003). In addition, hyperlipidemia has also been demonstrated to be a major risk factor of the progression of nephropathy (Arora et al., 2010). In our study the animals were fed on a high-fat diet for 9 weeks to induce hyperlipidemia. Besides, PAN was injected to induce ˜ nephrotic syndrome (Pena-Rico et al., 2006). After 9 weeks, the lipid levels of model group were increased accompanied with the renal function was deteriorated, however, PMT attenuated the tendency. Administration of PMT for 9 weeks lowered the levels of TC, TG and LDL, while the level of HDL was elevated. As expected, the renal function was improved. The results indicated that PMT could ameliorate the hyperlipidemia and protect the kidney against the impairments induced by PAN in hyperlipidemic mice. Furthermore, the possible mechanism was also investigated. It was reported that oxidative stress played a key role in the progress of NS (Ha and Kim, 1999). Taken into account that PMT could reduce the acute nephrotoxicity induced by K2 Cr2 O7 , which was closely associated with oxidative stress (Barrera et al., 2003), a possible mechanism emerged: the renal protective effect of PMT seemed related to its antioxidant activity. Therefore, the changes of the activities of renal antioxidant enzymes were investigated to evaluate the renal levels of oxidative stress. The level of MDA, which was the decomposed product of ox-LDL and an index for lipid peroxidation, was also studied. The activities of SOD, CAT and GPx in the model group
Fig. 4. Effects of PMT (200 or 100 mg/kg/d) on renal expression of VEGF. Values are given as means ± S.D. from 10 mice in each group. Values with different alphabets (a–d) are significantly different. The mean difference is significant at p < 0.05 (groups with different superscript alphabets are statistically significant).
were decreased and the level of MDA was elevated. Daily treatment of PMT for 9 weeks significantly increased the activities of these antioxidant enzymes. Similarly, the lowered level of MDA was observed. At this stage, a most possible hypothesis could be proposed: PMT could modulate the activities of kidney antioxidant enzymes, alleviate oxidative stress and decrease the levels of lipid profiles in mice, and finally improve the renal function. VEGF is closely related to the pathogenesis of NS (Cooper et al., 1999; Wasilewska and Zoch-Zwierz, 2006). Marked up-regulation of VEGF gene expression has been observed in NS. And VEGF excretion is significantly increased in accordance with the degree of renal injury (Libby et al., 2002; McKnight et al., 2007). In this study, the renal expression of VEGF was significantly enhanced. As expected, the level of VEGF was significantly increased in the model group accompanied with the levels of BUN, creatinine and urinary protein were notably enhanced. All of them were lowered by 9 weeks administration of PMT (both 200 mg/kg and 100 mg/kg). On the other hand, NO is reported to play an important role in the body and involve in the control of renal function (Plotnikov et al., 2009). Especially in the health status, the endothelial NOS (eNOS) is identified as a protective NO-generating enzyme and contributes to the maintenance of the normal renal structure (Sandovici et al., 2004). The NO derived from eNOS usually plays a protective effect, however, the one comes from iNOS might be implicated in the renal injury (Tain et al., 2008). Indeed, over-expression of iNOS and excessive NO participate in the development of NS (Becker et al., 2009; Milsom et al., 2010). Furthermore, increased level of NO can interact with oxygen radicals and finally lead to the deterioration of acute renal failure (ARF) (Traylor and Mayeux, 1997; Plotnikov et al., 2009). In our study, an elevated level of NO was observed in the model group. In the meantime, the activity of iNOS was significantly enhanced. However, administration of PMT attenuated the tendency. Additionally, report shows that the generation of NO can be stimulated by VEGF (Fan et al., 2008). It is suggested that increased level of VEGF would enhance the NO level and finally lead to the renal dysfunction (de Vriese et al., 2001). VEGF is suggested to be a potential regulator of NO generation. It has the capacity of regulating PKC signaling pathway (Wang et al., 2004; Farhangkhoee et al., 2006). Summarily, the VEGF-NO pathway is also involved in the NS. The whole findings indicated that PMT could suppress the enhancement of the renal VEGF expression in NS mice. And the decreased level of VEGF was observed in both PMT treated group. Taking into account that the levels of NO and iNOS were decreased in PMT treated groups, another possible hypothesis could be given: The renal protective effect of PMT was also associated with the activity of regulating the NO signaling pathway following the VEGF stimulation. Some herb extracts had been demonstrated could obviously inhibit oxidative stress and decrease NO level in rats, such as grape seed extract and Nigella sativa (Mattoo and Kovacevic, 2003; Yaman and Balikci, 2010). Our findings in this work were in line with the other reports about flavonoids and polyphenols were able to significantly enhance the activities of GPx, SOD and CAT, and prevented
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the overexpression of iNOS in rats suffered with kidney diseases (Badary et al., 2005; Morales et al., 2006). 5. Conclusions In summary, PMT could modulate the levels of renal oxidative stress and the VEGF-NO pathway, and finally attenuate the K2 Cr2 O7 -induced ARF and the PAN-induced chronic NS in hyperlipidemic mice. These results suggested that PMT had renoprotective potential, and represented a potential resource for the treatment of renal injury. Further investigations on detailed analysis of the mechanisms of PMT using longer times and varied dosages are required. And further toxicological studies also have a considerable impact on future clinical treatments of patients with renal disease to test the value of PMT as potential agents. Acknowledgements The authors thank Prof. Ceming Tan from the Jiujiang Forest Plants Specimen Mansion for the identification of the plant, the financial support from the State Natural Sciences Fund (NO30973864), the Ministry of Education Fund for Doctoral Program of higher subjects (NO20090142110021) and the Key Natural Science Fund of Hubei Province (NO2009CDA067). References Ainsworth, E.A., Gillespie, K.M., 2007. Estimation of total phenolic content and other oxidation substrates in plant tissues using Folin-Ciocalteu reagent. Nature Protocols 2, 875–877. Arora, M.K., Reddy, K., Balakumar, P., 2010. The low dose combination of fenofibrate and rosiglitazone halts the progression of diabetes-induced experimental nephropathy. European Journal of Pharmacology 636, 137–144. Badary, O.A., Abdel-Maksoud, S., Ahmed, W.A., Owieda, G.H., 2005. Naringenin attenuates cisplatin nephrotoxicity in rats. Life Sciences 76, 2125–2135. Barrera, D., Maldonado, P.D., Medina-Campos, O.N., Hernández-Pando, R., IbarraRubio, M.E., Pedraza-Chaverrí, J., 2003. Protective effect of SnCl2 on K2 Cr2 O7 induced nephrotoxicty in rats: the indispensability of HO-1 preinduction and lack of association with some antioxidant enzymes. Life Sciences 73, 3027–3041. Becker, T., Mevius, I., de Vries, D.K., Schaapherder, A.F., zu Vilsendorf, A.M., Klempnauer, J., Frölich, J.C., Tsikas, D., 2009. The l-arginine/NO pathway in end-stage liver disease and during orthotopic liver and kidney transplantation: biological and analytical ramifications. Nitric Oxide 20, 61–67. Bhatia, S., Shukla, R., Venkata-Madhu, S., Kaur-Gambhir, J., Madhava-Prabhu, K., 2003. Antioxidant status, lipid peroxidation and nitric oxide end products in patients of type 2 diabetes mellitus with nephropathy. Clinical Biochemistry 36, 557–562. Chen, X.H., Bai, X., Liu, Y.H., Tian, L.Y., Zhou, J.Q., Zhou, Q., Fang, J.B., Chen, J.C., 2009. Anti-diabetic effects of water extract and crude polysaccharides from tuberous root of Liriope spicata var. prolifera in mice. Journal of Ethnopharmacology 122, 205–209. Cooper, M.E., Vranes, D., Youssef, S., Stacker, S.A., Cox, A.J., Rizkalla, B., Casley, D.J., Bach, L.A., Kelly, D.J., Gilbert, R.E., 1999. Increased renal expression of vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 in experimental diabetes. Diabetes 48, 2229–2239. de Vriese, A.S., Tilton, R.G., Elger, M., Stephan, C.C., Kriz, W., Lameire, N.H., 2001. Antibodies against vascular endothelial growth factor improve early renal dysfunction in experimental diabetes. Journal of the American Society of Nephrology 12, 993–1000. Fan, H., Sulochana, K.N., Chong, Y.S., Ge, R., 2008. Decorin derived antiangiogenic peptide LRR5 inhibits endothelial call migration by interfering with VEGFstimulated NO release. The International Journal of Biochemistry & Cell Biology 40, 2120–2128. Farhangkhoee, H., Khan, Z.A., Kaur, H., Xin, X., Chen, S., Chakrabarti, S., 2006. Vascular endothelial dysfunction in diabetic cardiomyopathy: pathogenesis and potential treatment targets. Pharmacology & Therapeutics 111, 384–399. Fu, W., Fang, W., Ruan, J.L., 2009. Two new flavanone glycosides from Macrothelypteris torresiana (Gaud.) Ching. Chinese Chemical Letters 20, 579–581. Godard, M., Décordé, K., Ventura, E., Soteras, G., Baccou, J.C., Cristol, J.P., Rouanet, J.M., 2009. Polysaccharides from the green alga Ulva rigida improve the antioxidant status and prevent fatty streak lesions in the high cholesterol fed hamster, an animal model of nutritionally-induced atherosclerosis. Food Chemistry 115, 176–180. Ha, H., Kim, K.H., 1999. Pathogenesis of diabetic nephropathy: the role of oxidative stress and protein kinase C. Diabetes Research and Clinical Practice 45, 147– 151. Jeong, S.C., Jeong, Y.T., Yang, B.K., Islam, R., Koyyalamudi, S.R., Pang, G., Cho, K.Y., Song, C.H., 2010. White button mushroom (Agaricus bisporus) lowers blood
glucose and cholesterol levels in diabetic and hypercholesterolemic rats. Nutrition Research 30, 49–56. Kasetti, R.B., Rajasekhar, M.D., Kondeti, V.K., Fatima, S.S., Kumar, E.G., Swapna, S., Ramesh, B., Rao, C.A., 2010. Antihyperglycemic and antihyperlipidemic activities of methanol:water (4:1) fraction isolated from aqueous extract of Syzygium alternifolium seeds in streptozotocin induced diabetic rats. Food and Chemical Toxicology 48, 1078–1084. Khan, S.A., Priyamvada, S., Farooq, N., Khan, S., Khan, M.W., Yusufi, A.N., 2009. Protective effect of green tea extract on gentamicin-induced nephrotoxicity and oxidative damage in rat kidney. Pharmacological Research 59, 254–262. Li, S.Y., Chang, C.Q., Ma, F.Y., Yu, C.L., 2009. Modulating effects of chlorogenic acid on lipids and glucose metabolism and expression of hepatic peroxisome proliferator-activated receptor-␣ in golden hamsters fed on high fat diet. Biomedical and Environmental Sciences 22, 122–129. Libby, P., Ridker, P.M., Maseri, A., 2002. Inflammation and atherosclerosis. Circulation 105, 1135–1143. Mattoo, T.K., Kovacevic, L., 2003. Effect of grape seed extract on puromycinaminonucleoside-induced nephrosis in rats. Pediatric Nephrology 18, 872– 877. McKnight, A.J., Maxwell, A.P., Patterson, C.C., Brady, H.R., Savage, D.A., 2007. Association of VEGF-1499C → T polymorphism with diabetic nephropathy in type 1 diabetes mellitus. Journal of Diabetes and Its Complications 21, 242– 245. Milsom, A.B., Patel, N.S., Mazzon, E., Tripatara, P., Storey, A., Mota-Filipe, H., Sepodes, B., Webb, A.J., Cuzzocrea, S., Hobbs, A.J., Thiemermann, C., Ahluwalia, A., 2010. Role for endothelial nitric oxide synthase in nitrite-induced protection against renal ischemia–reperfusion injury in mice. Nitric Oxide 22, 141–148. Morales, A.I., Vicente-Sánchez, C., Jerkic, M., Santiago, J.M., Sánchez-González, P.D., Pérez-Barriocanal, F., López-Novoa, J.M., 2006. Effect of quercetin on metallothionein, nitric oxide synthases and cyclooxygenase-2 expression on experimental chronic cadmium nephrotoxicity in rats. Toxicology and Applied Pharmacology 210, 128–135. Parlakpinar, H., Tasdemir, S., Polat, A., Bay-Karabulut, A., Vardi, N., Ucar, M., Yanilmaz, M., Kavakli, A., Acet, A., 2006. Protective effect of chelerythrine on gentamicininduced nephrotoxicity. Cell Biochemistry and Function 24, 41–48. Pedraza-Chaverrí, J., Barrera, D., Hernández-Pando, R., Medina-Campos, O.N., Cruz, C., Murguía, F., Juárez-Nicolás, C., Correa-Rotter, R., Torres, N., Tovar, A.R., 2004. Soy protein diet ameliorates renal nitrotyrosine formation and chronic nephropathy induced by puromycin aminonucleoside. Life Sciences 74, 987–999. Pedraza-Chaverrí, J., González-Orozco, A.E., Maldonado, P.D., Barrera, D., MedinaCampos, O.N., Hernández-Pando, R., 2003. Diallyl disulfide ameliorates gentamicin-induced oxidative stress and nephropathy in rats. European Journal of Pharmacology 473, 71–78. ˜ M., Guadalupe Ortiz-Lápez, M., Camacho-Castillo, L., Cárdenas, M., Pena-Rico, Pedraza-Chaverri, J., Menjívar, M., 2006. Steroidogenic impairment due to reduced ovarian transcription of cytochrome P450 side-chain-cleavage (P450scc) and steroidogenic acute regulatory protein (StAR) during experimental nephrotic syndrome. Life Sciences 79, 702–708. Plotnikov, E.Y., Chupyrkina, A.A., Pevzner, I.B., Isaev, N.K., Zorov, D.B., 2009. Myoglobin causes oxidative stress, increase of NO production and dysfunction of kidney’s mitochondria. Biochimica et Biophysica Acta 1792, 796–803. Ratheesh, M., Shyni, G.L., Sindhu, G., Helen, A., 2011. Inhibitory effect of Ruta graveolens L. on oxidative damage, inflammation and aortic pathology in hypercholesteromic rats. Experimental and Toxicologic Pathology 63, 285– 290. Sandovici, M., Henning, R.H., Hut, R.A., Strijkstra, A.M., Epema, A.H., van Goor, H., Deelman, L.E., 2004. Differential regulation of glomerular and interstitial endothelial nitric oxide synthase expression in the kidney of hibernating ground squirrel. Nitric Oxide 11, 194–200. Sato, S., Yamate, J., Hori, Y., Hatai, A., Nozawa, M., Sagai, M., 2005. Protective effect of polyphenol-containing azuki bean (Vigna angularis) seed coats on the renal cortex in streptozotocin-induced diabetic rats. Journal of Nutritional Biochemistry 16, 547–553. Tain, Y.L., Muller, V., Szabo, A.J., Erdely, A., Smith, C., Baylis, C., 2008. Renal cortex neuronal nitric oxide synthase in response to rapamycin in kidney transplantation. Nitric Oxide 18, 80–86. Tang, Y., Fang, W., Ma, Y.T., Cai, Y.L., Ruan, J.L., 2009. A novel flavonoid from the root of Macrothelypteris torresiana (Gaud.) Ching. Chinese Chemical Letters 20, 815–816. Traylor, L.A., Mayeux, P.R., 1997. Superoxide generation by renal proximal tubule nitric oxide synthase. Nitric Oxide 1, 432–438. Vitor, R.F., Mota-Filipe, H., Teixeira, G., Borges, C., Rodrigues, A.I., Teixeira, A., Paulo, A., 2004. Flavonoids of an extract of Pterospartum tridentatum showing endothelial protection against oxidative injury. Journal of Ethnopharmacology 93, 363–370. Wang, Y., Nagase, S., Koyama, A., 2004. Stimulatory effect of IGF-I and VEGF on eNOS message, protein expression, eNOS phosphorylation and nitric oxide production in rat glomeruli, and the involvement of PI3-K signaling pathway. Nitric Oxide 10, 25–35. Wang, Y., Wang, Y., Yang, Q., Yan, J.T., Zhao, C.X., Cianflone, K., Wang, D.W., 2006. Effects of bezafibrate on the expression of endothelial nitric oxide synthase gene and its mechanisms in cultured bovine endothelial cells. Atherosclerosis 187, 265–273.
J. Chen et al. / Journal of Ethnopharmacology 139 (2012) 207–213 Wasilewska, A., Zoch-Zwierz, W., 2006. Glucocorticoid receptor and vascular endothelial growth factor in nephrotic syndrome. Acta Paediatrica 95, 587– 593. Xu, S.Y., Bian, L., Chen, X., 2003. Experimental Methodology of Pharmacology. People’s Medical Publishing House, Beijing, pp. 155–163.
213
Yaman, I., Balikci, E., 2010. Protective effects of Nigella sativa against gentamicininduced nephrotoxicity in rat. Experimental and Toxicologic Pathology 62, 183–190. Zhao, N.W., Zhang, J.J., Zhao, H.J., Pan, L.T., 2009. From Guizhou Thelypteridaceae the types and distribution of medicinal plants research. Lishizhen Medicine and Materia Medica Research 20, 1743–1745.