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Superior renoprotective effects of the combination of breviscapine with enalapril and its mechanism in diabetic rats
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ARTICLE IN PRESS Phytomedicine xxx (2013) xxx–xxx
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Superior renoprotective effects of the combination of breviscapine with enalapril and its mechanism in diabetic rats Xing-Xin Xu a , Wei Zhang a , Pei Zhang a , Xiang-Ming Qi a , Yong-Gui Wu a,∗ , Ji-Jia Shen b a b
Department of Nephrology, The First Affiliated Hospital, Anhui Medical University, Hefei, Anhui, PR China Department of Pathobiology, Anhui Medical University, Hefei, Anhui, PR China
a r t i c l e
i n f o
Keywords: Diabetic nephropathy Protein kinase C Breviscapine Enalapril Transforming growth factor 1
a b s t r a c t Breviscapine is a flavonoid extracted from a Chinese herb Erigeron breviscapus, previously it was shown that treatment with breviscapine attenuated renal injury in the diabetic rats. The purpose of this study was to investigate whether breviscapine combined with enalapril (an ACE inhibitor) have superior renoprotective effects against diabetic nephropathy. Rats were randomly separated into five groups: control, diabetes, diabetes treated with enalapril, diabetes treated with breviscapine, or diabetes treated with combined enalapril with breviscapine. Twenty-four hours urinary AER and the levels of 3-NT in renal tissue and MDA in renal tissue and urine as well as activities and expression of PKC in renal tissue were determined, and renal tissue morphology were observed by light microscopy after 8 weeks. Expression of TGF1 protein was performed by immunohistochemistry method. Increased AER and kidney pathologic injury were attenuated by treatment with either enalapril or breviscapine and further reduced by the combination of the two. Elevated 3-NT in renal tissue and MDA levels in renal tissue and urine were reduced by enalapril or breviscapine and, more effectively, by combined enalapril with breviscapine. PKC activities and expression were higher in renal tissue in diabetic rats than those of the control group, which were reduced by both monotherapies, and further abrogated by combination therapy in both cases. Overexpression of TGF1 protein observed in the glomeruli and tubulointerstitium of diabetic rats was attenuated by enalapril or breviscapine to a similar lever and further reduced by the combination of the two. The combination of enalapril and breviscapine confers superiority over monotherapies on renoprotection, which mechanism may be at least partly correlated with synergetic suppression on increased oxidative stress and PKC activities as well as overexpression of TGF1 in renal tissue. © 2013 Elsevier GmbH. All rights reserved.
Introduction DN, which is one of the most serious complications of diabetes and the number one cause of renal failure in the industrialized world, is characterized by albuminuria and enlargement of the glomerular mesangium due to the accumulation of extracellular matrix proteins (Najafian et al. 2011). Experimental and clinical studies in subjects with type 1 and type 2 diabetes clearly link hyperglycemia to vascular complications, including diabetic nephropathy. Hyperglycemia is responsible for the development
Abbreviations: DN, diabetic nephropathy; STZ, streptozotocin; MDA, malondialdehyde; 3-NT, 3-nitrotyrosine; AER, albumin excretion rate; PKC, protein kinase C; TGF1, transforming growth factor 1; ACE, Angiotensin-converting enzyme; ROS, Reactive oxygen species; ICAM-1, intercellular adhesion molecule-1; MCP1, monocyte chemoattractant protein-1; AG , glomerular cross-sectional area; AM , mesangial area; VG , glomerular volume; KW, kidney weight; BW, body weight; TII, tubulointerstital injury; SABC, Streptavidin-biotin-peroxidase complex. ∗ Corresponding author. Tel.: +86 551 6292 2450; fax: +86 551 6363 3742. E-mail address: [email protected] (Y.-G. Wu).
and progression of diabetic nephropathy through metabolic derangements, including increased oxidative stress, renal polyol formation, activation of PKC-mitogen-activated protein kinases, and accumulation of advanced glycation end products, as well as such hemodynamic factors as systemic hypertension and increased intraglomerular pressure (Brownlee 2001; JandeleitDahm and Cooper 2006; Schena and Gesualdo 2005; Tan et al. 2007; Wolf 2004). Amelioration of albuminuria and glomerular mesangium enlargement are now considered one of the targets in the prevention and retardation of DN (Turgut and Bolton 2010). ACE inhibitors as an accepted renoprotection drug, can attenuate hyperfiltration and hyperpressure in glomeruli and has been proved effective in reducing albuminuria, diminishing loss of kidney function and improving survival both in patients with DN and animal models through a hemodynamic pathway. The advent of ACE inhibitors has changed profoundly the therapeutic approach to diabetic nephropathy, and may improve the survival and the quality of life of diabetic patients significantly. Breviscapine extracted from a Chinese herb Erigeron breviscapus have been previously demonstrated in diabetic patients the ability to reduce albuminuria.
0944-7113/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymed.2013.03.027
Please cite this article in press as: Xu, X.-X., et al., Superior renoprotective effects of the combination of breviscapine with enalapril and its mechanism in diabetic rats. Phytomedicine (2013), http://dx.doi.org/10.1016/j.phymed.2013.03.027
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Fig. 1. The chemical structure of Breviscapine.
The active ingredient is a mixture as shown in Fig. 1 (Zhang et al. 1988). Breviscapine possesses a variety of pharmacological functions beyond hemodynamic effects, especially as anti-oxidative stress agent and PKC inhibitor (Ali et al. 2003; Chen et al. 1998; Shuai and Dong 1998). ROS generation and PKC activation are elevated in mesangial cells cultured under high glucose conditions (Ha et al. 2002), the kidneys of experimental diabetic animals also exhibit increased lipid peroxidation, which is a marker of increased ROS generation and PKC activation (Ferroni et al. 2004). Furthermore, antioxidants and PKC inhibitor have been reported to prevent or attenuate both glucose-induced mesangial cell activation and renal injury in diabetes (Lu et al. 2010; Noh and King 2007; Waisundara et al. 2008). We have proved that breviscapine can ameliorate renal injury in STZ diabetic rat in vivo. Several of our past studies have shown clearly that breviscapine can regulate expression of ICAM-1, MCP-1 and antagonize the activity of PKC (Qi et al. 2006). Renal protection with either an ACE inhibitor or an anti-oxidative stress agent and PKC inhibitor as monotherapy is suboptimal. The observation that a substantial fraction of diabetic subjects responds poorly to these treatments underscores the need for additional therapeutic maneuvers capable of halting the progression to end-stage renal failure. In a rat model of passive Heymann nephritis, addition of simvastatin to the ACE inhibitor, enalapril, resulted in greater reduction in proteinuria than was seen with enalapril alone (Zoja et al. 2002). Recently, we and others studies demonstrated that combination therapy is superior than either therapy alone in STZ diabetic rat (Jia et al. 2007; Wu et al. 2006a,b). These data suggest that a therapy combining an ACE inhibitor with an anti-oxidative stress agent and PKC inhibitor may be useful in progressive renal disease. In the present study, we evaluated the renal effects of a combination of an ACE inhibitor with breviscapine in experimental diabetes. In addition, we explored the effects of these therapies on protein expression of TGF-1, which was considered to be important mediators of injury leading to kidney injury in diabetes.
Reagents Breviscapine (containing flavone 4.5 mg/20 mg) was supplied by Yuxi Pharmaceutical Co. Ltd. (Kunming, China). STZ was purchased from Sigma Chemical Co. (St. Louis, Mo, USA). Microalbumin assay kit was purchased from Exocell Inc. (Philadelphia, PA, USA). MDA assay kits were the product of Nanjing Jiancheng Bioengineering Institute (Nanjing, China). PKC activities assay kit was purchased from Calbiochem Co. (La Jolla, CA, USA). [␥-32 P]ATP was purchased from Beijing YaHui Bioengineering Institute (Beijing, China). Anti 3-NT monoclonal antibody was purchased from Upstate Technology (Lake Placid, NY, USA). Anti-PKC monoclonal antibody and anti-TGF-1 polyclonal antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Chemiluminescence kit was from Amersham Life Science (Little Chalfont, UK). SABC kit was from Bosten Biotechnique Co. (Wuhan, China). Experimental protocol Diabetic was induced by single injection of STZ at a dose of 65 mg/kg IP, diluted in citrate buffer 0.1 mol/l (pH 4.0). Two days later, the diabetic state was confirmed by measurement of tail blood glucose (BG) level. Diabetic rats received daily injections of NPH insulin, in doses adjusted individually (ranging from 1 to 4 units) to maintain BG levels between 200 mg/dl and 400 mg/dl, and to avoid ketonuria. BG level were measured twice a week. Five experimental groups were studied and treated for 8 weeks. The groups are as follows: control with no treatment (group C, n = 10); diabetes with no treatment (group DM, n = 10); diabetes treated with enalapril, at a dose of 10 mg/kg body weight/day by gavage (group DM + E, n = 10), diabetes treated with breviscapine, at a dose of 20 mg/kg body weight/day by gavage (group DM + B, n = 10), diabetic rats treated with the combination of both enalapril and breviscapine at the doses described above (group DM + E + B, n = 10). In preliminary experiments, these were found to be the suitable dose that the rats would tolerate without losing weight or showing deterioration of their general condition.
Materials and methods Metabolic parameters and tissue collection Animals Adult male munich-wistar rats, with initial weights of 180–200 g (Grade II, Certificate No. 01) were obtained from Experimental Animal Center of Anhui Medical University. The research protocol was in accordance with the principles approved by the animal ethics committee of Anhui Medical University. Animals were housed at a temperature of 24 ± 1 ◦ C and humidity of 65–70%, and were submitted to a 12 h light/dark cycle, and allowed free access to standard laboratory chow and tap water.
BW was measured at the conclusion of the experiment. Rats were then anesthetized with sodium pentobarbital (50 mg/kg IP) and placed on a temperature-regulated table, the right jugular artery was catheterized, this arterial catheter was used for blood sampling. BG levels were determined with a glucose analyzer. The kidneys were perfused in vivo via the abdominal aorta with 100 ml of normal saline at 4 ◦ C, while the left renal vein was punctured to permit the perfusate to drain, the kidneys were removed immediately and fixed in 10% formalin and processed in paraffin for
Please cite this article in press as: Xu, X.-X., et al., Superior renoprotective effects of the combination of breviscapine with enalapril and its mechanism in diabetic rats. Phytomedicine (2013), http://dx.doi.org/10.1016/j.phymed.2013.03.027
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subsequent histologic assessment and immunohiatochemical studies. The remaining kidney was stored at −70 ◦ C for evaluation of oxidative stress parameters, PKC activities and Western blot analysis. Urinary albumin excretion Prior to sacrifice, animals were placed in metabolic cages for collection of urine over 24 h for measurement of albumin concentrations. Urinary albumin concentrations were measured by enzyme-linked immunoabsorbent assay using an anti-rat albumin antibody and 24 h urinary albumin excretion was calculated by multiplying the urinary protein excretion by the 24 h urine volume. Renal pathology Formalin-fixed kidney sections (2 m) were stained with PAS reagent to identify kidney structure and hematoxylin to distinguish cell nuclei. Digital images of glomeruli and interstitial areas were obtained from mioroscopy (magnification, 400×). The AG and AM were measured in 50 glomerutar profiles per rat by using computerized image analysis system (Beijing Aeronautic and Aerospace University, Beijing, China). The VG was then calculated as: VG = ˇ/K[AG I3/2 ], where ˇ = 1.38 is the size distribution coefficient and K = 1.1 is the shape coefficient for glomeruli idealized as a sphere. Tubulointerstitial area in the cortex was evaluated and graded as: 0, normal; 1, the area of interstitial inflammation and fibosis, tubular atrophy and dilation with cast formation involving <25% of the field; 2, lesion area between 25% and 50% of the field; and 3, lesions involving >50% of the field. The indices for TII were calculated by averaging the grades assigned to all tubule fields. All measurements and scoring were performed on blinded slides. Determination of MDA MDA was determined according to the manufacturer’s protocol. Results in urinary MDA were expressed as nmol of MDA in 24 h, results in renal MDA levels were expressed as nmol MDA per milligram protein (nmol/mg prot). The protein content was estimated by the dye binding assay of Bradford, with bovine serum albumin used as a standard.
3
sulfate (SDS), 100 g/ml aprotinin, 100 g/ml phenylmethylsulfonyl fluoride (PMSF), sodium orthovanadate] at 4 ◦ C throughout all procedures, and sonicated for 70 s, then add 300 g PMSF per gram of tissue and incubate on ice for 30 min, followed by centrifugation at 15,000 rpm for 20 min at 4 ◦ C. The protein content was estimated by the dye binding assay of Bradford, with bovine serum albumin used as a standard. Protein samples (50 g) were boiled with 2× sample buffer containing 5% -mercaptoethanol for 5 min, separated by size on 15% polyacrylamide gel under SDS denaturing conditions, and transferred to a nitrocellucose membrane at 90 V for 2 h. Non-specific binding was blocked by incubation in block buffer (5% non-fat dry milk, 0.05% Tween-20, 1 × TBS) overnight at 4 ◦ C, the membranes were hybridized with a 1:1000 dilution of monoclonal antibody mouse anti-rat 3-NT and anti-rat PKC, then incubated with a horseradish peroxidase-labeled rabbit antimouse IgG (1:500). The bound secondary antibody was detected by enhanced chemiluminescence. Positive immunoreactive bands were quantified densitometrically (Leica Q500IW image analysis system). Relative quantities were compared normalized to control values, arbitrarily assigned as 100%. Housekeeping protein -actin was used as a loading control. Positive immunoreactive bands were quantified densitometrically (Leica Q500IW image analysis system) and expressed as ratio of 3-NT and PKC to -actin in optical density units. Immunohistochemistry Immunostaining of TGF-1 in renal tissue sections was conducted using SABC method. The primary antibodies that were used a polyclonal rabbit anti-TGF-1 antibody (diluted 1:200). Imunostaining of TGF-1 in glomeruli was evaluated using the following semiquantitative scale: 0 = diffuse, very weak or absent staining; 1 = staining involving less than 25%; 2 = staining involving 25–50%; 3 = staining involving 50–75% and 4 = staining involving 75–100%. Immunostaining of TGF-1 in tubulointerstitium was quantified using computerized image analysis system (Beijing Aeronautic and Aerospace University, Beijing, China) by evaluating the positively stained area of the sections under the same light intensity for microscopy. All scoring was performed on blinded slides. Statistical analysis
Assay for PKC activities Membrane and cytosolic frctions in renal tissue were obtained by the method described by Kikkawa (Kikkawa et al., 1994). PKC activities was determined with a method described by Heasley and Johonson (Heasley et al., 1989). Briefly, membrane and cytosolic fractions were preincubated with the salt solution for 10 min at 37 ◦ C and incubated for another 15 min in the presence or absence of 100 M PKC-specific peptide substrate, subsequently with 50 g/ml digitonin and 100 M ATP mixed with [␥-32 P]ATP (<1500 cpm/pmol). The reaction was terminated with 5% TCA (final concentration). Aliquots of the reaction mixture were spotted on 3 cm × 3 cm phosphocellulose papers and washed with three changes of 75 mM phosphoric acid and one change of 75 mM sodium phosphate (pH 7.5). The radioactivities of phosphorylated substrate was determined by liquid scintillation counting. Results in PKC activities were expressed as pmol/mg min. The protein content was estimated by the dye binding assay of Bradford (Bradford et al., 1976), with bovine serum albumin used as a standard. Western blot analysis Kidney samples were homogenized in RIPA buffer [PBS, 1% nonidet P-40 (NP-40), 0.5% sodium deoxycholate, 0.1% sodium dodecyl
Data were expressed as the mean ± S.E.M. One-way analysis of variance (ANOVA) with pairwise comparisons according to the Tukey method was used in this study. Since urinary AER followed a nonnormal distribution, log transformation was performed prior to statistical analysis of this parameter. Differences were considered significant if the p value was less than 0.05. Results Clinical and metabolic parameters Rats in group DM had increased BG levels. No effects on BG were observed with monotherapy treatment or enalapril and breviscapine combination treatment. As shown previously, kidney enlargement as increased KW and KW/BW was observed in group DM, which was reduced by treatment with both enalapril and breviscapine, but its level was higher than that in control rats. The level of KW/BW in group DM treated with combination of enalapril and brevisapine was significantly lower than that in the groups DM treated with enalapril or breviscapine alone. Treatment with both enalapril and breviscapine attenuated the increase in albuminuria in the diabetic rats, but this level was still higher than that observed in control rats. The combination of enalapril and breviscapine was
Please cite this article in press as: Xu, X.-X., et al., Superior renoprotective effects of the combination of breviscapine with enalapril and its mechanism in diabetic rats. Phytomedicine (2013), http://dx.doi.org/10.1016/j.phymed.2013.03.027
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Table 1 ¯ ± s, n = 10). Change of clinical and metabolic parameters in five groups ( Groups
Blood glucose (mmol/l)
C DM DM + E DM + B DM + E + B
6.03 28.20 26.37 25.94 24.55
± ± ± ± ±
0.42 1.26** 1.59 2.52 1.50
Body weight (g) 458.75 221.13 329.87 253.38 321.27
± ± ± ± ±
Kidney weight (g)
27.32 22.81** 15.39 37.59 23.21
1.81 2.29 1.98 1.85 1.78
± ± ± ± ±
0.07 0.12* 0.10# 0.09# 0.07#
Relative kidney (×10−3 ) 4.20 9.38 6.09 7.04 5.52
± ± ± ± ±
0.42 0.84** 0.36# 0.35# 0.20## ,
AER (mg/24 h) 0.48 ×/÷ 1.3 1.26 ×/÷ 1.1** 0.86 ×/÷ 1.1# 0.68 ×/÷ 1.1# 0.54 ×/÷ 1.2## ,
AER was expressed as geometric mean ×/÷ tolerance factor. * p < 0.05. ** p < 0.01 vs C. # p < 0.05. ## p < 0.01 vs DM. p < 0.05 vs DM + B or DM + E.
Table 2 ¯ ± s, n = 10). Change of morphologic parameters in the glomeruli and tubulointerstitial injury index in five groups ( Groups
AG (m2 )
C DM DM + E DM + B DM + E + B
3962.30 5786.22 4611.26 4701.26 4082.30
* ** # ##
± ± ± ± ±
193.18 219.37** 237.58# 224.87# 226.84## ,
VG (106 m3 )
AM (m2 )
± ± ± ± ±
676.82 1366.33 968.00 1066.08 854.17
1.02 1.52 1.21 1.23 1.06
0.11 0.11* 0.12# 0.11# 0.08## ,
± ± ± ± ±
TII 13.48 23.32** 17.63# 18.16# 22.48## ,
0.38 0.84 0.59 0.62 0.48
± ± ± ± ±
0.14 0.25* 0.19 0.18 0.14#
p < 0.05. p < 0.01 vs C. p < 0.05. p < 0.01 vs DM. p < 0.05 vs DM + B or DM + E.
associated with a further reduction in albuminuria than was seen with either drug administrated alone, the similar AER level to that observed in control animals (Table 1). Renal histology Rats in group DM had an increase in AG , VG and AM when compared with the values in group C, treatment with enalapril and breviscapine significantly ameliorated the increase of AG , VG and
AM . The combination of enalapril and breviscapine was associated with a further reduction in AG , VG and AM than was seen with either drug administrated alone. Rats in group DM had an increase in TII when compared to group C, treatment with enalapril and breviscapine was associated with a reduction in TII as compared with group DM, but this did not reach statistical significance. However, the combination of enalapril and breviscapine was associated with a further reduction in TII when compared to untreated diabetic rats (Fig. 2 and Table 2).
Fig. 2. A representative microphotograph of kidney tissue stained with Periodic acid-Schiff from C (A), DM (B), DM + E (C), DM + B (D) and DM + E + B (E) rats. Original magnification 400×.
Please cite this article in press as: Xu, X.-X., et al., Superior renoprotective effects of the combination of breviscapine with enalapril and its mechanism in diabetic rats. Phytomedicine (2013), http://dx.doi.org/10.1016/j.phymed.2013.03.027
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Fig. 3. Western blot analysis of PKC protein (A) and densitometric analysis (B) in renal tissue in C, DM, DM + E, DM + B and DM + E + B. Values are the means ± S.E.M. **p < 0.01 vs C, # p < 0.05, ## p < 0.01 vs DM, p < 0.05 (s DM + B or DM + E.
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Fig. 4. Western blot analysis of 3-NT protein (A) and densitometric analysis (B) in renal tissue in C, DM, DM + E, DM + B and DM + E + B. Values are the means ± S.E.M. **p < 0.01 vs C, # p < 0.05, ## p < 0.01 vs DM, p < 0.05 (s DM + B or DM + E.
PKC activities
Determination of MDA The concentration of MDA in renal tissue and urine was significantly higher in the group DM when compared with group C, treatment with enalapril and breviscapine significantly ameliorated the increase of MDA in renal tissue and urine. The combination of enalapril and breviscapine was associated with a further reduction in MDA in renal tissue and urine than with either drug administrated alone (Table 3).
Compared with group C, PKC activities in the cytosolic fraction (PKCc), the membrane fraction (PKCm) and the ratio of the PKC activities in the membrane fraction to cytosolic fraction (PKCm/PKCc) were significantly elevated in renal tissue from group DM, suggesting that diabetic state induced renal PKC activation. Increased PKC activities was effectively inhibited by enalapril or breviscapine administration. The combination of enalapril and breviscapine was associated with a further reduction in PKC activities in renal tissue than with either drug administrated alone (Table 4).
Renal 3-NT expression Renal PKC expression Fig. 3 showed that renal 3-NT expression in diabetic rats was increased compared with control animals. This increased expression of 3-NT in diabetic animals was significantly reduced by treatment with enalapril and breviscapine. The combination of enalapril and breviscapine was associated with a reduction in 3-NT expression when compared to diabetic rats treated with enalapril or breviscapine.
TGF-ˇ1 immunohistochemistry
Table 3 ¯ ± s, n = 10). Change of MDA level in renal tissue and urine in five groups ( Groups
Renal MDA (nmol/mg prot)
C DM DM + E DM + B DM + E + B
1.74 3.60 2.72 2.51 2.17
** # ##
± ± ± ± ±
0.07 0.08** 0.04# 0.04# 0.05## ,
TGF-1 immunostaining was observed in the glomerulus and tubulointerstitium in control rats. Minimal immunostainable TGF1 was present in the kidneys of control rats. In contrast, abundant TGF-1 was expressed in the kidney of diabetic rats. This overexpression was attenuated in diabetic rats treated with enalapril and breviscapine. The combination of enalapril and breviscapine result in further reduction in renal TGF-1 expression when compared to diabetic rats treated with enalapril or breviscapine (Fig. 5 and Table 5).
Urine MDA (nmol/24 h) 240.66 769.47 554.62 580.36 344.72
± ± ± ± ±
Western blot analysis demonstrated increased PKC expression in the kidneys of diabetic compared with control animals. This increased expression of PKC in diabetic animals was significantly reduced by treatment with enalapril and breviscapine. The combination of enalapril and breviscapine was associated with a reduction in PKC expression when compared to diabetic rats treated with enalapril or breviscapine (Fig. 4).
11.37 58.64** 47.067# 25.71# 26.77## ,
p < 0.01 vs C. p < 0.05. p < 0.01 vs DM. p < 0.05 vs DM + B or DM + E.
Table 4 ¯ ± s, n = 10). Change of PKC activity in renal tissue in five groups ( Groups C DM DM + E DM + B DM + E + B * ** # ##
PKCt (pmol/min mg) 9.83 34.34 18.45 14.38 12.46
± ± ± ± ±
0.97 4.75** 1.82# 1.70# 1.23#
PKCc (pmol/min mg) 7.57 16.70 13.19 9.50 9.73
± ± ± ± ±
0.95 2.44** 1.86# 1.63# 1.74#
PKCm (pmol/min mg) 2.26 17.65 5.26 4.88 2.72
± ± ± ± ±
0.33 2.25** 0.54# 0.41# 0.36## ,
PKCm/PKCc 0.23 0.51 0.40 0.34 0.28
± ± ± ± ±
0.03 0.02* 0.03# 0.04# 0.02## ,
p < 0.05. p < 0.01 vs C. p < 0.05. p < 0.01 vs DM. p < 0.05 vs DM + B or DM + E.
Please cite this article in press as: Xu, X.-X., et al., Superior renoprotective effects of the combination of breviscapine with enalapril and its mechanism in diabetic rats. Phytomedicine (2013), http://dx.doi.org/10.1016/j.phymed.2013.03.027
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Table 5 ¯ ± s, n = 10). Change of TGF1 immunohistochemistry in renal tissue in five groups ( Groups
Glomeruli score/gcs
C DM DM + E DM + B DM + E + B
0.66 2.18 1.46 1.32 0.84
** # ##
± ± ± ± ±
0.12 0.18** 0.13# 0.16# 0.14## ,
tubulointerstitium % 5.8 21.2 12.8 13.8 7.8
± ± ± ± ±
0.6 2.4** 1.6# 01.4# 0.7## ,
p < 0.01 vs C. p < 0.05. p < 0.01 vs DM. p < 0.05 vs DM + B or DM + E.
Discussion Our study shows that the combination of an agent that interrupts the renin-angiotensin system with breviscapine provides superior renoprotection in a model of experimental diabetes than either agent alone. This has been shown for a range of functional and structural parameters including albuminuria, glomerular and tubulointerstitial injury. These observations should be considered in the context of the beneficial effects of both ACE and breviscapine on the progression of renal damage in the experimental context. The pathogenesis of diabetic nephropathy is complex and involves both glucose-dependent and glucose-independent pathways. In both type 1 and type 2 diabetes, the degree of hyperglycemia influences both the likelihood of developing nephropathy and the rate of its progression (Nalysnyk et al. 2010; Stratton et al. 2000). High intracellular glucose concentrations, per se, may lead to activation of PKC and in particular the ( isoform, which has been shown to be activated in the glomeruli in experimental diabetes (Kelly et al., 2003; Wu et al., 2009). However, in addition to these glucose-dependent mechanisms, other glucose-independent components of the diabetic state contribute to the development and progression of diabetic nephropathy. In particular, both experimental and clinical studies indicate that
hypertension and inappropriate activation of the RAS are likely key contributors. Both Ang II, the effector molecule of the RAS, and cell stretch, the in vitro counterpart of hypertension, activate PKC (Feng et al. 1998). Previous studies showed that increasing PKC activation by PDBu, a diacylglycerol analogue, mimicked the effect of high glucose on Ang II production and that a PKC inhibitor totally abolished the effect of glucose on Ang II without a significant change in Ang II production in normal glocose (Ikehara et al. 2003). These results suggest that high glucose levels stimulate Ang II production through glucose induced PKC activation in human MCs. Since it is well established that Ang II increases inositol triphosphate and diacylglycerol via activation of phospholipase C, leading to stimulate PKC activity. It has been reported that high glucose milieu of diabetes increases Ang II generation via PKC activation, in turn increased Ang II synergistically stimulates PKC activation in an autocrine fashion in MCs. In the present study, enalapril and breviscapine significantly attenuated the structural and functional manifestations of diabetic renal injury along with a reduction in the overexpression of the profibrotic growth factor TGF-1. Furthermore, the combination of enalapril and breviscapine was associated with a further attenuation of reduction in albuminuria, MDA, PKC and TGF-1 in the kidney of experimental diabetes. The finding that these beneficial changes occurred despite the continued presence of hyperglycemia is consistent with PKC activation and activation of the RAS as a final common pathway for these pathogenetic attributes of the diabetic milieu. Elevated oxidative stress plays an important role in the progression of diabetes and its complications. High glucose increases ROS to enhance oxidative stress in tissue via a series of pathwaysg (Forbes et al. 2008; Gao and Mann 2009; Kashihara et al. 2010), ROS may attack the unsaturated fatty acid in the biomembrane to yield lipid peroxidation such as MDA, ketone and hydroxyl which amplify oxidative stress to promote the progression of diabetic complications (Zhu et al. 2005). ROS may also increase NF- B and TGF-1 which lead to deposition of extracellular matrix and tissue fibrosis. Increased lipid peroxidation and decreased antioxidant enzymes have been reported in kidney tissue of STZ-induced diabetic rats (Kitada et al. 2011). However, due to the abundant blood flow in
Fig. 5. A representative microphotograph of immunostaining for TGF1 in renal tissue from C (A), DM (B), DM + E (C), DM + B (D) and DM + E + B (E) rats. Negative control (F). Original magnification 400×.
Please cite this article in press as: Xu, X.-X., et al., Superior renoprotective effects of the combination of breviscapine with enalapril and its mechanism in diabetic rats. Phytomedicine (2013), http://dx.doi.org/10.1016/j.phymed.2013.03.027
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kidney tissue, oxidative substance and antioxidant enzymes in blood may affect glomerular endothelial cells directly, infiltrate into the mesangial area, tubular interstitial area and other parts of renal tissue, which may play an important role in oxidative injury to kidney tissue. A previous report has demonstrated that increased lipid peroxidation and decreased antioxidant enzymes which may impair kidney tissue existed in plasma of patients with chronic glomerulonephritis. The results presented in our study imply that in STZ-induced diabetic rats MDAr and MDAu are increased. A growing body of study have shown oxidant stress is increased in clinical and experimental diabetic nephropathy (Giacco and Brownlee 2010), and our present study, in addition to confirming their findings, have demonstrated elevated MDA level in renal tissue was reduced by enalapril or breviscapine and, more effectively, by combined enalapril with breviscapine in diabetic rats. This effect of combined enalapril with breviscapine was likely the result of suppression of PKC. Lately, the reactive nitrogen species originate from NO have attracted vast attention as a new pathway of oxidative stress. NO has a high affinity for SOD, and their interaction form the extremely strong and reactive oxidant peroxynitrite (ONOO ). Because the production of ONOO is difficult to determine, the assay of 3-NT in protein has been proposed as an indirect marker of ONOO production. Our present study showed the elevated 3-NT in diabetic rats was reduced by enalapril or breviscapine and, more effectively, by combined enalapril with breviscapine in diabetic rats. The renal structural injury observed in the diabetic rats, as assessed in both the glomerulus and tubulointerstitium, was reduced by both treatments as monotherapy, with an additional effect observed with the combination when compared to either enalapril or breviscapine treatment alone. The reduction in glomerular and tubulointerstitial injury was observed in association with a reduction in the expression of the prosclerotic cytokine, TGF1 in all three treatment groups. Increased ROS generation, protein kinase C activation, angiotensin II in association with increased glucose metabolism and hemodynamic disorder are considered to be the main upstream signaling molecules of diabetic-induced renal injury. Both ACE and PKC inhibition reduce the degree of glomerulosclerosis possibly via a reduction in the expression of prosclerotic cytokines such as TGF1. Our results further support to the concept that both AII and PKC formation are implicated in the progression of glomerular structural injury via a common cytokine pathway such as TGF1. Because combination therapy was correlated with synergetic suppression on overexpression of PKC, 3-NT, MDA and TGF1 in renal tissue in diabetic rats, the production of TGF-ß1 may have more effectively been inhibited, resulting in prevention of diabetic nephropathy progression. The magnitude of tubulointerstitial injury is an important prognostic marker of renal outcome in many forms of renal disease. In human diabetic nephropathy, the extent of interstitial fibrosis is strongly associated with mesangial expansion, falling GFR, and increasing proteinuria. In experimental diabetic nephropathy, investigation has focused almost exclusively on the glomerulus and particularly on the mesangial cell, although tubulointerstitial disease also develops in the streptozotocin model. Accumulation of extracellular matrix, first recognized as thickening of capillary basement membranes, is a characteristic pathologic feature of diabetes and is present in the tubulointerstitial as well as the glomerulus Enalapril and breviscapine were not only associated with a decrease in glomerulosclerosis, but also had effects on reducing tubulointerstitial injury. The tubulointerstitium is now considered a major site of diabetes-related injury and is an important determinant of the rate of decline in renal function in diabetic nephropathy. Combination therapy was associated with a further reduction in tubulointerstitial injury.
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This study contributes to our understanding of the multifactorial nature of diabetic nephropathy and implies that therapeutic intervention for this condition may require an approach involving different agents to inhibit the various putative pathogenic pathways. Presumably such an approach could include both PKC and ACE inhibition. Acknowledgments The research presented in this paper was financially supported by Doctoral Fund of Ministry of Education of China (No. 20093420110003) and Anhui Provincial University Key Project of Natural Science (No. KJ2010A191). References Ali, A., Gan, L., Liu, Q., Liu, H., Xu, H.B., 2003. Antioxidant effect of scutellarin and its antagonistic action on the liver toxicity of se in rats. Chinese Pharmacological Bulletin 19, 113–115. Brownlee, M., 2001. Biochemistry and molecular cell biology of diabetic complications. Nature 414, 813–820. Chen, X.X., He, B., Chen, Y.Y., 1998. Mechanisms of inhibitory effects of breviscapine on lipid peroxidation in rat brain mitochondria. Journal of Chinese Pharmaceutical Science 7, 209–213. Feng, X., Zhang, J., Barak, L.S., Meyer, T., Caron, M.G., Hannun, Y.A., 1998. Visualization of dynamic trafficking of a protein kinase c betaii/green fluorescent protein conjugate reveals differences in g protein-coupled receptor activation and desensitization. Journal of Biological Chemistry 273, 10755–10762. Ferroni, P., Basili, S., Falco, A., Davi, G., 2004. Platelet activation in type 2 diabetes mellitus. Journal of Thrombosis and Haemostasis 2, 1282–1291. Forbes, J.M., Coughlan, M.T., Cooper, M.E., 2008. Oxidative stress as a major culprit in kidney disease in diabetes. Diabetes 57, 1446–1454. Gao, L., Mann, G.E., 2009. Vascular nad(p)h oxidase activation in diabetes: a doubleedged sword in redox signaling. Cardiovascular Research 82, 9–20. Giacco, F., Brownlee, M., 2010. Oxidative stress and diabetic complications. Circulation Research 107, 1058–1070. Ha, H., Yu, M.R., Choi, Y.J., Kitamura, M., Lee, H.B., 2002. Role of high glucose-induced nuclear factor-kappab activation in monocyte chemoattractant protein-1 expression by mesangial cells. Journal of the American Society of Nephrology 13, 894–902. Ikehara, K., Tada, H., Kuboki, K., Inokuchi, T., 2003. Role of protein kinase cangiotensin ii pathway for extracellular matrix production in cultured human mesangial cells exposed to high glucose levels. Diabetes Research and Clinical Practice 59, 25–30. Jandeleit-Dahm, K., Cooper, M.E., 2006. Hypertension and diabetes: role of the reninangiotensin system. Endocrinology and Metabolism Clinics of North America 35, 469–490, vii. Jia, Z.H., Liu, Z.H., Zheng, J.M., Zeng, C.H., Li, L.S., 2007. Combined therapy of rhein and benazepril on the treatment of diabetic nephropathy in db/db mice. Experimental and Clinical Endocrinology & Diabetes 115, 571–576. Kashihara, N., Haruna, Y., Kondeti, V.K., Kanwar, Y.S., 2010. Oxidative stress in diabetic nephropathy. Current Medicinal Chemistry 17, 4256–4269. Kelly, D.J., Zhang, Y., Hepper, C., Gow, R.M., Jaworski, K., Kemp, B.E., WilkinsonBerka, J.L., Gilbert, R.E., 2003. Protein kinase c beta inhibition attenuates the progression of experimental diabetic nephropathy in the presence of continued hypertension. Diabetes 52, 512–518. Kitada, M., Kume, S., Imaizumi, N., Koya, D., 2011. Resveratrol improves oxidative stress and protects against diabetic nephropathy through normalization of mnsod dysfunction in ampk/sirt1-independent pathway. Diabetes 60, 634–643. Lu, Y.X., Zhang, Q., Li, J., Sun, Y.X., Wang, L.Y., Cheng, W.M., Hu, X.Y., 2010. Antidiabetic effects of total flavonoids from litsea coreana leve on fat-fed, streptozotocin-induced type 2 diabetic rats. American Journal of Chinese Medicine 38, 713–725. Najafian, B., Alpers, C.E., Fogo, A.B., 2011. Pathology of human diabetic nephropathy. Contributions to Nephrology 170, 36–47. Nalysnyk, L., Hernandez-Medina, M., Krishnarajah, G., 2010. Glycaemic variability and complications in patients with diabetes mellitus: evidence from a systematic review of the literature. Diabetes, Obesity & Metabolism 12, 288–298. Noh, H., King, G.L., 2007. The role of protein kinase c activation in diabetic nephropathy. Kidney International. Supplement, S49–S53. Qi, X.M., Wu, G.Z., Wu, Y.G., Lin, H., Shen, J.J., Lin, S.Y., 2006. Renoprotective effect of breviscapine through suppression of renal macrophage recruitment in streptozotocin-induced diabetic rats. Nephron. Experimental Nephrology 104, e147–e157. Schena, F.P., Gesualdo, L., 2005. Pathogenetic mechanisms of diabetic nephropathy. Journal of the American Society of Nephrology 16 (Suppl. 1), S30–S33. Shuai, J., Dong, W.W., 1998. Experimental research of pkc inhibitor, erigeron breviscapus on the ischemic/reperfusion brain injury. China Pharmacology Bulletin 14, 75–77.
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Superior renoprotective effects of the combination of breviscapine with enalapril and its mechanism in diabetic rats Xing-Xin Xu a , Wei Zhang a , Pei Zhang a , Xiang-Ming Qi a , Yong-Gui Wu a,∗ , Ji-Jia Shen b a b
Department of Nephrology, The First Affiliated Hospital, Anhui Medical University, Hefei, Anhui, PR China Department of Pathobiology, Anhui Medical University, Hefei, Anhui, PR China
a r t i c l e
i n f o
Keywords: Diabetic nephropathy Protein kinase C Breviscapine Enalapril Transforming growth factor 1
a b s t r a c t Breviscapine is a flavonoid extracted from a Chinese herb Erigeron breviscapus, previously it was shown that treatment with breviscapine attenuated renal injury in the diabetic rats. The purpose of this study was to investigate whether breviscapine combined with enalapril (an ACE inhibitor) have superior renoprotective effects against diabetic nephropathy. Rats were randomly separated into five groups: control, diabetes, diabetes treated with enalapril, diabetes treated with breviscapine, or diabetes treated with combined enalapril with breviscapine. Twenty-four hours urinary AER and the levels of 3-NT in renal tissue and MDA in renal tissue and urine as well as activities and expression of PKC in renal tissue were determined, and renal tissue morphology were observed by light microscopy after 8 weeks. Expression of TGF1 protein was performed by immunohistochemistry method. Increased AER and kidney pathologic injury were attenuated by treatment with either enalapril or breviscapine and further reduced by the combination of the two. Elevated 3-NT in renal tissue and MDA levels in renal tissue and urine were reduced by enalapril or breviscapine and, more effectively, by combined enalapril with breviscapine. PKC activities and expression were higher in renal tissue in diabetic rats than those of the control group, which were reduced by both monotherapies, and further abrogated by combination therapy in both cases. Overexpression of TGF1 protein observed in the glomeruli and tubulointerstitium of diabetic rats was attenuated by enalapril or breviscapine to a similar lever and further reduced by the combination of the two. The combination of enalapril and breviscapine confers superiority over monotherapies on renoprotection, which mechanism may be at least partly correlated with synergetic suppression on increased oxidative stress and PKC activities as well as overexpression of TGF1 in renal tissue. © 2013 Elsevier GmbH. All rights reserved.
Introduction DN, which is one of the most serious complications of diabetes and the number one cause of renal failure in the industrialized world, is characterized by albuminuria and enlargement of the glomerular mesangium due to the accumulation of extracellular matrix proteins (Najafian et al. 2011). Experimental and clinical studies in subjects with type 1 and type 2 diabetes clearly link hyperglycemia to vascular complications, including diabetic nephropathy. Hyperglycemia is responsible for the development
Abbreviations: DN, diabetic nephropathy; STZ, streptozotocin; MDA, malondialdehyde; 3-NT, 3-nitrotyrosine; AER, albumin excretion rate; PKC, protein kinase C; TGF1, transforming growth factor 1; ACE, Angiotensin-converting enzyme; ROS, Reactive oxygen species; ICAM-1, intercellular adhesion molecule-1; MCP1, monocyte chemoattractant protein-1; AG , glomerular cross-sectional area; AM , mesangial area; VG , glomerular volume; KW, kidney weight; BW, body weight; TII, tubulointerstital injury; SABC, Streptavidin-biotin-peroxidase complex. ∗ Corresponding author. Tel.: +86 551 6292 2450; fax: +86 551 6363 3742. E-mail address: [email protected] (Y.-G. Wu).
and progression of diabetic nephropathy through metabolic derangements, including increased oxidative stress, renal polyol formation, activation of PKC-mitogen-activated protein kinases, and accumulation of advanced glycation end products, as well as such hemodynamic factors as systemic hypertension and increased intraglomerular pressure (Brownlee 2001; JandeleitDahm and Cooper 2006; Schena and Gesualdo 2005; Tan et al. 2007; Wolf 2004). Amelioration of albuminuria and glomerular mesangium enlargement are now considered one of the targets in the prevention and retardation of DN (Turgut and Bolton 2010). ACE inhibitors as an accepted renoprotection drug, can attenuate hyperfiltration and hyperpressure in glomeruli and has been proved effective in reducing albuminuria, diminishing loss of kidney function and improving survival both in patients with DN and animal models through a hemodynamic pathway. The advent of ACE inhibitors has changed profoundly the therapeutic approach to diabetic nephropathy, and may improve the survival and the quality of life of diabetic patients significantly. Breviscapine extracted from a Chinese herb Erigeron breviscapus have been previously demonstrated in diabetic patients the ability to reduce albuminuria.
0944-7113/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymed.2013.03.027
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Fig. 1. The chemical structure of Breviscapine.
The active ingredient is a mixture as shown in Fig. 1 (Zhang et al. 1988). Breviscapine possesses a variety of pharmacological functions beyond hemodynamic effects, especially as anti-oxidative stress agent and PKC inhibitor (Ali et al. 2003; Chen et al. 1998; Shuai and Dong 1998). ROS generation and PKC activation are elevated in mesangial cells cultured under high glucose conditions (Ha et al. 2002), the kidneys of experimental diabetic animals also exhibit increased lipid peroxidation, which is a marker of increased ROS generation and PKC activation (Ferroni et al. 2004). Furthermore, antioxidants and PKC inhibitor have been reported to prevent or attenuate both glucose-induced mesangial cell activation and renal injury in diabetes (Lu et al. 2010; Noh and King 2007; Waisundara et al. 2008). We have proved that breviscapine can ameliorate renal injury in STZ diabetic rat in vivo. Several of our past studies have shown clearly that breviscapine can regulate expression of ICAM-1, MCP-1 and antagonize the activity of PKC (Qi et al. 2006). Renal protection with either an ACE inhibitor or an anti-oxidative stress agent and PKC inhibitor as monotherapy is suboptimal. The observation that a substantial fraction of diabetic subjects responds poorly to these treatments underscores the need for additional therapeutic maneuvers capable of halting the progression to end-stage renal failure. In a rat model of passive Heymann nephritis, addition of simvastatin to the ACE inhibitor, enalapril, resulted in greater reduction in proteinuria than was seen with enalapril alone (Zoja et al. 2002). Recently, we and others studies demonstrated that combination therapy is superior than either therapy alone in STZ diabetic rat (Jia et al. 2007; Wu et al. 2006a,b). These data suggest that a therapy combining an ACE inhibitor with an anti-oxidative stress agent and PKC inhibitor may be useful in progressive renal disease. In the present study, we evaluated the renal effects of a combination of an ACE inhibitor with breviscapine in experimental diabetes. In addition, we explored the effects of these therapies on protein expression of TGF-1, which was considered to be important mediators of injury leading to kidney injury in diabetes.
Reagents Breviscapine (containing flavone 4.5 mg/20 mg) was supplied by Yuxi Pharmaceutical Co. Ltd. (Kunming, China). STZ was purchased from Sigma Chemical Co. (St. Louis, Mo, USA). Microalbumin assay kit was purchased from Exocell Inc. (Philadelphia, PA, USA). MDA assay kits were the product of Nanjing Jiancheng Bioengineering Institute (Nanjing, China). PKC activities assay kit was purchased from Calbiochem Co. (La Jolla, CA, USA). [␥-32 P]ATP was purchased from Beijing YaHui Bioengineering Institute (Beijing, China). Anti 3-NT monoclonal antibody was purchased from Upstate Technology (Lake Placid, NY, USA). Anti-PKC monoclonal antibody and anti-TGF-1 polyclonal antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Chemiluminescence kit was from Amersham Life Science (Little Chalfont, UK). SABC kit was from Bosten Biotechnique Co. (Wuhan, China). Experimental protocol Diabetic was induced by single injection of STZ at a dose of 65 mg/kg IP, diluted in citrate buffer 0.1 mol/l (pH 4.0). Two days later, the diabetic state was confirmed by measurement of tail blood glucose (BG) level. Diabetic rats received daily injections of NPH insulin, in doses adjusted individually (ranging from 1 to 4 units) to maintain BG levels between 200 mg/dl and 400 mg/dl, and to avoid ketonuria. BG level were measured twice a week. Five experimental groups were studied and treated for 8 weeks. The groups are as follows: control with no treatment (group C, n = 10); diabetes with no treatment (group DM, n = 10); diabetes treated with enalapril, at a dose of 10 mg/kg body weight/day by gavage (group DM + E, n = 10), diabetes treated with breviscapine, at a dose of 20 mg/kg body weight/day by gavage (group DM + B, n = 10), diabetic rats treated with the combination of both enalapril and breviscapine at the doses described above (group DM + E + B, n = 10). In preliminary experiments, these were found to be the suitable dose that the rats would tolerate without losing weight or showing deterioration of their general condition.
Materials and methods Metabolic parameters and tissue collection Animals Adult male munich-wistar rats, with initial weights of 180–200 g (Grade II, Certificate No. 01) were obtained from Experimental Animal Center of Anhui Medical University. The research protocol was in accordance with the principles approved by the animal ethics committee of Anhui Medical University. Animals were housed at a temperature of 24 ± 1 ◦ C and humidity of 65–70%, and were submitted to a 12 h light/dark cycle, and allowed free access to standard laboratory chow and tap water.
BW was measured at the conclusion of the experiment. Rats were then anesthetized with sodium pentobarbital (50 mg/kg IP) and placed on a temperature-regulated table, the right jugular artery was catheterized, this arterial catheter was used for blood sampling. BG levels were determined with a glucose analyzer. The kidneys were perfused in vivo via the abdominal aorta with 100 ml of normal saline at 4 ◦ C, while the left renal vein was punctured to permit the perfusate to drain, the kidneys were removed immediately and fixed in 10% formalin and processed in paraffin for
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subsequent histologic assessment and immunohiatochemical studies. The remaining kidney was stored at −70 ◦ C for evaluation of oxidative stress parameters, PKC activities and Western blot analysis. Urinary albumin excretion Prior to sacrifice, animals were placed in metabolic cages for collection of urine over 24 h for measurement of albumin concentrations. Urinary albumin concentrations were measured by enzyme-linked immunoabsorbent assay using an anti-rat albumin antibody and 24 h urinary albumin excretion was calculated by multiplying the urinary protein excretion by the 24 h urine volume. Renal pathology Formalin-fixed kidney sections (2 m) were stained with PAS reagent to identify kidney structure and hematoxylin to distinguish cell nuclei. Digital images of glomeruli and interstitial areas were obtained from mioroscopy (magnification, 400×). The AG and AM were measured in 50 glomerutar profiles per rat by using computerized image analysis system (Beijing Aeronautic and Aerospace University, Beijing, China). The VG was then calculated as: VG = ˇ/K[AG I3/2 ], where ˇ = 1.38 is the size distribution coefficient and K = 1.1 is the shape coefficient for glomeruli idealized as a sphere. Tubulointerstitial area in the cortex was evaluated and graded as: 0, normal; 1, the area of interstitial inflammation and fibosis, tubular atrophy and dilation with cast formation involving <25% of the field; 2, lesion area between 25% and 50% of the field; and 3, lesions involving >50% of the field. The indices for TII were calculated by averaging the grades assigned to all tubule fields. All measurements and scoring were performed on blinded slides. Determination of MDA MDA was determined according to the manufacturer’s protocol. Results in urinary MDA were expressed as nmol of MDA in 24 h, results in renal MDA levels were expressed as nmol MDA per milligram protein (nmol/mg prot). The protein content was estimated by the dye binding assay of Bradford, with bovine serum albumin used as a standard.
3
sulfate (SDS), 100 g/ml aprotinin, 100 g/ml phenylmethylsulfonyl fluoride (PMSF), sodium orthovanadate] at 4 ◦ C throughout all procedures, and sonicated for 70 s, then add 300 g PMSF per gram of tissue and incubate on ice for 30 min, followed by centrifugation at 15,000 rpm for 20 min at 4 ◦ C. The protein content was estimated by the dye binding assay of Bradford, with bovine serum albumin used as a standard. Protein samples (50 g) were boiled with 2× sample buffer containing 5% -mercaptoethanol for 5 min, separated by size on 15% polyacrylamide gel under SDS denaturing conditions, and transferred to a nitrocellucose membrane at 90 V for 2 h. Non-specific binding was blocked by incubation in block buffer (5% non-fat dry milk, 0.05% Tween-20, 1 × TBS) overnight at 4 ◦ C, the membranes were hybridized with a 1:1000 dilution of monoclonal antibody mouse anti-rat 3-NT and anti-rat PKC, then incubated with a horseradish peroxidase-labeled rabbit antimouse IgG (1:500). The bound secondary antibody was detected by enhanced chemiluminescence. Positive immunoreactive bands were quantified densitometrically (Leica Q500IW image analysis system). Relative quantities were compared normalized to control values, arbitrarily assigned as 100%. Housekeeping protein -actin was used as a loading control. Positive immunoreactive bands were quantified densitometrically (Leica Q500IW image analysis system) and expressed as ratio of 3-NT and PKC to -actin in optical density units. Immunohistochemistry Immunostaining of TGF-1 in renal tissue sections was conducted using SABC method. The primary antibodies that were used a polyclonal rabbit anti-TGF-1 antibody (diluted 1:200). Imunostaining of TGF-1 in glomeruli was evaluated using the following semiquantitative scale: 0 = diffuse, very weak or absent staining; 1 = staining involving less than 25%; 2 = staining involving 25–50%; 3 = staining involving 50–75% and 4 = staining involving 75–100%. Immunostaining of TGF-1 in tubulointerstitium was quantified using computerized image analysis system (Beijing Aeronautic and Aerospace University, Beijing, China) by evaluating the positively stained area of the sections under the same light intensity for microscopy. All scoring was performed on blinded slides. Statistical analysis
Assay for PKC activities Membrane and cytosolic frctions in renal tissue were obtained by the method described by Kikkawa (Kikkawa et al., 1994). PKC activities was determined with a method described by Heasley and Johonson (Heasley et al., 1989). Briefly, membrane and cytosolic fractions were preincubated with the salt solution for 10 min at 37 ◦ C and incubated for another 15 min in the presence or absence of 100 M PKC-specific peptide substrate, subsequently with 50 g/ml digitonin and 100 M ATP mixed with [␥-32 P]ATP (<1500 cpm/pmol). The reaction was terminated with 5% TCA (final concentration). Aliquots of the reaction mixture were spotted on 3 cm × 3 cm phosphocellulose papers and washed with three changes of 75 mM phosphoric acid and one change of 75 mM sodium phosphate (pH 7.5). The radioactivities of phosphorylated substrate was determined by liquid scintillation counting. Results in PKC activities were expressed as pmol/mg min. The protein content was estimated by the dye binding assay of Bradford (Bradford et al., 1976), with bovine serum albumin used as a standard. Western blot analysis Kidney samples were homogenized in RIPA buffer [PBS, 1% nonidet P-40 (NP-40), 0.5% sodium deoxycholate, 0.1% sodium dodecyl
Data were expressed as the mean ± S.E.M. One-way analysis of variance (ANOVA) with pairwise comparisons according to the Tukey method was used in this study. Since urinary AER followed a nonnormal distribution, log transformation was performed prior to statistical analysis of this parameter. Differences were considered significant if the p value was less than 0.05. Results Clinical and metabolic parameters Rats in group DM had increased BG levels. No effects on BG were observed with monotherapy treatment or enalapril and breviscapine combination treatment. As shown previously, kidney enlargement as increased KW and KW/BW was observed in group DM, which was reduced by treatment with both enalapril and breviscapine, but its level was higher than that in control rats. The level of KW/BW in group DM treated with combination of enalapril and brevisapine was significantly lower than that in the groups DM treated with enalapril or breviscapine alone. Treatment with both enalapril and breviscapine attenuated the increase in albuminuria in the diabetic rats, but this level was still higher than that observed in control rats. The combination of enalapril and breviscapine was
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Table 1 ¯ ± s, n = 10). Change of clinical and metabolic parameters in five groups ( Groups
Blood glucose (mmol/l)
C DM DM + E DM + B DM + E + B
6.03 28.20 26.37 25.94 24.55
± ± ± ± ±
0.42 1.26** 1.59 2.52 1.50
Body weight (g) 458.75 221.13 329.87 253.38 321.27
± ± ± ± ±
Kidney weight (g)
27.32 22.81** 15.39 37.59 23.21
1.81 2.29 1.98 1.85 1.78
± ± ± ± ±
0.07 0.12* 0.10# 0.09# 0.07#
Relative kidney (×10−3 ) 4.20 9.38 6.09 7.04 5.52
± ± ± ± ±
0.42 0.84** 0.36# 0.35# 0.20## ,
AER (mg/24 h) 0.48 ×/÷ 1.3 1.26 ×/÷ 1.1** 0.86 ×/÷ 1.1# 0.68 ×/÷ 1.1# 0.54 ×/÷ 1.2## ,
AER was expressed as geometric mean ×/÷ tolerance factor. * p < 0.05. ** p < 0.01 vs C. # p < 0.05. ## p < 0.01 vs DM. p < 0.05 vs DM + B or DM + E.
Table 2 ¯ ± s, n = 10). Change of morphologic parameters in the glomeruli and tubulointerstitial injury index in five groups ( Groups
AG (m2 )
C DM DM + E DM + B DM + E + B
3962.30 5786.22 4611.26 4701.26 4082.30
* ** # ##
± ± ± ± ±
193.18 219.37** 237.58# 224.87# 226.84## ,
VG (106 m3 )
AM (m2 )
± ± ± ± ±
676.82 1366.33 968.00 1066.08 854.17
1.02 1.52 1.21 1.23 1.06
0.11 0.11* 0.12# 0.11# 0.08## ,
± ± ± ± ±
TII 13.48 23.32** 17.63# 18.16# 22.48## ,
0.38 0.84 0.59 0.62 0.48
± ± ± ± ±
0.14 0.25* 0.19 0.18 0.14#
p < 0.05. p < 0.01 vs C. p < 0.05. p < 0.01 vs DM. p < 0.05 vs DM + B or DM + E.
associated with a further reduction in albuminuria than was seen with either drug administrated alone, the similar AER level to that observed in control animals (Table 1). Renal histology Rats in group DM had an increase in AG , VG and AM when compared with the values in group C, treatment with enalapril and breviscapine significantly ameliorated the increase of AG , VG and
AM . The combination of enalapril and breviscapine was associated with a further reduction in AG , VG and AM than was seen with either drug administrated alone. Rats in group DM had an increase in TII when compared to group C, treatment with enalapril and breviscapine was associated with a reduction in TII as compared with group DM, but this did not reach statistical significance. However, the combination of enalapril and breviscapine was associated with a further reduction in TII when compared to untreated diabetic rats (Fig. 2 and Table 2).
Fig. 2. A representative microphotograph of kidney tissue stained with Periodic acid-Schiff from C (A), DM (B), DM + E (C), DM + B (D) and DM + E + B (E) rats. Original magnification 400×.
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Fig. 3. Western blot analysis of PKC protein (A) and densitometric analysis (B) in renal tissue in C, DM, DM + E, DM + B and DM + E + B. Values are the means ± S.E.M. **p < 0.01 vs C, # p < 0.05, ## p < 0.01 vs DM, p < 0.05 (s DM + B or DM + E.
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Fig. 4. Western blot analysis of 3-NT protein (A) and densitometric analysis (B) in renal tissue in C, DM, DM + E, DM + B and DM + E + B. Values are the means ± S.E.M. **p < 0.01 vs C, # p < 0.05, ## p < 0.01 vs DM, p < 0.05 (s DM + B or DM + E.
PKC activities
Determination of MDA The concentration of MDA in renal tissue and urine was significantly higher in the group DM when compared with group C, treatment with enalapril and breviscapine significantly ameliorated the increase of MDA in renal tissue and urine. The combination of enalapril and breviscapine was associated with a further reduction in MDA in renal tissue and urine than with either drug administrated alone (Table 3).
Compared with group C, PKC activities in the cytosolic fraction (PKCc), the membrane fraction (PKCm) and the ratio of the PKC activities in the membrane fraction to cytosolic fraction (PKCm/PKCc) were significantly elevated in renal tissue from group DM, suggesting that diabetic state induced renal PKC activation. Increased PKC activities was effectively inhibited by enalapril or breviscapine administration. The combination of enalapril and breviscapine was associated with a further reduction in PKC activities in renal tissue than with either drug administrated alone (Table 4).
Renal 3-NT expression Renal PKC expression Fig. 3 showed that renal 3-NT expression in diabetic rats was increased compared with control animals. This increased expression of 3-NT in diabetic animals was significantly reduced by treatment with enalapril and breviscapine. The combination of enalapril and breviscapine was associated with a reduction in 3-NT expression when compared to diabetic rats treated with enalapril or breviscapine.
TGF-ˇ1 immunohistochemistry
Table 3 ¯ ± s, n = 10). Change of MDA level in renal tissue and urine in five groups ( Groups
Renal MDA (nmol/mg prot)
C DM DM + E DM + B DM + E + B
1.74 3.60 2.72 2.51 2.17
** # ##
± ± ± ± ±
0.07 0.08** 0.04# 0.04# 0.05## ,
TGF-1 immunostaining was observed in the glomerulus and tubulointerstitium in control rats. Minimal immunostainable TGF1 was present in the kidneys of control rats. In contrast, abundant TGF-1 was expressed in the kidney of diabetic rats. This overexpression was attenuated in diabetic rats treated with enalapril and breviscapine. The combination of enalapril and breviscapine result in further reduction in renal TGF-1 expression when compared to diabetic rats treated with enalapril or breviscapine (Fig. 5 and Table 5).
Urine MDA (nmol/24 h) 240.66 769.47 554.62 580.36 344.72
± ± ± ± ±
Western blot analysis demonstrated increased PKC expression in the kidneys of diabetic compared with control animals. This increased expression of PKC in diabetic animals was significantly reduced by treatment with enalapril and breviscapine. The combination of enalapril and breviscapine was associated with a reduction in PKC expression when compared to diabetic rats treated with enalapril or breviscapine (Fig. 4).
11.37 58.64** 47.067# 25.71# 26.77## ,
p < 0.01 vs C. p < 0.05. p < 0.01 vs DM. p < 0.05 vs DM + B or DM + E.
Table 4 ¯ ± s, n = 10). Change of PKC activity in renal tissue in five groups ( Groups C DM DM + E DM + B DM + E + B * ** # ##
PKCt (pmol/min mg) 9.83 34.34 18.45 14.38 12.46
± ± ± ± ±
0.97 4.75** 1.82# 1.70# 1.23#
PKCc (pmol/min mg) 7.57 16.70 13.19 9.50 9.73
± ± ± ± ±
0.95 2.44** 1.86# 1.63# 1.74#
PKCm (pmol/min mg) 2.26 17.65 5.26 4.88 2.72
± ± ± ± ±
0.33 2.25** 0.54# 0.41# 0.36## ,
PKCm/PKCc 0.23 0.51 0.40 0.34 0.28
± ± ± ± ±
0.03 0.02* 0.03# 0.04# 0.02## ,
p < 0.05. p < 0.01 vs C. p < 0.05. p < 0.01 vs DM. p < 0.05 vs DM + B or DM + E.
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Table 5 ¯ ± s, n = 10). Change of TGF1 immunohistochemistry in renal tissue in five groups ( Groups
Glomeruli score/gcs
C DM DM + E DM + B DM + E + B
0.66 2.18 1.46 1.32 0.84
** # ##
± ± ± ± ±
0.12 0.18** 0.13# 0.16# 0.14## ,
tubulointerstitium % 5.8 21.2 12.8 13.8 7.8
± ± ± ± ±
0.6 2.4** 1.6# 01.4# 0.7## ,
p < 0.01 vs C. p < 0.05. p < 0.01 vs DM. p < 0.05 vs DM + B or DM + E.
Discussion Our study shows that the combination of an agent that interrupts the renin-angiotensin system with breviscapine provides superior renoprotection in a model of experimental diabetes than either agent alone. This has been shown for a range of functional and structural parameters including albuminuria, glomerular and tubulointerstitial injury. These observations should be considered in the context of the beneficial effects of both ACE and breviscapine on the progression of renal damage in the experimental context. The pathogenesis of diabetic nephropathy is complex and involves both glucose-dependent and glucose-independent pathways. In both type 1 and type 2 diabetes, the degree of hyperglycemia influences both the likelihood of developing nephropathy and the rate of its progression (Nalysnyk et al. 2010; Stratton et al. 2000). High intracellular glucose concentrations, per se, may lead to activation of PKC and in particular the ( isoform, which has been shown to be activated in the glomeruli in experimental diabetes (Kelly et al., 2003; Wu et al., 2009). However, in addition to these glucose-dependent mechanisms, other glucose-independent components of the diabetic state contribute to the development and progression of diabetic nephropathy. In particular, both experimental and clinical studies indicate that
hypertension and inappropriate activation of the RAS are likely key contributors. Both Ang II, the effector molecule of the RAS, and cell stretch, the in vitro counterpart of hypertension, activate PKC (Feng et al. 1998). Previous studies showed that increasing PKC activation by PDBu, a diacylglycerol analogue, mimicked the effect of high glucose on Ang II production and that a PKC inhibitor totally abolished the effect of glucose on Ang II without a significant change in Ang II production in normal glocose (Ikehara et al. 2003). These results suggest that high glucose levels stimulate Ang II production through glucose induced PKC activation in human MCs. Since it is well established that Ang II increases inositol triphosphate and diacylglycerol via activation of phospholipase C, leading to stimulate PKC activity. It has been reported that high glucose milieu of diabetes increases Ang II generation via PKC activation, in turn increased Ang II synergistically stimulates PKC activation in an autocrine fashion in MCs. In the present study, enalapril and breviscapine significantly attenuated the structural and functional manifestations of diabetic renal injury along with a reduction in the overexpression of the profibrotic growth factor TGF-1. Furthermore, the combination of enalapril and breviscapine was associated with a further attenuation of reduction in albuminuria, MDA, PKC and TGF-1 in the kidney of experimental diabetes. The finding that these beneficial changes occurred despite the continued presence of hyperglycemia is consistent with PKC activation and activation of the RAS as a final common pathway for these pathogenetic attributes of the diabetic milieu. Elevated oxidative stress plays an important role in the progression of diabetes and its complications. High glucose increases ROS to enhance oxidative stress in tissue via a series of pathwaysg (Forbes et al. 2008; Gao and Mann 2009; Kashihara et al. 2010), ROS may attack the unsaturated fatty acid in the biomembrane to yield lipid peroxidation such as MDA, ketone and hydroxyl which amplify oxidative stress to promote the progression of diabetic complications (Zhu et al. 2005). ROS may also increase NF- B and TGF-1 which lead to deposition of extracellular matrix and tissue fibrosis. Increased lipid peroxidation and decreased antioxidant enzymes have been reported in kidney tissue of STZ-induced diabetic rats (Kitada et al. 2011). However, due to the abundant blood flow in
Fig. 5. A representative microphotograph of immunostaining for TGF1 in renal tissue from C (A), DM (B), DM + E (C), DM + B (D) and DM + E + B (E) rats. Negative control (F). Original magnification 400×.
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kidney tissue, oxidative substance and antioxidant enzymes in blood may affect glomerular endothelial cells directly, infiltrate into the mesangial area, tubular interstitial area and other parts of renal tissue, which may play an important role in oxidative injury to kidney tissue. A previous report has demonstrated that increased lipid peroxidation and decreased antioxidant enzymes which may impair kidney tissue existed in plasma of patients with chronic glomerulonephritis. The results presented in our study imply that in STZ-induced diabetic rats MDAr and MDAu are increased. A growing body of study have shown oxidant stress is increased in clinical and experimental diabetic nephropathy (Giacco and Brownlee 2010), and our present study, in addition to confirming their findings, have demonstrated elevated MDA level in renal tissue was reduced by enalapril or breviscapine and, more effectively, by combined enalapril with breviscapine in diabetic rats. This effect of combined enalapril with breviscapine was likely the result of suppression of PKC. Lately, the reactive nitrogen species originate from NO have attracted vast attention as a new pathway of oxidative stress. NO has a high affinity for SOD, and their interaction form the extremely strong and reactive oxidant peroxynitrite (ONOO ). Because the production of ONOO is difficult to determine, the assay of 3-NT in protein has been proposed as an indirect marker of ONOO production. Our present study showed the elevated 3-NT in diabetic rats was reduced by enalapril or breviscapine and, more effectively, by combined enalapril with breviscapine in diabetic rats. The renal structural injury observed in the diabetic rats, as assessed in both the glomerulus and tubulointerstitium, was reduced by both treatments as monotherapy, with an additional effect observed with the combination when compared to either enalapril or breviscapine treatment alone. The reduction in glomerular and tubulointerstitial injury was observed in association with a reduction in the expression of the prosclerotic cytokine, TGF1 in all three treatment groups. Increased ROS generation, protein kinase C activation, angiotensin II in association with increased glucose metabolism and hemodynamic disorder are considered to be the main upstream signaling molecules of diabetic-induced renal injury. Both ACE and PKC inhibition reduce the degree of glomerulosclerosis possibly via a reduction in the expression of prosclerotic cytokines such as TGF1. Our results further support to the concept that both AII and PKC formation are implicated in the progression of glomerular structural injury via a common cytokine pathway such as TGF1. Because combination therapy was correlated with synergetic suppression on overexpression of PKC, 3-NT, MDA and TGF1 in renal tissue in diabetic rats, the production of TGF-ß1 may have more effectively been inhibited, resulting in prevention of diabetic nephropathy progression. The magnitude of tubulointerstitial injury is an important prognostic marker of renal outcome in many forms of renal disease. In human diabetic nephropathy, the extent of interstitial fibrosis is strongly associated with mesangial expansion, falling GFR, and increasing proteinuria. In experimental diabetic nephropathy, investigation has focused almost exclusively on the glomerulus and particularly on the mesangial cell, although tubulointerstitial disease also develops in the streptozotocin model. Accumulation of extracellular matrix, first recognized as thickening of capillary basement membranes, is a characteristic pathologic feature of diabetes and is present in the tubulointerstitial as well as the glomerulus Enalapril and breviscapine were not only associated with a decrease in glomerulosclerosis, but also had effects on reducing tubulointerstitial injury. The tubulointerstitium is now considered a major site of diabetes-related injury and is an important determinant of the rate of decline in renal function in diabetic nephropathy. Combination therapy was associated with a further reduction in tubulointerstitial injury.
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