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Effect of cinnamon and its procyanidin-B2 enriched fraction on diabetic nephropathy in rats
Chemico-Biological Interactions 222 (2014) 68–76
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Effect of cinnamon and its procyanidin-B2 enriched fraction on diabetic nephropathy in rats P. Muthenna, G. Raghu, P. Anil Kumar, M.V. Surekha, G. Bhanuprakash Reddy ⇑ National Institute of Nutrition, Hyderabad, India
a r t i c l e
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Article history: Received 13 February 2014 Received in revised form 23 August 2014 Accepted 27 August 2014 Available online 6 September 2014 Keywords: Diabetic nephropathy Cinnamon Procyanidin-B2 AGE CML Rat
a b s t r a c t Non-enzymatic protein glycation and resultant accumulation of advanced glycation endproducts (AGE) are implicated in the pathogenesis of diabetic complications including diabetic nephropathy (DN). It is considered that antiglycating agents offer protection against AGE mediated pathologies including DN. Earlier we characterized procyanidin-B2 (PCB2) as the active component from cinnamon (Cinnamomum zeylanicum) that inhibits AGE formation in vitro. In this study, we have investigated the potential of PCB2-enriched fraction of cinnamon to prevent in vivo accumulation of AGE and to ameliorate renal changes in diabetic rats. Streptozotocin-induced diabetic rats were fed with either 3% cinnamon or 0.002% PCB2-fraction in diet for 12 weeks. Biochemical analysis of blood and urine was performed at the end of experiment. Evaluation of glomerular markers that serve as indicators of renal function was done by immunohistochemistry, immunoblotting and qRT-PCR. Supplementation of diabetic rats with cinnamon and PCB2-fraction prevented glycation mediated RBC-IgG cross-links and HbA1c accumulation in diabetes rats. Cinnamon and PCB2-fraction also inhibited the accumulation of N-carboxy methyl lysine (CML), a prominent AGE in diabetic kidney. Interestingly, cinnamon and its PCB2-fraction prevented the AGE mediated loss of expression of glomerular podocyte proteins; nephrin and podocin. Inhibition of AGE by cinnamon and PCB2-fraction ameliorated the diabetes mediated renal malfunction in rats as evidenced by reduced urinary albumin and creatinine. In conclusion, PCB2 from cinnamon inhibited AGE accumulation in diabetic rat kidney and ameliorated AGE mediated pathogenesis of DN. Ó 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Diabetes mellitus is the most common metabolic disease and a leading cause of end-stage renal disease (ESRD). Considerable evidence suggests that chronic hyperglycemia is the major culprit for microvascular complications of diabetes including diabetic nephropathy (DN) that develops in 20–40% of patients with type 1 and type 2 diabetes [1]. During nephropathy, glomerulus shows both morphological and functional changes in all elements that constitute glomerular filtration barrier (GFB) of the kidney: endothelium, glomerular basement membrane (GBM), and glomerular podocytes. Alterations in GFB results in reduced glomerular filtration rate with poor renal outcome ranging from microalbuminuria to overt proteinuria. The clinical manifestations of DN include thickening glomerular basement membrane, glomerular hypertrophy, mesangial cell expansion and loss of podocytes [2]. Glomerular podocytes are highly specialized visceral epithelial cells that ⇑ Corresponding author. Address: National Institute of Nutrition, Hyderabad 500007, India. Tel.: +91 40 27197252; fax: +91 40 27019074. E-mail address: [email protected] (G.B. Reddy). http://dx.doi.org/10.1016/j.cbi.2014.08.013 0009-2797/Ó 2014 Elsevier Ireland Ltd. All rights reserved.
play a vital role in glomerular filtration. A reduction in the podocyte density has been documented in the kidneys of diabetic patients as a result of either substantial podocyte damage leading to podocyte apoptosis or as a result of podocyte detachment from the basement membrane [3]. Human biopsy studies have provided the evidence that podocytes are functionally and structurally injured very early in the pathogenesis of DN [3]. Decreased expression of podocyte proteins such as nephrin and podocin was reported in experimental models of nephropathy [4]. While several mechanisms that may either manifest in podocyte damage or cause glomerular changes in diabetes have been discussed, the precise molecular mechanism that mediates hyperglycemic podocyte loss remains largely unknown. Among several biochemical and molecular events that manifest during diabetes, formation of advanced glycation end-products (AGE) has been suggested as a major mechanism in pathogenesis of DN [5,6]. Advanced glycation end-products are chemically heterogeneous compounds formed non-enzymatically through an interaction of reducing sugars with the free amino group of biological macromolecules including proteins. The initial product of this reaction is called as Schiff base, which spontaneously rearranges itself
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into an Amadori product. A series of subsequent reactions and rearrangements lead to the formation of AGE. N-carboxymethyllysine (CML), pentosidine and methylglyoxal derivatives are examples of a few well-characterized AGE. AGE, particularly CML and pentosidine are present in all renal compartments in diabetic patients [7]. The characteristic structural changes of DN such as thickening of GBM and mesangial expansion are accompanied by accumulation of AGE, leading to glomerulosclerosis and interstitial fibrosis. Infusion of control rats with AGE resulted in the development of similar morphological changes and significant proteinuria to that of diabetic rats [8]. Experimental and human studies suggest that AGE promote inflammation and aggravate diabetic complications by inducing secretion of cytokines and growth factors including MCP-1, TGF-b and VEGF. Alternatively, pathological effects of AGE are mediated by their interaction with intracellular receptors for AGE (RAGE) [9]. Activation of RAGE by AGE has been implicated in promoting renal damage and development of DN via activating an array of cellular signaling cascades [5]. In rodents, either administration of RAGE neutralizing antibodies [10], or genetic deficiency in RAGE [9] protected against renal injury in diabetes. Taken together, accumulation of AGE and their interaction with RAGE is likely to have a pivotal role in the development and progression of DN. The efficacy of AGE inhibitors such as aminoguanidine, pyridoxamine, thiamine, OPB-9195, ALT-946 and LR-90 in either preventing and/or improving experimental DN suggest the significance of anti-AGE agents in offering protection against AGE mediated renal injury [11]. Hence, identification and testing of novel anti-AGE agents with greater efficacy is much warranted. In our earlier work, we have demonstrated that aqueous extract of cinnamon (Cinnamomum zeylanicum) has significant inhibitory potential against the AGE formation under in vitro conditions [12]. Subsequently we characterized procyanidin-B2 (PCB2) as the active component of cinnamon extract that is involved in AGE inhibition using bioassay-guided fractionation with eye lens proteins under in vitro conditions and showed its potential to delay diabetic cataract in rats [13]. While our study is under progress Zhang et al., reported that oral administration of grape seed PCB2 attenuated renal dysfunction in db/db mice by preventing the expression of milk fat globule EGF-8 expression [14]. In the present study, the effect of cinnamon and its PCB2-enriched fraction on the AGE formation and development of early renal manifestation (albuminuria, creatinuria) in experimental diabetic rats was investigated. Dietary supplementation of cinnamon and its PCB2-fraction attenuated CML accumulation in glomerular region of diabetic rats and alleviated pathology of streptozotocin (STZ) induced DN. The renoprotective effects of cinnamon and its active component in experimental diabetic model suggest potential pharmacological ability of these components to treat diabetic complications including nephropathy where AGE mediated pathology prevails.
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with 0.045 M glyoxalic acid and 0.15 M sodium cyanoborohydride in 0.2 M sodium phosphate buffer (pH 7.8) for 24 h at 37 °C for the preparation of CML-KLH. Low molecular weight reactants were removed by extensive dialysis. Antibodies were produced against CML-KLH by immunizing 3-months old female New Zealand white rabbits. The anti-serum was partially purified by ammonium sulfate fractionation followed by DEAE-Sepharose anion exchange chromatography to obtain IgG rich fraction. 2.3. Extraction and isolation of PCB2 from cinnamon Extraction and separation of PCB2-enriched fraction from cinnamon (C. zeylanicum) was carried out according to the methods described earlier [13]. In brief, cinnamon bark was powdered and extracted with absolute ethanol. The ethanol-extract was partitioned with ethyl acetate and the ethyl acetate-soluble fraction was fractionated on a Sephadex LH-20 column. The bound fractions were eluted with 95% ethanol followed by 50% acetone and the eluent was collected in various fractions. The active fraction was characterized as PCB2-enriched fraction by LC–MS/MS and HPLC analysis. 2.4. Experimental design and dietary regimen Two-month-old male Wistar NIN (WNIN) rats with an average bodyweight of 220 ± 17 g were used in the study. All the animals were fed with AIN-93 diet ad libitum. The control (group I) rats received sham consists of 0.1 M citrate buffer, pH 4.5 while the experimental rats received a single i.p injection of STZ (35 mg/ kg) in citrate buffer. 72 h post-injection with STZ, fasting blood glucose levels were monitored. Animals having blood glucose levels <150 mg/dl were excluded from the experiment and the rest were distributed into three groups (groups II–IV). Animals in group II received AIN-93 diet alone whereas group III animals received the AIN-93 diet supplemented with 3% cinnamon powder whereas group IV animals received AIN-93 diet containing 0.002% PCB2fraction. Each group consists of six animals. A pilot study was conducted to find an optimal dose of cinnamon and was found to be 3% in the diet. Approximately 3 g cinnamon bark powder yields 0.002 g PCB2 according to the extraction procedure described above. Based on our earlier study, we fed diabetic animals either with 3% cinnamon or 0.002% PCB2-fraction in the diet [13]. Appropriate controls were maintained that were fed with either cinnamon or PCB2. Animal care and experimental protocols were in accordance with institutional animal ethical committee. Animals were housed in individual cages in a temperature (22 °C) and humidity-controlled room with a 12 h light/dark cycle. All the animals had free access to water. Food intake (daily) and body weights (weekly) were monitored.
2. Materials and methods
2.5. Biochemical estimations
2.1. Materials
Serum glucose was measured by the glucose oxidase–peroxidase (GOD–POD) method using a commercially available kit (BioSystems, Barcelona, Spain). HbA1c (RBC), albumin, creatinine (urine) and urea (plasma) were estimated using commercially available kits (BioSystems, Spain).
Streptozotocin (STZ), EDTA, BSA, methylglyoxal (MGO), Freund’s complete adjuvant, Freund’s incomplete adjuvant, glyoxylic acid and sodium cyanoborohydride were purchased from sigma (MO, USA). Immobilon-NC membrane was from Millipore (Bedford, MA). All other chemicals and solvents were of analytical grade and were obtained from local companies. 2.2. Preparation of CML antigens and production of polyclonal antiCML antibodies Carboxy methyl lysine (CML) antigens were prepared as described earlier [12,15]. Briefly, KLH (50 mg/ml) was incubated
2.6. Blood, kidney collection and processing Blood was collected once a week from the retro-orbital plexus for estimation of glucose. 24 h urine was collected from experimental animals by placing them in metabolic cages. At the end of 12 weeks duration of diabetes, the animals were sacrificed by CO2 asphyxiation. Kidneys were perfused via abdominal aorta with 100 ml of normal saline. The left renal vein was punctured to
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permit the perfusate to drain and the kidney was removed immediately and placed in 4% paraformaldehyde for subsequent histologic studies. The remaining kidney was snap frozen in liquid nitrogen and stored at 80 °C for isolation of either RNA or protein for further studies. 2.7. Analysis of RBC-IgG cross-linking Blood was collected from the rats in heparinized tubes and plasma is carefully removed. Then, RBC was washed 4–5 times with ice-cold PBS and 0.2 ml of packed RBC is collected from the bottom of the tube and diluted in PBS to desirable dilution and used for further analysis. The amount of IgG bound to RBC was quantified using ELISA as described in Vasan et al. [16]. The bound IgG was calculated (100 (OD410 control OD410 treated)/ OD410 control). 2.8. Quantitative real-time PCR Total RNA was extracted using tri-reagent (Invitrogen). Isolated RNA was further purified by RNeasy Mini Kit (Qiagen) and quantified by measuring the absorbance at 260 nm on NanoDrop (ND 1000). Four micrograms of total RNA was reverse transcribed using high capacity cDNA Reverse Transcription kit (ABI). Reverse transcription reaction was carried out with thermocycler (ABI 9700) and reaction conditions were as follows: initial RT for 10 min at 25 °C, followed by 37 °C for 120 min and inactivation of reverse transcriptase at 84 °C for 5 min. Real-time PCR (ABI-7500) was performed in triplicates with 25 ng cDNA templates using SYBR green RT-PCR kit with gene specific primers (Table 1). Normalization and validation of data were carried using b-actin as an internal control and data were compared between control and diabetic samples according to comparative CT (2DDct) method. The reaction conditions were as follows: 40 cycles of initial denaturation temperature at 95 °C for 30 s followed by annealing at 52 °C for 40 s and extension at 72 °C for 1 min and product specificity was analyzed by melt curve analysis. 2.9. Immunohistochemistry Kidneys were collected at the end of animal experiment and fixed in 4% paraformaldehyde in sodium phosphate buffer (pH 7.2), followed by embedding and sectioning using standard protocols. Immunolocalization of nephrin, podocin, CML, PKC-a and VEGF were carried out on 4 lm thick paraffin sections of WNIN rat kidneys taken on chrome alum gelatin coated slides. The kidney sections were deparaffinized by incubating the slides in xylene for 10 min and same was repeated three times. Afterwards, the
Table 1 List of primers and their sequence used in the study. Gene
Sequence
b-Actin
50 CGA CAA CGG CTC CGG CAT GT 30 50 GGG GCC ACA CGC AGC TCA TT 30
ICAM
50 TGG GCA CCC AGC AGA AGT TCT T 30 50 ACT GAG GCA GTG GCT GAC ACA A 30
VCAM
50 TCT TCG GAG CCT CAA CGG TAC T 30 50 TGG TGC TGC AAG TCA GGA GCA T 30 0
0
MCP1
5 GCT GTC TCA GCC AGA TGC AGT T3 50 AGC TTC TTT GGG ACA CCT GCT G30
Nephrin
50 ACA GCG TGC TGG TGA TGA CTG T30 50 TGG TAA TGG CGC TTG GGG GAA A30
Podocin
50 AGC CAT CCA GTT CCT GGT GCA A30 50 TGC CCC AAA CAC AGG TCA CTG A30
sections were dehydrated in decreasing grades of isopropyl alcohol (90%, 70% and 50%). Antigen retrieval was done by heating the slides in 0.1 M sodium citrate, pH 6.0 for 10 min at 60 °C in microwave oven. The endogenous peroxidase activity was quenched by incubating the slides in 3% H2O2 for 30 min. Two washes of 5 min interval each time were given in phosphate buffer saline (PBS). To prevent non-specific binding of the antibody, blocking was done by incubating the slides in 10% normal goat serum in PBS at room temperature for 1 h. Later the slides were incubated overnight at 4 °C with primary antibody in 1% normal goat serum in PBS. After incubation slides were washed with PBS and incubated with biotinylated secondary antibody for 30 min and followed by incubation of slides for 30 min with Vectastain elite ABC reagent (ABC elite kit, Vector Laboratories). The protein was localized by brown staining in the kidney sections by addition of DAB solution containing H2O2. Slides were observed under an epifluorescence microscope (E800; Nikon, Tokyo, Japan). Images were captured using appropriate filters. 2.10. Western blot analysis Tissue lysates were prepared in homogenization buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS) containing protease inhibitor (Complete mini, Roche, Mannheim, Germany), 1 mM sodium orthovanadate and 50 mM NaF. Lysates were centrifuged at 12,000g and aliquots of the supernatants were separated by 12% SDS–PAGE and transferred to nitrocellulose membrane (Millipore). After probing with corresponding primary antibodies antigen–antibody complexes were detected with horseradish peroxidase-labeled secondary antibodies, respectively, and visualized using enhanced chemiluminescence reagents (Pierce, Rockford, IL) according to the manufacturer’s protocol. 2.11. Statistical analysis Data were expressed as mean ± SEM unless otherwise stated. One-way analysis of variance (ANOVA) with pairwise comparisons according to the Tukey method was used in this study. Differences were considered significant if the p-value was less than 0.05. 3. Results 3.1. Animal characteristics As reported by us previously [13,15,43], diabetic rats showed increase in food intake compared with the control animals. Despite increased food intake, the bodyweight of diabetic animals decreased significantly compared with non-diabetic control animals (Table 2). Fasting blood glucose levels were elevated in STZ treated diabetic rats and persisted for 12 weeks of the study compared with control animals (Table 2). While, feeding of cinnamon and PCB2-fraction to diabetic rats has no effect on the altered body weights, the blood glucose levels were marginally lowered (but statistically significant) (Table 2). The toxic effects of feeding cinnamon or PCB2-fraction to control rats was not noticed and are similar to untreated control animals. 3.2. Diabetes induced protein glycation was inhibited by cinnamon and PCB2 Glycated hemoglobin (HbA1c) levels in untreated diabetic rats were significantly higher compared with control animals (Table 2). While feeding of PCB2-fraction ameliorated HbA1c accumulation in experimental diabetic rats, cinnamon has no significant effect in preventing HbA1c accumulation in diabetic rats (Table 2).
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Table 2 Effect of cinnamon and PCB2 on body weight, blood glucose, glycosylated haemoglobin, plasma urea, and albumin to creatinine ratio in diabetic rats. These parameters were measured at the end of 12 weeks of chronic hyperglycemia. Data were mean ± SE (n = 6) and superscript (*) denotes significantly different from control and superscript (**) denotes significantly different from diabetes (p < 0.05). Parameter
Control
Diabetic (D)
D + cinnamon
D+PCB2
Body weight (g) Blood glucose (mg/dl) Glycosylated haemoglobin, HbA1c (%) Urea in plasma (mg/dl) Albumin/creatinine (lg/mg)
264 ± 5.0 89 ± 2.0 6.72 ± 0.54 35.3 ± 6 1760 ± 135
217 ± 4.9⁄ 365 ± 13.0⁄ 11.9 ± 0.72⁄ 82 ± 5⁄ 5870 ± 225⁄
234 ± 4.9 329 ± 9.2⁄ 10.8 ± 0.61 71 ± 7 3920 ± 236⁄⁄
241 ± 6.2 311 ± 10.2⁄ 9.00 ± 0.36⁄⁄ 44 ± 4⁄⁄ 1710 ± 214⁄⁄
Further, we have investigated the ability of cinnamon and PCB2 to prevent cross-linking of IgG on red blood cell surface (RBC-IgG). During diabetic conditions, there is a considerable increase in RBC-IgG cross-linking that provide an index of AGE mediated protein cross-linking. We assayed the extent of RBC-IgG cross-linking in experimental and treated rats. Dietary supplementation with cinnamon and PCB2 significantly decreased RBC-IgG cross-linking to 45% and 40% respectively compared to that of diabetic rats (70%) (Fig. 1A). Further, we investigated whether cinnamon and PCB2 prevent the accumulation of CML, an AGE that is abundant in renal tissues of diabetic rats. Immunohistochemical examination revealed that cinnamon and PCB2 prevented the CML formation in glomeruli of diabetic rats (Fig. 1B). Immunoblotting also substantiate immunohistochemical findings that cinnamon and PCB2 prevented the CML formation in kidney of diabetic rats (Fig. 2). The cytotoxicity of AGE in cells is mediated via cell-surface receptor for AGE (RAGE). Therefore, we measured expression of RAGE in these experimental animals. Increased expression of RAGE was observed in diabetic rat kidney compared with control rats. Interestingly, cinnamon and PCB2 prevented hyperglycemia induced expression of RAGE in diabetic rat kidney (Fig. 2). 3.3. Diabetes induced PKCa expression is prevented by dietary cinnamon and PCB2 intervention The activation of protein kinase C-a (PKC-a) in the kidney from diabetic animal is well known and increased expression in podocytes in renal biopsies of patients with DN has been reported. It is
evident from earlier studies that AGE induce activation of PKC-a [17]. This high glucose inducible kinase orchestrates nephrin internalization in podocytes that consequences into impaired glomerular filtration barrier and proteinuria. Therefore we measured PKC-a in experimental rats. In accordance with earlier studies, immunohistochemical staining revealed that PKC-a expression is increased in glomeruli from diabetic rat (Fig. 3). Interestingly, cinnamon and PCB2 feeding prevented the hyperglycemia mediated expression of PKC-a in diabetic rat kidney (Fig. 3). To elucidate possible mediators of PKC-a in renal damage under hyperglycemia conditions, we analyzed glomerular expression of VEGF. In untreated diabetic rats, increase in VEGF expression was observed (Fig. 3). In contrast, this increase in VEGF expression is significantly reduced in diabetic rats fed with PCB2-fraction, but not completely prevented in cinnamon fed rats (Fig. 3). 3.4. Anti-AGE effect of cinnamon and PCB2 prevented diabetes induced expression of cell adhesion and inflammatory molecules Mononuclear cells such as monocytes/macrophages and T-cells are considered to be involved in the progression of DN, although the mechanism of their recruitment into diabetic glomeruli is unclear. Cell adhesion molecules promote endothelial dysfunction through perturbations of coagulation, permeability, vasomotor function and cell adhesion, leading to the development of macro and microvascular renal lesions. In the present study, we investigated the expression of intercellular adhesion molecule-1 (ICAM) in the glomeruli of STZ-induced experimental diabetic rats. The
Fig. 1. Cinnamon and PCB2 inhibit protein glycation and AGE formation in diabetic rats. The in vivo anti-AGE effect of cinnamon and PCB2 was assessed by estimation of AGE mediated RBC-IgG cross-linking (A), immunohistochemical analysis of glomerular accumulation of CML (B). Data in panel A were presented as mean ± SE (n = 6). (⁄p < 0.01, vs. control rats; ⁄⁄p < 0.01, vs. diabetic rats) and data in panel B are representative of three independent analyses. The images in panel B were taken at 400 magnification.
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3.5. Cinnamon and PCB2 prevented depletion of podocyte slitdiaphragm proteins We analyzed expression of nephrin and podocin in diabetic rats. When RT-PCR studies were performed, it was found that mRNA expression of both nephrin and podocin were decreased 40% in diabetic rat glomerulus (Fig. 5). Dietary supplementation of cinnamon and its active PCB2-fraction attenuated the hyperglycemia mediated loss of glomerular nephrin and podocin expression. In concurrence with RT-PCR analysis, immunohistochemical analysis found diminished expression of nephrin and podocin in untreated diabetic rats whereas cinnamon and PCB2 supplementation mitigated the loss of glomerular nephrin and podocin expression (Fig. 6). Further, immunoblotting analysis also supports the diminished expression of nephrin in untreated diabetic rats and its mitigation by cinnamon and PCB2 supplementation (Fig. 6B). 3.6. Cinnamon and PCB2 ameliorate proteinuria in diabetic rats
Fig. 2. Panel A – immunoblotting analysis of CML and RAGE from control, diabetic and diabetic rats treated with cinnamon or PCB2. Panel B – densitometric quantification of expression of CML and RAGE normalized to b-actin. Data in panel B were presented as mean ± SEM, n = 6. (⁄p < 0.01, vs. control rats; ⁄⁄p < 0.01, vs. diabetic rats).
expression of ICAM was not induced during diabetes (Fig. 4). Alternatively we have also measured vascular cell adhesion molecule-1 (VCAM) in diabetic rats. There was increased expression of VCAM in untreated diabetic rats (Fig. 4). Though dietary supplementation of cinnamon had meager effect on VCAM expression in diabetic rats, PCB2-fraction significantly prevented the hyperglycemia induced VCAM expression (Fig. 4). Monocyte chemoattractant protein-1 (MCP-1), an inflammatory cytokine, levels are elevated in response to AGE [18] and activates the macrophage infiltration into renal tissue. Tissue macrophages mediate variety of pathologies including obesity, insulin resistance and DN. Elevated expression of MCP-1 in untreated diabetic rats was prevented with dietary supplementation of cinnamon and PCB2 (Fig. 4).
To assess renal function, we estimated urinary albumin and creatinine content in diabetic rats. Diabetic rats excreted significantly elevated levels of albumin compared to control rats (117.4 vs. 14.08 mg/24 h). Urinary albumin content is significantly decreased in rats fed with cinnamon and PCB2-fraction (62.72 & 17.1 mg/ 24 h). Diabetic rats excreted greater amounts of creatinine compared with non-diabetic control rats (20 vs. 8 mg/24 h) whereas feeding diabetic rats with cinnamon and PCB2-fraction (16.0 & 10.0 mg/24 h) manifested in the diminished excretion of creatinine which is also reflected in albumin to creatinine ratio (Table 2). Noticeably, PCB2 is more effective in preventing creatinine excretion in diabetic rats than cinnamon. Furthermore, we estimated urea content in the plasma. Diabetic animals displayed 82 ± 5 mg/ml urea in plasma against 35 ± 6 mg/ml in control rats. Plasma urea content is restricted to 71 ± 7 and 44 ± 4 mg/ml in cinnamon and PCB2 fed rats, respectively (Table 2). 4. Discussion Diabetes mellitus is associated with a myriad of deviations from normal homeostasis that results in an array of complications. The accelerated accumulation of AGE cross-links is a predominant factor in the pathogenesis of diabetic complications. The adverse consequences of AGE formation were first described in the renal and cardiovascular systems of humans and diabetic rats [19,20]. AGE formation is associated with renal injury and activation of diverse signal transduction systems in various kidney cells including podocytes that eventually results in proteinuria, a hall mark feature of
Fig. 3. Cinnamon and PCB2 mitigate the glomerular PKC-a and VEGF expression in diabetic rats: Immunohistochemical staining using anti-PKC-a (upper panel) and antiVEGF antibodies (lower panel) on paraffin sections of kidney cortex of control, diabetic, diabetic rats treated with cinnamon and PCB2. Original images were taken at 400 magnification. Data are representative of three independent analyses.
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Fig. 5. Inhibition of AGE by cinnamon and PCB2 preserve nephrin and podocin expression in glomeruli from diabetic rats: Quantitative RT-PCR analysis of nephrin (A) and podocin (B) in renal extract from four different experimental groups. Data were mean ± SEM, n = 6. (⁄p < 0.01, vs. control rats; ⁄⁄p < 0.01, vs. diabetic rats.)
Fig. 4. Cinnamon and PCB2 prevented renal expression of cell adhesion and inflammatory molecules in diabetic rats: quantitative RT-PCR analysis of steady state expression of ICAM (A), VCAM (B) and MCP-1 (C) in control, diabetic and diabetic rats treated with either cinnamon or PCB2. Data were mean ± SEM, n = 6. (⁄p < 0.01, vs. control rats; ⁄⁄p < 0.01, vs. diabetic rats.)
renal diseases [5,21]. The cardinal finding of our study was cinnamon and in particular, its active component PCB2 prevented the accumulation of CML, predominant AGE in renal tissues of diabetic rats. AGE dependent expression of RAGE and inflammatory cytokine, MCP-1 was concealed with cinnamon and PCB2. Our studies also highlight that cinnamon and PCB2 mitigate AGE induced loss of nephrin, the podocyte slit diaphragm protein and offer salutary effects in improving the proteinuria in diabetic rats. However, unlike earlier studies [22] we have not noticed a significant hypoglycemic effect of cinnamon. Podocyte foot processes, GBM, and the fenestrated endothelial cells together form the GFB. GFB ensures the filtration of low molecular weight solutes and thus preventing the loss of proteins such as albumin into urine. The cell–cell junctions between the podocyte foot processes are bridged by slit diaphragm, and the functional integrity of slit diaphragm is critical for preserving normal renal function. Proteinuria ensues when the structure of podocytes is destroyed by disruption of the slit diaphragm. Nephrin, a key component of podocyte slit-diaphragm, plays a determinative role in the integrity of GFB. The essential role of nephrin in the renal function was evident from the studies showing that
mutations in the nephrin gene (NPHS1) associated with congenital nephrotic syndrome [23]. A positive correlation between altered nephrin expression and proteinuria has been shown in experimental animals injected with mAb nephrin [24] and in case of puromycin aminonucleoside-induced nephrosis [25]. It was shown that diabetic hypertensive rats develop albuminuria concomitance with a reduction in nephrin expression [26]. Similarly, in rats with STZinduced diabetes and in nonobese diabetic mice, nephrin got redistributed and lost in the urine [4,27]. Also, in patients with type 1 and type 2 diabetes and nephropathy, expression of nephrin was markedly reduced [28]. Plethora of evidence suggests that nephrin loss could be a critical event in the progression of DN, thus conditioning the onset of proteinuria [4,27–31]. Analogously, in the current study, we have noticed diminished expression of nephrin in diabetic rats; interestingly, cinnamon and PCB2 ameliorated the expression of nephrin in diabetic conditions. There could be two possible explanations for observed loss of nephrin expression in STZ-diabetic rats that are mutually inclusive. One explanation comes from reported studies that non-enzymatically glycated proteins via activation of their receptors (RAGE) repress nephrin expression in diabetic kidney [28]. Furthermore, AGE also induces MCP-1 [32] and its induction has been implicated in the development and progression of DN as evidenced by both human and experimental studies [33,34]. MCP-1 secreted by mesangial cells plays a key role in migration of monocytes and macrophages into kidney which is an index of tissue damage during progression of DN. Furthermore, architecture of slit-diaphragm is altered by MCP-1 via down regulating nephrin expression [35]. Patients with diabetes show gradual increase in urinary levels of MCP-1 that correlate with clinical severity of the disease [36]. In
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Fig. 6. Panel A – immunohistochemical analysis of glomerular expression of nephrin (upper panel) and podocin (bottom panel); immunoblotting analysis of nephrin (B) and densitometric quantification of expression of nephrin (C). Data in panel A are representative of three independent analyses. Data in panel C were mean ± SEM, n = 6. (⁄p < 0.01, vs. control rats; ⁄⁄p < 0.01, vs. diabetic rats.)
the current study, as expected MCP-1 expression was elevated in diabetic rats. Interestingly, dietary supplementation of cinnamon and PCB2 inhibited the renal expression of MCP-1 in diabetic rats. AGE dependent loss of expression is not only pertinent to nephrin but AGE also interact with a-actinin-4 (ACTN-4), a component of slit diaphragm. In the presence of AGE, ACTN-4 expression by podocytes is significantly decreased [37]. Therefore, the interventions that can slacken the AGE formation in kidney and prevent AGE mediated manifestation on proteins that constitute slit-diaphragm offer the pharmacological promise. Based on the results, we propose a model (Fig. 7) that cinnamon and PCB2 mitigate formation of AGE (CML) and suppress the consequent events that abate the nephrin expression. Thus, antiglycating potential of PCB2-fraction offers salutary effect in preventing proteinuria in experimental diabetic rats. Alternatively, hyperglycemia induced transcriptional repression of nephrin could also be regulated by PKC-a [38]. PKC-a, a major intracellular mediator of hyperglycemia induced glomerular permeability represses a transcription factor WT1, which has the binding site in the nephrin promoter [39]. Animals devoid of PKC-a are protected against the development of albuminuria [40]. In the present study, histological examination revealed elevated expression of PKC-a in diabetic rat kidney is concomitant with decreased expression of nephrin. Interestingly, PCB2-fraction feeding ameliorated the expression of PKC-a. Noticeably, PCB2-enriched fraction of cinnamon was more potent than cinnamon in inhibiting PCK-a expression in diabetic rat kidney. The ability of PCB2 in preventing PCK-a expression greater than its source cinnamon, argue for the improved renoprotective effect of PCB2 over cinnamon. Also, glomerular expression of adhesion molecules such as VCAM was shown to increase in diabetic human and rodent kidneys. It was reported that AGE induce expression of these adhesion molecules that promote progression of DN by facilitating infiltration of macrophages into kidney [41]. Interestingly, in the current study, elevated expression of AGE and VCAM was abrogated by PCB2.
Fig. 7. Proposed model for the in vivo role of cinnamon and PCB2 in combating proteinuria in diabetes: cinnamon and its active component PCB2 effectively inhibited CML formation in diabetic rats, which in turn prevented AGE dependent activation of PKC-a and elevated expression of inflammatory molecule MCP1. Via sequel of events anti-AGE effect of cinnamon and PCB2 in diabetic rats preserved the nephrin expression thereby prevented proteinuria and nephropathy.
Nephrin expression is being considered as an index of glomerular function, an effort to preserve its expression in clinical conditions such as DN display a great therapeutic promise against an array of renal diseases during which loss of nephrin is a predisposing factor. In an earlier study, the loss of nephrin expression induced by glycated albumin was prevented with neutralizing antibodies to RAGE, suggesting the involvement of RAGE in the regulation of nephrin expression [28]. It was also shown that inhibition of PKC-a stabilizes nephrin expression and prevents proteinuria in streptozotocin induced diabetes in mice [42]. Recently, we have
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also reported that a dietary flavonoid, ellagic acid, which has antiglycating potential was effective in reducing atherosclerotic process by blocking proliferation of vascular smooth muscle cells [43]. However, in our study, nephrin expression was preserved with diet based ingredients that serve as antiglycating agents. To best of our knowledge, this is a first study to report dietary interventions ameliorate nephrin expression in a clinical condition and it could be a promising therapeutic target for diabetic nephropathy. 5. Conclusions In conclusion, the results obtained in the present study suggest that cinnamon and particularly its active principle PCB2 ameliorated STZ-induced proteinuria and podocyte injury in rats. This effect, at least in part, could be attributed to suppressing renal AGE-RAGE stimulated MCP-1 and PKC-a expression, thereby modulating slit diaphragm proteins nephrin and podocin expression (Fig. 7). Therefore, preventing the loss of nephrin expression impedes diabetes induced proteinuria in experimental animals that fed with either cinnamon or its PCB2-fraction. Furthermore, this study emphasizes the pivotal role that AGE inhibitors could play in the prevention of DN and offers an important nutraceutical based therapeutic approach to combat diabetic kidney diseases.
[5]
[6]
[7] [8]
[9]
[10]
[11] [12]
[13]
[14]
Conflicts of Interest G.B.R – received grants from the Department of Biotechnology, India under 7th FP of Indo-EU collaborative grant on functional foods (Grant agreement #245030). P.M. – received a research fellowship from University Grants Commission, India; PAK – received INSPIRE-Faculty fellowship from Department of Science & Technology, India. The authors have received nephrin antibody as gratis from Dr. Rakesh Verma, University of Michigan, Ann Arbor, USA. The work presented in this manuscript forms a part of the patent titled ‘Antiglycating potential of procyanidin-B2 isolated from cinnamon bark: Prospects for prevention or treatment of diabetic complications (cataract, retinopathy & nephropathy)’. Patent application (No. 1452/DEL/2012) with complete specifications was submitted on 21.11.2013 at Patent Office, New Delhi, India. But for the above disclosures, all the authors declare no conflict of interests.
[15]
[16]
[17]
[18]
[19]
[20]
[21]
Transparency Document [22]
The 10.1016/j.cbi.2014.08.013 associated with this article can be found in the online version.
Acknowledgements The authors thank Ms. K. Sharadha (Pathology Department, National Institute of Nutrition) for her help in preparing the kidney sections and Dr. Rakesh Verma, University of Michigan, Ann Arbor for providing nephrin antibody.
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References [27] [1] N.I.o.H. US Renal Data System. USRDS 2009 Annual Data Report: Atlas of ESRD, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD, 2009. [2] H. Cheng, R.C. Harris, The glomerulus – a view from the outside – the podocyte, Int. J. Biochem. Cell Biol. 42 (2010) 1380–1387. [3] G. Wolf, S. Chen, F.N. Ziyadeh, From the periphery of the glomerular capillary wall toward the center of disease: podocyte injury comes of age in diabetic nephropathy, Diabetes 54 (2005) 1626–1634. [4] P. Aaltonen, P. Luimula, E. Astrom, T. Palmen, T. Gronholm, E. Palojoki, I. Jaakkola, H. Ahola, I. Tikkanen, H. Holthofer, Changes in the expression of
[28]
[29]
75
nephrin gene and protein in experimental diabetic nephropathy, Lab. Invest. 81 (2001) 1185–1190. T. Wendt, N. Tanji, J. Guo, B.I. Hudson, A. Bierhaus, R. Ramasamy, B. Arnold, P.P. Nawroth, S.F. Yan, V. D’Agati, A.M. Schmidt, Glucose, glycation, and RAGE: implications for amplification of cellular dysfunction in diabetic nephropathy, J. Am. Soc. Nephrol. 14 (2003) 1383–1395. M. Daroux, G. PrÃÓvost, H. Maillard-Lefebvre, C. Gaxatte, E. Boulanger, V.D. D’Agati, A.M. Schmidt, Advanced glycation end-products: implications for diabetic and non-diabetic nephropathies, Diabetes Metab. 36 (2010) 1–10. R. Singh, A. Barden, T. Mori, L. Beilin, Advanced glycation end-products: a review, Diabetologia 44 (2001) 129–146. H. Vlassara, L.J. Striker, S. Teichberg, H. Fuh, Y.M. Li, M. Steffes, Advanced glycation end products induce glomerular sclerosis and albuminuria in normal rats, Proc. Natl. Acad. Sci. U.S.A. 91 (1994) 11704–11708. T.M. Wendt, N. Tanji, J. Guo, T.R. Kislinger, W. Qu, Y. Lu, L.G. Bucciarelli, L.L. Rong, B. Moser, G.S. Markowitz, G. Stein, A. Bierhaus, B. Liliensiek, B. Arnold, P.P. Nawroth, D.M. Stern, V.D. D’Agati, A.M. Schmidt, RAGE drives the development of glomerulosclerosis and implicates podocyte activation in the pathogenesis of diabetic nephropathy, Am. J. Pathol. 162 (2003) 1123–1137. L.J. Jensen, L. Denner, B.F. Schrijvers, R.G. Tilton, R. Rasch, A. Flyvbjerg, Renal effects of a neutralising RAGE-antibody in long-term streptozotocin-diabetic mice, J. Endocrinol. 188 (2006) 493–501. E. Stitt-Cavanagh, L. MacLeod, C. Kennedy, The podocyte in diabetic kidney disease, Sci. World J. 9 (2009) 1127–1139. M. Saraswat, P.Y. Reddy, P. Muthenna, G.B. Reddy, Prevention of non-enzymic glycation of proteins by dietary agents: prospects for alleviating diabetic complications, Br. J. Nutr. 101 (2009) 1714–1721. P. Muthenna, G. Raghu, C. Akileshwari, S.N. Sinha, P. Suryanarayana, G.B. Reddy, Inhibition of protein glycation by procyanidin-B2 enriched fraction of cinnamon: delay of diabetic cataract in rats, IUBMB Life 65 (2013) 941–950. Z. Zhang, B.Y. Li, X.L. Li, M. Cheng, F. Yu, W.D. Lu, Q. Cai, J.F. Wang, R.H. Zhou, H.Q. Gao, L. Shen, Proteomic analysis of kidney and protective effects of grape seed procyanidin B2 in db/db mice indicate MFG-E8 as a key molecule in the development of diabetic nephropathy, Biochim. Biophys. Acta (1832) 805–816. M. Saraswat, P. Suryanarayana, P.Y. Reddy, M.A. Patil, N. Balakrishna, G.B. Reddy, Antiglycating potential of Zingiber officinalis and delay of diabetic cataract in rats, Mol. Vis. 16 (2010) 1525–1537. S. Vasan, X. Zhang, A. Kapurniotu, J. Bernhagen, S. Teichberg, J. Basgen, D. Wagle, D. Shih, I. Terlecky, R. Bucala, A. Cerami, J. Egan, P. Ulrich, An agent cleaving glucose-derived protein crosslinks in vitro and in vivo, Nature 382 (1996) 275–278. V. Scivittaro, M.B. Ganz, M.F. Weiss, AGEs induce oxidative stress and activate protein kinase C-beta(II) in neonatal mesangial cells, Am. J. Physiol. Renal Physiol. 278 (2000) F676–683. Y. Ishibashi, T. Matsui, M. Takeuchi, S. Yamagishi, Beneficial effects of metformin and irbesartan on advanced glycation end products (AGEs)RAGE-induced proximal tubular cell injury, Pharmacol. Res. 65 (2012) 297– 302. M. Brownlee, A. Cerami, H. Vlassara, Advanced glycosylation end products in tissue and the biochemical basis of diabetic complications, N. Engl. J. Med. 318 (1988) 1315–1321. M. Brownlee, A. Cerami, H. Vlassara, Advanced products of nonenzymatic glycosylation and the pathogenesis of diabetic vascular disease, Diabetes Metab. Rev. 4 (1988) 437–451. Z. Makita, S. Radoff, E.J. Rayfield, Z. Yang, E. Skolnik, V. Delaney, E.A. Friedman, A. Cerami, H. Vlassara, Advanced glycosylation end products in patients with diabetic nephropathy, N. Engl. J. Med. 325 (1991) 836–842. R. Deng, A review of the hypoglycemic effects of five commonly used herbal food supplements, Recent Pat. Food Nutr. Agric. 4 (2012) 50–60. M. KestilÃ, U. Lenkkeri, M. MÃnnikkÃ, J. Lamerdin, P. McCready, H. Putaala, V. Ruotsalainen, T. Morita, M. Nissinen, R. Herva, C.E. Kashtan, L. Peltonen, C. Holmberg, A. Olsen, K. Tryggvason, Positionally cloned gene for a novel glomerular protein nephrin is mutated in congenital nephrotic syndrome, Mol. Cell 1 (1998) 575–582. P.S. Topham, H. Kawachi, S.A. Haydar, S. Chugh, T.A. Addona, K.B. Charron, L.B. Holzman, M. Shia, F. Shimizu, D.J. Salant, Nephritogenic mAb 5-1-6 is directed at the extracellular domain of rat nephrin, J. Clin. Invest. 104 (1999) 1559– 1566. M. Hosoyamada, K. Yan, Y. Nishibori, Y. Takiue, A. Kudo, H. Kawakami, T. Shibasaki, H. Endou, Nephrin and podocin expression around the onset of puromycin aminonucleoside nephrosis, J. Pharm. Sci. 97 (2005) 234–241. F. Bonnet, M.E. Cooper, H. Kawachi, T.J. Allen, G. Boner, Z. Cao, Irbesartan normalises the deficiency in glomerular nephrin expression in a model of diabetes and hypertension, Diabetologia 44 (2001) 874–877. J.M. Forbes, F. Bonnet, L.M. Russo, W.C. Burns, Z. Cao, R. Candido, H. Kawachi, T.J. Allen, M.E. Cooper, G. Jerums, T.M. Osicka, Modulation of nephrin in the diabetic kidney: association with systemic hypertension and increasing albuminuria, J. Hypertens. 20 (2002) 985–992. S. Doublier, G. Salvidio, E. Lupia, V. Ruotsalainen, D. Verzola, G. Deferrari, G. Camussi, Nephrin expression is reduced in human diabetic nephropathy: evidence for a distinct role for glycated albumin and angiotensin II, Diabetes 52 (2003) 1023–1030. Y. Wu, J. Dong, L. Yuan, C. Liang, K. Ren, W. Zhang, F. Fang, J. Shen, Nephrin and podocin loss is prevented by mycophenolate mofetil in early experimental diabetic nephropathy, Cytokine 44 (2008) 85–91.
76
P. Muthenna et al. / Chemico-Biological Interactions 222 (2014) 68–76
[30] R.G. Langham, D.J. Kelly, A.J. Cox, N.M. Thomson, H. Holthofer, P. Zaoui, N. Pinel, D.J. Cordonnier, R.E. Gilbert, Proteinuria and the expression of the podocyte slit diaphragm protein, nephrin, in diabetic nephropathy: effects of angiotensin converting enzyme inhibition, Diabetologia 45 (2002) 1572–1576. [31] D.J. Kelly, P. Aaltonen, A.J. Cox, J.R. Rumble, R. Langham, S. Panagiotopoulos, G. Jerums, H. Holthofer, R.E. Gilbert, Expression of the slit-diaphragm protein, nephrin, in experimental diabetic nephropathy: differing effects of antiproteinuric therapies, Nephrol. Dial. Transplant. 17 (2002) 1327–1332. [32] S. Yamagishi, Y. Inagaki, T. Okamoto, S. Amano, K. Koga, M. Takeuchi, Z. Makita, Advanced glycation end product-induced apoptosis and overexpression of vascular endothelial growth factor and monocyte chemoattractant protein-1 in human-cultured mesangial cells, J. Biol. Chem. 277 (2002) 20309–20315. [33] N. Banba, T. Nakamura, M. Matsumura, H. Kuroda, Y. Hattori, K. Kasai, Possible relationship of monocyte chemoattractant protein-1 with diabetic nephropathy, Kidney Int. 58 (2000) 684–690. [34] F.Y. Chow, D.J. Nikolic-Paterson, E. Ozols, R.C. Atkins, B.J. Rollin, G.H. Tesch, Monocyte chemoattractant protein-1 promotes the development of diabetic renal injury in streptozotocin-treated mice, Kidney Int. 69 (2006) 73–80. [35] E. Tarabra, S. Giunti, F. Barutta, G. Salvidio, D. Burt, G. Deferrari, R. Gambino, D. Vergola, S. Pinach, P.C. Perin, G. Camussi, G. Gruden, Effect of the monocyte chemoattractant protein-1/CC chemokine receptor 2 system on nephrin expression in streptozotocin-treated mice and human cultured podocytes, Diabetes 58 (2009) 2109–2118. [36] G.H. Tesch, MCP-1/CCL2: a new diagnostic marker and therapeutic target for progressive renal injury in diabetic nephropathy, Am. J. Physiol. Renal Physiol. 294 (2008) F697–701.
[37] T.S. Ha, High glucose and advanced glycosylated end-products affect the expression of alpha-actinin-4 in glomerular epithelial cells, Nephrology (Carlton) 11 (2006) 435–441. [38] I. Quack, M. Woznowski, S.A. Potthoff, R. Palmer, E. Konigshausen, S. Sivritas, M. Schiffer, J. Stegbauer, O. Vonend, L.C. Rump, L. Sellin, PKC alpha mediates beta-arrestin2-dependent nephrin endocytosis in hyperglycemia, J. Biol. Chem. 286 (2011) 12959–12970. [39] J. Menne, M. Meier, J.K. Park, M. Boehne, T. Kirsch, C. Lindschau, R. Ociepka, M. Leitges, J. Rinta-Valkama, H. Holthofer, H. Haller, Nephrin loss in experimental diabetic nephropathy is prevented by deletion of protein kinase C alpha signaling in-vivo, Kidney Int. 70 (2006) 1456–1462. [40] M. Meier, J.K. Park, D. Overheu, T. Kirsch, C. Lindschau, F. Gueler, M. Leitges, J. Menne, H. Haller, Deletion of protein kinase C-beta isoform in vivo reduces renal hypertrophy but not albuminuria in the streptozotocin-induced diabetic mouse model, Diabetes 56 (2007) 346–354. [41] H. Vlassara, H. Fuh, T. Donnelly, M. Cybulsky, Advanced glycation endproducts promote adhesion molecule (VCAM-1, ICAM-1) expression and atheroma formation in normal rabbits, Mol. Med. 1 (1995) 447–456. [42] I. Tossidou, B. Teng, J. Menne, N. Shushakova, J.K. Park, J.U. Becker, F. Modde, M. Leitges, H. Haller, M. Schiffer, Podocytic PKC-alpha is regulated in murine and human diabetes and mediates nephrin endocytosis, PLoS One 5 (2010) e10185. [43] P.U. Rani, R. Kesavan, R. Ganugula, P.U. Kumar, G.B. Reddy, M. Dixit, Ellagic acid inhibits PDGF-BB-induced vascular smooth muscle cell proliferation and prevents atheroma formation in streptozotocin-induced diabetic rats, J. Nutr. Biochem. 24 (2013) 1830–1839.
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Effect of cinnamon and its procyanidin-B2 enriched fraction on diabetic nephropathy in rats P. Muthenna, G. Raghu, P. Anil Kumar, M.V. Surekha, G. Bhanuprakash Reddy ⇑ National Institute of Nutrition, Hyderabad, India
a r t i c l e
i n f o
Article history: Received 13 February 2014 Received in revised form 23 August 2014 Accepted 27 August 2014 Available online 6 September 2014 Keywords: Diabetic nephropathy Cinnamon Procyanidin-B2 AGE CML Rat
a b s t r a c t Non-enzymatic protein glycation and resultant accumulation of advanced glycation endproducts (AGE) are implicated in the pathogenesis of diabetic complications including diabetic nephropathy (DN). It is considered that antiglycating agents offer protection against AGE mediated pathologies including DN. Earlier we characterized procyanidin-B2 (PCB2) as the active component from cinnamon (Cinnamomum zeylanicum) that inhibits AGE formation in vitro. In this study, we have investigated the potential of PCB2-enriched fraction of cinnamon to prevent in vivo accumulation of AGE and to ameliorate renal changes in diabetic rats. Streptozotocin-induced diabetic rats were fed with either 3% cinnamon or 0.002% PCB2-fraction in diet for 12 weeks. Biochemical analysis of blood and urine was performed at the end of experiment. Evaluation of glomerular markers that serve as indicators of renal function was done by immunohistochemistry, immunoblotting and qRT-PCR. Supplementation of diabetic rats with cinnamon and PCB2-fraction prevented glycation mediated RBC-IgG cross-links and HbA1c accumulation in diabetes rats. Cinnamon and PCB2-fraction also inhibited the accumulation of N-carboxy methyl lysine (CML), a prominent AGE in diabetic kidney. Interestingly, cinnamon and its PCB2-fraction prevented the AGE mediated loss of expression of glomerular podocyte proteins; nephrin and podocin. Inhibition of AGE by cinnamon and PCB2-fraction ameliorated the diabetes mediated renal malfunction in rats as evidenced by reduced urinary albumin and creatinine. In conclusion, PCB2 from cinnamon inhibited AGE accumulation in diabetic rat kidney and ameliorated AGE mediated pathogenesis of DN. Ó 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Diabetes mellitus is the most common metabolic disease and a leading cause of end-stage renal disease (ESRD). Considerable evidence suggests that chronic hyperglycemia is the major culprit for microvascular complications of diabetes including diabetic nephropathy (DN) that develops in 20–40% of patients with type 1 and type 2 diabetes [1]. During nephropathy, glomerulus shows both morphological and functional changes in all elements that constitute glomerular filtration barrier (GFB) of the kidney: endothelium, glomerular basement membrane (GBM), and glomerular podocytes. Alterations in GFB results in reduced glomerular filtration rate with poor renal outcome ranging from microalbuminuria to overt proteinuria. The clinical manifestations of DN include thickening glomerular basement membrane, glomerular hypertrophy, mesangial cell expansion and loss of podocytes [2]. Glomerular podocytes are highly specialized visceral epithelial cells that ⇑ Corresponding author. Address: National Institute of Nutrition, Hyderabad 500007, India. Tel.: +91 40 27197252; fax: +91 40 27019074. E-mail address: [email protected] (G.B. Reddy). http://dx.doi.org/10.1016/j.cbi.2014.08.013 0009-2797/Ó 2014 Elsevier Ireland Ltd. All rights reserved.
play a vital role in glomerular filtration. A reduction in the podocyte density has been documented in the kidneys of diabetic patients as a result of either substantial podocyte damage leading to podocyte apoptosis or as a result of podocyte detachment from the basement membrane [3]. Human biopsy studies have provided the evidence that podocytes are functionally and structurally injured very early in the pathogenesis of DN [3]. Decreased expression of podocyte proteins such as nephrin and podocin was reported in experimental models of nephropathy [4]. While several mechanisms that may either manifest in podocyte damage or cause glomerular changes in diabetes have been discussed, the precise molecular mechanism that mediates hyperglycemic podocyte loss remains largely unknown. Among several biochemical and molecular events that manifest during diabetes, formation of advanced glycation end-products (AGE) has been suggested as a major mechanism in pathogenesis of DN [5,6]. Advanced glycation end-products are chemically heterogeneous compounds formed non-enzymatically through an interaction of reducing sugars with the free amino group of biological macromolecules including proteins. The initial product of this reaction is called as Schiff base, which spontaneously rearranges itself
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into an Amadori product. A series of subsequent reactions and rearrangements lead to the formation of AGE. N-carboxymethyllysine (CML), pentosidine and methylglyoxal derivatives are examples of a few well-characterized AGE. AGE, particularly CML and pentosidine are present in all renal compartments in diabetic patients [7]. The characteristic structural changes of DN such as thickening of GBM and mesangial expansion are accompanied by accumulation of AGE, leading to glomerulosclerosis and interstitial fibrosis. Infusion of control rats with AGE resulted in the development of similar morphological changes and significant proteinuria to that of diabetic rats [8]. Experimental and human studies suggest that AGE promote inflammation and aggravate diabetic complications by inducing secretion of cytokines and growth factors including MCP-1, TGF-b and VEGF. Alternatively, pathological effects of AGE are mediated by their interaction with intracellular receptors for AGE (RAGE) [9]. Activation of RAGE by AGE has been implicated in promoting renal damage and development of DN via activating an array of cellular signaling cascades [5]. In rodents, either administration of RAGE neutralizing antibodies [10], or genetic deficiency in RAGE [9] protected against renal injury in diabetes. Taken together, accumulation of AGE and their interaction with RAGE is likely to have a pivotal role in the development and progression of DN. The efficacy of AGE inhibitors such as aminoguanidine, pyridoxamine, thiamine, OPB-9195, ALT-946 and LR-90 in either preventing and/or improving experimental DN suggest the significance of anti-AGE agents in offering protection against AGE mediated renal injury [11]. Hence, identification and testing of novel anti-AGE agents with greater efficacy is much warranted. In our earlier work, we have demonstrated that aqueous extract of cinnamon (Cinnamomum zeylanicum) has significant inhibitory potential against the AGE formation under in vitro conditions [12]. Subsequently we characterized procyanidin-B2 (PCB2) as the active component of cinnamon extract that is involved in AGE inhibition using bioassay-guided fractionation with eye lens proteins under in vitro conditions and showed its potential to delay diabetic cataract in rats [13]. While our study is under progress Zhang et al., reported that oral administration of grape seed PCB2 attenuated renal dysfunction in db/db mice by preventing the expression of milk fat globule EGF-8 expression [14]. In the present study, the effect of cinnamon and its PCB2-enriched fraction on the AGE formation and development of early renal manifestation (albuminuria, creatinuria) in experimental diabetic rats was investigated. Dietary supplementation of cinnamon and its PCB2-fraction attenuated CML accumulation in glomerular region of diabetic rats and alleviated pathology of streptozotocin (STZ) induced DN. The renoprotective effects of cinnamon and its active component in experimental diabetic model suggest potential pharmacological ability of these components to treat diabetic complications including nephropathy where AGE mediated pathology prevails.
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with 0.045 M glyoxalic acid and 0.15 M sodium cyanoborohydride in 0.2 M sodium phosphate buffer (pH 7.8) for 24 h at 37 °C for the preparation of CML-KLH. Low molecular weight reactants were removed by extensive dialysis. Antibodies were produced against CML-KLH by immunizing 3-months old female New Zealand white rabbits. The anti-serum was partially purified by ammonium sulfate fractionation followed by DEAE-Sepharose anion exchange chromatography to obtain IgG rich fraction. 2.3. Extraction and isolation of PCB2 from cinnamon Extraction and separation of PCB2-enriched fraction from cinnamon (C. zeylanicum) was carried out according to the methods described earlier [13]. In brief, cinnamon bark was powdered and extracted with absolute ethanol. The ethanol-extract was partitioned with ethyl acetate and the ethyl acetate-soluble fraction was fractionated on a Sephadex LH-20 column. The bound fractions were eluted with 95% ethanol followed by 50% acetone and the eluent was collected in various fractions. The active fraction was characterized as PCB2-enriched fraction by LC–MS/MS and HPLC analysis. 2.4. Experimental design and dietary regimen Two-month-old male Wistar NIN (WNIN) rats with an average bodyweight of 220 ± 17 g were used in the study. All the animals were fed with AIN-93 diet ad libitum. The control (group I) rats received sham consists of 0.1 M citrate buffer, pH 4.5 while the experimental rats received a single i.p injection of STZ (35 mg/ kg) in citrate buffer. 72 h post-injection with STZ, fasting blood glucose levels were monitored. Animals having blood glucose levels <150 mg/dl were excluded from the experiment and the rest were distributed into three groups (groups II–IV). Animals in group II received AIN-93 diet alone whereas group III animals received the AIN-93 diet supplemented with 3% cinnamon powder whereas group IV animals received AIN-93 diet containing 0.002% PCB2fraction. Each group consists of six animals. A pilot study was conducted to find an optimal dose of cinnamon and was found to be 3% in the diet. Approximately 3 g cinnamon bark powder yields 0.002 g PCB2 according to the extraction procedure described above. Based on our earlier study, we fed diabetic animals either with 3% cinnamon or 0.002% PCB2-fraction in the diet [13]. Appropriate controls were maintained that were fed with either cinnamon or PCB2. Animal care and experimental protocols were in accordance with institutional animal ethical committee. Animals were housed in individual cages in a temperature (22 °C) and humidity-controlled room with a 12 h light/dark cycle. All the animals had free access to water. Food intake (daily) and body weights (weekly) were monitored.
2. Materials and methods
2.5. Biochemical estimations
2.1. Materials
Serum glucose was measured by the glucose oxidase–peroxidase (GOD–POD) method using a commercially available kit (BioSystems, Barcelona, Spain). HbA1c (RBC), albumin, creatinine (urine) and urea (plasma) were estimated using commercially available kits (BioSystems, Spain).
Streptozotocin (STZ), EDTA, BSA, methylglyoxal (MGO), Freund’s complete adjuvant, Freund’s incomplete adjuvant, glyoxylic acid and sodium cyanoborohydride were purchased from sigma (MO, USA). Immobilon-NC membrane was from Millipore (Bedford, MA). All other chemicals and solvents were of analytical grade and were obtained from local companies. 2.2. Preparation of CML antigens and production of polyclonal antiCML antibodies Carboxy methyl lysine (CML) antigens were prepared as described earlier [12,15]. Briefly, KLH (50 mg/ml) was incubated
2.6. Blood, kidney collection and processing Blood was collected once a week from the retro-orbital plexus for estimation of glucose. 24 h urine was collected from experimental animals by placing them in metabolic cages. At the end of 12 weeks duration of diabetes, the animals were sacrificed by CO2 asphyxiation. Kidneys were perfused via abdominal aorta with 100 ml of normal saline. The left renal vein was punctured to
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permit the perfusate to drain and the kidney was removed immediately and placed in 4% paraformaldehyde for subsequent histologic studies. The remaining kidney was snap frozen in liquid nitrogen and stored at 80 °C for isolation of either RNA or protein for further studies. 2.7. Analysis of RBC-IgG cross-linking Blood was collected from the rats in heparinized tubes and plasma is carefully removed. Then, RBC was washed 4–5 times with ice-cold PBS and 0.2 ml of packed RBC is collected from the bottom of the tube and diluted in PBS to desirable dilution and used for further analysis. The amount of IgG bound to RBC was quantified using ELISA as described in Vasan et al. [16]. The bound IgG was calculated (100 (OD410 control OD410 treated)/ OD410 control). 2.8. Quantitative real-time PCR Total RNA was extracted using tri-reagent (Invitrogen). Isolated RNA was further purified by RNeasy Mini Kit (Qiagen) and quantified by measuring the absorbance at 260 nm on NanoDrop (ND 1000). Four micrograms of total RNA was reverse transcribed using high capacity cDNA Reverse Transcription kit (ABI). Reverse transcription reaction was carried out with thermocycler (ABI 9700) and reaction conditions were as follows: initial RT for 10 min at 25 °C, followed by 37 °C for 120 min and inactivation of reverse transcriptase at 84 °C for 5 min. Real-time PCR (ABI-7500) was performed in triplicates with 25 ng cDNA templates using SYBR green RT-PCR kit with gene specific primers (Table 1). Normalization and validation of data were carried using b-actin as an internal control and data were compared between control and diabetic samples according to comparative CT (2DDct) method. The reaction conditions were as follows: 40 cycles of initial denaturation temperature at 95 °C for 30 s followed by annealing at 52 °C for 40 s and extension at 72 °C for 1 min and product specificity was analyzed by melt curve analysis. 2.9. Immunohistochemistry Kidneys were collected at the end of animal experiment and fixed in 4% paraformaldehyde in sodium phosphate buffer (pH 7.2), followed by embedding and sectioning using standard protocols. Immunolocalization of nephrin, podocin, CML, PKC-a and VEGF were carried out on 4 lm thick paraffin sections of WNIN rat kidneys taken on chrome alum gelatin coated slides. The kidney sections were deparaffinized by incubating the slides in xylene for 10 min and same was repeated three times. Afterwards, the
Table 1 List of primers and their sequence used in the study. Gene
Sequence
b-Actin
50 CGA CAA CGG CTC CGG CAT GT 30 50 GGG GCC ACA CGC AGC TCA TT 30
ICAM
50 TGG GCA CCC AGC AGA AGT TCT T 30 50 ACT GAG GCA GTG GCT GAC ACA A 30
VCAM
50 TCT TCG GAG CCT CAA CGG TAC T 30 50 TGG TGC TGC AAG TCA GGA GCA T 30 0
0
MCP1
5 GCT GTC TCA GCC AGA TGC AGT T3 50 AGC TTC TTT GGG ACA CCT GCT G30
Nephrin
50 ACA GCG TGC TGG TGA TGA CTG T30 50 TGG TAA TGG CGC TTG GGG GAA A30
Podocin
50 AGC CAT CCA GTT CCT GGT GCA A30 50 TGC CCC AAA CAC AGG TCA CTG A30
sections were dehydrated in decreasing grades of isopropyl alcohol (90%, 70% and 50%). Antigen retrieval was done by heating the slides in 0.1 M sodium citrate, pH 6.0 for 10 min at 60 °C in microwave oven. The endogenous peroxidase activity was quenched by incubating the slides in 3% H2O2 for 30 min. Two washes of 5 min interval each time were given in phosphate buffer saline (PBS). To prevent non-specific binding of the antibody, blocking was done by incubating the slides in 10% normal goat serum in PBS at room temperature for 1 h. Later the slides were incubated overnight at 4 °C with primary antibody in 1% normal goat serum in PBS. After incubation slides were washed with PBS and incubated with biotinylated secondary antibody for 30 min and followed by incubation of slides for 30 min with Vectastain elite ABC reagent (ABC elite kit, Vector Laboratories). The protein was localized by brown staining in the kidney sections by addition of DAB solution containing H2O2. Slides were observed under an epifluorescence microscope (E800; Nikon, Tokyo, Japan). Images were captured using appropriate filters. 2.10. Western blot analysis Tissue lysates were prepared in homogenization buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS) containing protease inhibitor (Complete mini, Roche, Mannheim, Germany), 1 mM sodium orthovanadate and 50 mM NaF. Lysates were centrifuged at 12,000g and aliquots of the supernatants were separated by 12% SDS–PAGE and transferred to nitrocellulose membrane (Millipore). After probing with corresponding primary antibodies antigen–antibody complexes were detected with horseradish peroxidase-labeled secondary antibodies, respectively, and visualized using enhanced chemiluminescence reagents (Pierce, Rockford, IL) according to the manufacturer’s protocol. 2.11. Statistical analysis Data were expressed as mean ± SEM unless otherwise stated. One-way analysis of variance (ANOVA) with pairwise comparisons according to the Tukey method was used in this study. Differences were considered significant if the p-value was less than 0.05. 3. Results 3.1. Animal characteristics As reported by us previously [13,15,43], diabetic rats showed increase in food intake compared with the control animals. Despite increased food intake, the bodyweight of diabetic animals decreased significantly compared with non-diabetic control animals (Table 2). Fasting blood glucose levels were elevated in STZ treated diabetic rats and persisted for 12 weeks of the study compared with control animals (Table 2). While, feeding of cinnamon and PCB2-fraction to diabetic rats has no effect on the altered body weights, the blood glucose levels were marginally lowered (but statistically significant) (Table 2). The toxic effects of feeding cinnamon or PCB2-fraction to control rats was not noticed and are similar to untreated control animals. 3.2. Diabetes induced protein glycation was inhibited by cinnamon and PCB2 Glycated hemoglobin (HbA1c) levels in untreated diabetic rats were significantly higher compared with control animals (Table 2). While feeding of PCB2-fraction ameliorated HbA1c accumulation in experimental diabetic rats, cinnamon has no significant effect in preventing HbA1c accumulation in diabetic rats (Table 2).
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Table 2 Effect of cinnamon and PCB2 on body weight, blood glucose, glycosylated haemoglobin, plasma urea, and albumin to creatinine ratio in diabetic rats. These parameters were measured at the end of 12 weeks of chronic hyperglycemia. Data were mean ± SE (n = 6) and superscript (*) denotes significantly different from control and superscript (**) denotes significantly different from diabetes (p < 0.05). Parameter
Control
Diabetic (D)
D + cinnamon
D+PCB2
Body weight (g) Blood glucose (mg/dl) Glycosylated haemoglobin, HbA1c (%) Urea in plasma (mg/dl) Albumin/creatinine (lg/mg)
264 ± 5.0 89 ± 2.0 6.72 ± 0.54 35.3 ± 6 1760 ± 135
217 ± 4.9⁄ 365 ± 13.0⁄ 11.9 ± 0.72⁄ 82 ± 5⁄ 5870 ± 225⁄
234 ± 4.9 329 ± 9.2⁄ 10.8 ± 0.61 71 ± 7 3920 ± 236⁄⁄
241 ± 6.2 311 ± 10.2⁄ 9.00 ± 0.36⁄⁄ 44 ± 4⁄⁄ 1710 ± 214⁄⁄
Further, we have investigated the ability of cinnamon and PCB2 to prevent cross-linking of IgG on red blood cell surface (RBC-IgG). During diabetic conditions, there is a considerable increase in RBC-IgG cross-linking that provide an index of AGE mediated protein cross-linking. We assayed the extent of RBC-IgG cross-linking in experimental and treated rats. Dietary supplementation with cinnamon and PCB2 significantly decreased RBC-IgG cross-linking to 45% and 40% respectively compared to that of diabetic rats (70%) (Fig. 1A). Further, we investigated whether cinnamon and PCB2 prevent the accumulation of CML, an AGE that is abundant in renal tissues of diabetic rats. Immunohistochemical examination revealed that cinnamon and PCB2 prevented the CML formation in glomeruli of diabetic rats (Fig. 1B). Immunoblotting also substantiate immunohistochemical findings that cinnamon and PCB2 prevented the CML formation in kidney of diabetic rats (Fig. 2). The cytotoxicity of AGE in cells is mediated via cell-surface receptor for AGE (RAGE). Therefore, we measured expression of RAGE in these experimental animals. Increased expression of RAGE was observed in diabetic rat kidney compared with control rats. Interestingly, cinnamon and PCB2 prevented hyperglycemia induced expression of RAGE in diabetic rat kidney (Fig. 2). 3.3. Diabetes induced PKCa expression is prevented by dietary cinnamon and PCB2 intervention The activation of protein kinase C-a (PKC-a) in the kidney from diabetic animal is well known and increased expression in podocytes in renal biopsies of patients with DN has been reported. It is
evident from earlier studies that AGE induce activation of PKC-a [17]. This high glucose inducible kinase orchestrates nephrin internalization in podocytes that consequences into impaired glomerular filtration barrier and proteinuria. Therefore we measured PKC-a in experimental rats. In accordance with earlier studies, immunohistochemical staining revealed that PKC-a expression is increased in glomeruli from diabetic rat (Fig. 3). Interestingly, cinnamon and PCB2 feeding prevented the hyperglycemia mediated expression of PKC-a in diabetic rat kidney (Fig. 3). To elucidate possible mediators of PKC-a in renal damage under hyperglycemia conditions, we analyzed glomerular expression of VEGF. In untreated diabetic rats, increase in VEGF expression was observed (Fig. 3). In contrast, this increase in VEGF expression is significantly reduced in diabetic rats fed with PCB2-fraction, but not completely prevented in cinnamon fed rats (Fig. 3). 3.4. Anti-AGE effect of cinnamon and PCB2 prevented diabetes induced expression of cell adhesion and inflammatory molecules Mononuclear cells such as monocytes/macrophages and T-cells are considered to be involved in the progression of DN, although the mechanism of their recruitment into diabetic glomeruli is unclear. Cell adhesion molecules promote endothelial dysfunction through perturbations of coagulation, permeability, vasomotor function and cell adhesion, leading to the development of macro and microvascular renal lesions. In the present study, we investigated the expression of intercellular adhesion molecule-1 (ICAM) in the glomeruli of STZ-induced experimental diabetic rats. The
Fig. 1. Cinnamon and PCB2 inhibit protein glycation and AGE formation in diabetic rats. The in vivo anti-AGE effect of cinnamon and PCB2 was assessed by estimation of AGE mediated RBC-IgG cross-linking (A), immunohistochemical analysis of glomerular accumulation of CML (B). Data in panel A were presented as mean ± SE (n = 6). (⁄p < 0.01, vs. control rats; ⁄⁄p < 0.01, vs. diabetic rats) and data in panel B are representative of three independent analyses. The images in panel B were taken at 400 magnification.
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3.5. Cinnamon and PCB2 prevented depletion of podocyte slitdiaphragm proteins We analyzed expression of nephrin and podocin in diabetic rats. When RT-PCR studies were performed, it was found that mRNA expression of both nephrin and podocin were decreased 40% in diabetic rat glomerulus (Fig. 5). Dietary supplementation of cinnamon and its active PCB2-fraction attenuated the hyperglycemia mediated loss of glomerular nephrin and podocin expression. In concurrence with RT-PCR analysis, immunohistochemical analysis found diminished expression of nephrin and podocin in untreated diabetic rats whereas cinnamon and PCB2 supplementation mitigated the loss of glomerular nephrin and podocin expression (Fig. 6). Further, immunoblotting analysis also supports the diminished expression of nephrin in untreated diabetic rats and its mitigation by cinnamon and PCB2 supplementation (Fig. 6B). 3.6. Cinnamon and PCB2 ameliorate proteinuria in diabetic rats
Fig. 2. Panel A – immunoblotting analysis of CML and RAGE from control, diabetic and diabetic rats treated with cinnamon or PCB2. Panel B – densitometric quantification of expression of CML and RAGE normalized to b-actin. Data in panel B were presented as mean ± SEM, n = 6. (⁄p < 0.01, vs. control rats; ⁄⁄p < 0.01, vs. diabetic rats).
expression of ICAM was not induced during diabetes (Fig. 4). Alternatively we have also measured vascular cell adhesion molecule-1 (VCAM) in diabetic rats. There was increased expression of VCAM in untreated diabetic rats (Fig. 4). Though dietary supplementation of cinnamon had meager effect on VCAM expression in diabetic rats, PCB2-fraction significantly prevented the hyperglycemia induced VCAM expression (Fig. 4). Monocyte chemoattractant protein-1 (MCP-1), an inflammatory cytokine, levels are elevated in response to AGE [18] and activates the macrophage infiltration into renal tissue. Tissue macrophages mediate variety of pathologies including obesity, insulin resistance and DN. Elevated expression of MCP-1 in untreated diabetic rats was prevented with dietary supplementation of cinnamon and PCB2 (Fig. 4).
To assess renal function, we estimated urinary albumin and creatinine content in diabetic rats. Diabetic rats excreted significantly elevated levels of albumin compared to control rats (117.4 vs. 14.08 mg/24 h). Urinary albumin content is significantly decreased in rats fed with cinnamon and PCB2-fraction (62.72 & 17.1 mg/ 24 h). Diabetic rats excreted greater amounts of creatinine compared with non-diabetic control rats (20 vs. 8 mg/24 h) whereas feeding diabetic rats with cinnamon and PCB2-fraction (16.0 & 10.0 mg/24 h) manifested in the diminished excretion of creatinine which is also reflected in albumin to creatinine ratio (Table 2). Noticeably, PCB2 is more effective in preventing creatinine excretion in diabetic rats than cinnamon. Furthermore, we estimated urea content in the plasma. Diabetic animals displayed 82 ± 5 mg/ml urea in plasma against 35 ± 6 mg/ml in control rats. Plasma urea content is restricted to 71 ± 7 and 44 ± 4 mg/ml in cinnamon and PCB2 fed rats, respectively (Table 2). 4. Discussion Diabetes mellitus is associated with a myriad of deviations from normal homeostasis that results in an array of complications. The accelerated accumulation of AGE cross-links is a predominant factor in the pathogenesis of diabetic complications. The adverse consequences of AGE formation were first described in the renal and cardiovascular systems of humans and diabetic rats [19,20]. AGE formation is associated with renal injury and activation of diverse signal transduction systems in various kidney cells including podocytes that eventually results in proteinuria, a hall mark feature of
Fig. 3. Cinnamon and PCB2 mitigate the glomerular PKC-a and VEGF expression in diabetic rats: Immunohistochemical staining using anti-PKC-a (upper panel) and antiVEGF antibodies (lower panel) on paraffin sections of kidney cortex of control, diabetic, diabetic rats treated with cinnamon and PCB2. Original images were taken at 400 magnification. Data are representative of three independent analyses.
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Fig. 5. Inhibition of AGE by cinnamon and PCB2 preserve nephrin and podocin expression in glomeruli from diabetic rats: Quantitative RT-PCR analysis of nephrin (A) and podocin (B) in renal extract from four different experimental groups. Data were mean ± SEM, n = 6. (⁄p < 0.01, vs. control rats; ⁄⁄p < 0.01, vs. diabetic rats.)
Fig. 4. Cinnamon and PCB2 prevented renal expression of cell adhesion and inflammatory molecules in diabetic rats: quantitative RT-PCR analysis of steady state expression of ICAM (A), VCAM (B) and MCP-1 (C) in control, diabetic and diabetic rats treated with either cinnamon or PCB2. Data were mean ± SEM, n = 6. (⁄p < 0.01, vs. control rats; ⁄⁄p < 0.01, vs. diabetic rats.)
renal diseases [5,21]. The cardinal finding of our study was cinnamon and in particular, its active component PCB2 prevented the accumulation of CML, predominant AGE in renal tissues of diabetic rats. AGE dependent expression of RAGE and inflammatory cytokine, MCP-1 was concealed with cinnamon and PCB2. Our studies also highlight that cinnamon and PCB2 mitigate AGE induced loss of nephrin, the podocyte slit diaphragm protein and offer salutary effects in improving the proteinuria in diabetic rats. However, unlike earlier studies [22] we have not noticed a significant hypoglycemic effect of cinnamon. Podocyte foot processes, GBM, and the fenestrated endothelial cells together form the GFB. GFB ensures the filtration of low molecular weight solutes and thus preventing the loss of proteins such as albumin into urine. The cell–cell junctions between the podocyte foot processes are bridged by slit diaphragm, and the functional integrity of slit diaphragm is critical for preserving normal renal function. Proteinuria ensues when the structure of podocytes is destroyed by disruption of the slit diaphragm. Nephrin, a key component of podocyte slit-diaphragm, plays a determinative role in the integrity of GFB. The essential role of nephrin in the renal function was evident from the studies showing that
mutations in the nephrin gene (NPHS1) associated with congenital nephrotic syndrome [23]. A positive correlation between altered nephrin expression and proteinuria has been shown in experimental animals injected with mAb nephrin [24] and in case of puromycin aminonucleoside-induced nephrosis [25]. It was shown that diabetic hypertensive rats develop albuminuria concomitance with a reduction in nephrin expression [26]. Similarly, in rats with STZinduced diabetes and in nonobese diabetic mice, nephrin got redistributed and lost in the urine [4,27]. Also, in patients with type 1 and type 2 diabetes and nephropathy, expression of nephrin was markedly reduced [28]. Plethora of evidence suggests that nephrin loss could be a critical event in the progression of DN, thus conditioning the onset of proteinuria [4,27–31]. Analogously, in the current study, we have noticed diminished expression of nephrin in diabetic rats; interestingly, cinnamon and PCB2 ameliorated the expression of nephrin in diabetic conditions. There could be two possible explanations for observed loss of nephrin expression in STZ-diabetic rats that are mutually inclusive. One explanation comes from reported studies that non-enzymatically glycated proteins via activation of their receptors (RAGE) repress nephrin expression in diabetic kidney [28]. Furthermore, AGE also induces MCP-1 [32] and its induction has been implicated in the development and progression of DN as evidenced by both human and experimental studies [33,34]. MCP-1 secreted by mesangial cells plays a key role in migration of monocytes and macrophages into kidney which is an index of tissue damage during progression of DN. Furthermore, architecture of slit-diaphragm is altered by MCP-1 via down regulating nephrin expression [35]. Patients with diabetes show gradual increase in urinary levels of MCP-1 that correlate with clinical severity of the disease [36]. In
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Fig. 6. Panel A – immunohistochemical analysis of glomerular expression of nephrin (upper panel) and podocin (bottom panel); immunoblotting analysis of nephrin (B) and densitometric quantification of expression of nephrin (C). Data in panel A are representative of three independent analyses. Data in panel C were mean ± SEM, n = 6. (⁄p < 0.01, vs. control rats; ⁄⁄p < 0.01, vs. diabetic rats.)
the current study, as expected MCP-1 expression was elevated in diabetic rats. Interestingly, dietary supplementation of cinnamon and PCB2 inhibited the renal expression of MCP-1 in diabetic rats. AGE dependent loss of expression is not only pertinent to nephrin but AGE also interact with a-actinin-4 (ACTN-4), a component of slit diaphragm. In the presence of AGE, ACTN-4 expression by podocytes is significantly decreased [37]. Therefore, the interventions that can slacken the AGE formation in kidney and prevent AGE mediated manifestation on proteins that constitute slit-diaphragm offer the pharmacological promise. Based on the results, we propose a model (Fig. 7) that cinnamon and PCB2 mitigate formation of AGE (CML) and suppress the consequent events that abate the nephrin expression. Thus, antiglycating potential of PCB2-fraction offers salutary effect in preventing proteinuria in experimental diabetic rats. Alternatively, hyperglycemia induced transcriptional repression of nephrin could also be regulated by PKC-a [38]. PKC-a, a major intracellular mediator of hyperglycemia induced glomerular permeability represses a transcription factor WT1, which has the binding site in the nephrin promoter [39]. Animals devoid of PKC-a are protected against the development of albuminuria [40]. In the present study, histological examination revealed elevated expression of PKC-a in diabetic rat kidney is concomitant with decreased expression of nephrin. Interestingly, PCB2-fraction feeding ameliorated the expression of PKC-a. Noticeably, PCB2-enriched fraction of cinnamon was more potent than cinnamon in inhibiting PCK-a expression in diabetic rat kidney. The ability of PCB2 in preventing PCK-a expression greater than its source cinnamon, argue for the improved renoprotective effect of PCB2 over cinnamon. Also, glomerular expression of adhesion molecules such as VCAM was shown to increase in diabetic human and rodent kidneys. It was reported that AGE induce expression of these adhesion molecules that promote progression of DN by facilitating infiltration of macrophages into kidney [41]. Interestingly, in the current study, elevated expression of AGE and VCAM was abrogated by PCB2.
Fig. 7. Proposed model for the in vivo role of cinnamon and PCB2 in combating proteinuria in diabetes: cinnamon and its active component PCB2 effectively inhibited CML formation in diabetic rats, which in turn prevented AGE dependent activation of PKC-a and elevated expression of inflammatory molecule MCP1. Via sequel of events anti-AGE effect of cinnamon and PCB2 in diabetic rats preserved the nephrin expression thereby prevented proteinuria and nephropathy.
Nephrin expression is being considered as an index of glomerular function, an effort to preserve its expression in clinical conditions such as DN display a great therapeutic promise against an array of renal diseases during which loss of nephrin is a predisposing factor. In an earlier study, the loss of nephrin expression induced by glycated albumin was prevented with neutralizing antibodies to RAGE, suggesting the involvement of RAGE in the regulation of nephrin expression [28]. It was also shown that inhibition of PKC-a stabilizes nephrin expression and prevents proteinuria in streptozotocin induced diabetes in mice [42]. Recently, we have
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also reported that a dietary flavonoid, ellagic acid, which has antiglycating potential was effective in reducing atherosclerotic process by blocking proliferation of vascular smooth muscle cells [43]. However, in our study, nephrin expression was preserved with diet based ingredients that serve as antiglycating agents. To best of our knowledge, this is a first study to report dietary interventions ameliorate nephrin expression in a clinical condition and it could be a promising therapeutic target for diabetic nephropathy. 5. Conclusions In conclusion, the results obtained in the present study suggest that cinnamon and particularly its active principle PCB2 ameliorated STZ-induced proteinuria and podocyte injury in rats. This effect, at least in part, could be attributed to suppressing renal AGE-RAGE stimulated MCP-1 and PKC-a expression, thereby modulating slit diaphragm proteins nephrin and podocin expression (Fig. 7). Therefore, preventing the loss of nephrin expression impedes diabetes induced proteinuria in experimental animals that fed with either cinnamon or its PCB2-fraction. Furthermore, this study emphasizes the pivotal role that AGE inhibitors could play in the prevention of DN and offers an important nutraceutical based therapeutic approach to combat diabetic kidney diseases.
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Conflicts of Interest G.B.R – received grants from the Department of Biotechnology, India under 7th FP of Indo-EU collaborative grant on functional foods (Grant agreement #245030). P.M. – received a research fellowship from University Grants Commission, India; PAK – received INSPIRE-Faculty fellowship from Department of Science & Technology, India. The authors have received nephrin antibody as gratis from Dr. Rakesh Verma, University of Michigan, Ann Arbor, USA. The work presented in this manuscript forms a part of the patent titled ‘Antiglycating potential of procyanidin-B2 isolated from cinnamon bark: Prospects for prevention or treatment of diabetic complications (cataract, retinopathy & nephropathy)’. Patent application (No. 1452/DEL/2012) with complete specifications was submitted on 21.11.2013 at Patent Office, New Delhi, India. But for the above disclosures, all the authors declare no conflict of interests.
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[16]
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Transparency Document [22]
The 10.1016/j.cbi.2014.08.013 associated with this article can be found in the online version.
Acknowledgements The authors thank Ms. K. Sharadha (Pathology Department, National Institute of Nutrition) for her help in preparing the kidney sections and Dr. Rakesh Verma, University of Michigan, Ann Arbor for providing nephrin antibody.
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[24]
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[26]
References [27] [1] N.I.o.H. US Renal Data System. USRDS 2009 Annual Data Report: Atlas of ESRD, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD, 2009. [2] H. Cheng, R.C. Harris, The glomerulus – a view from the outside – the podocyte, Int. J. Biochem. Cell Biol. 42 (2010) 1380–1387. [3] G. Wolf, S. Chen, F.N. Ziyadeh, From the periphery of the glomerular capillary wall toward the center of disease: podocyte injury comes of age in diabetic nephropathy, Diabetes 54 (2005) 1626–1634. [4] P. Aaltonen, P. Luimula, E. Astrom, T. Palmen, T. Gronholm, E. Palojoki, I. Jaakkola, H. Ahola, I. Tikkanen, H. Holthofer, Changes in the expression of
[28]
[29]
75
nephrin gene and protein in experimental diabetic nephropathy, Lab. Invest. 81 (2001) 1185–1190. T. Wendt, N. Tanji, J. Guo, B.I. Hudson, A. Bierhaus, R. Ramasamy, B. Arnold, P.P. Nawroth, S.F. Yan, V. D’Agati, A.M. Schmidt, Glucose, glycation, and RAGE: implications for amplification of cellular dysfunction in diabetic nephropathy, J. Am. Soc. Nephrol. 14 (2003) 1383–1395. M. Daroux, G. PrÃÓvost, H. Maillard-Lefebvre, C. Gaxatte, E. Boulanger, V.D. D’Agati, A.M. Schmidt, Advanced glycation end-products: implications for diabetic and non-diabetic nephropathies, Diabetes Metab. 36 (2010) 1–10. R. Singh, A. Barden, T. Mori, L. Beilin, Advanced glycation end-products: a review, Diabetologia 44 (2001) 129–146. H. Vlassara, L.J. Striker, S. Teichberg, H. Fuh, Y.M. Li, M. Steffes, Advanced glycation end products induce glomerular sclerosis and albuminuria in normal rats, Proc. Natl. Acad. Sci. U.S.A. 91 (1994) 11704–11708. T.M. Wendt, N. Tanji, J. Guo, T.R. Kislinger, W. Qu, Y. Lu, L.G. Bucciarelli, L.L. Rong, B. Moser, G.S. Markowitz, G. Stein, A. Bierhaus, B. Liliensiek, B. Arnold, P.P. Nawroth, D.M. Stern, V.D. D’Agati, A.M. Schmidt, RAGE drives the development of glomerulosclerosis and implicates podocyte activation in the pathogenesis of diabetic nephropathy, Am. J. Pathol. 162 (2003) 1123–1137. L.J. Jensen, L. Denner, B.F. Schrijvers, R.G. Tilton, R. Rasch, A. Flyvbjerg, Renal effects of a neutralising RAGE-antibody in long-term streptozotocin-diabetic mice, J. Endocrinol. 188 (2006) 493–501. E. Stitt-Cavanagh, L. MacLeod, C. Kennedy, The podocyte in diabetic kidney disease, Sci. World J. 9 (2009) 1127–1139. M. Saraswat, P.Y. Reddy, P. Muthenna, G.B. Reddy, Prevention of non-enzymic glycation of proteins by dietary agents: prospects for alleviating diabetic complications, Br. J. Nutr. 101 (2009) 1714–1721. P. Muthenna, G. Raghu, C. Akileshwari, S.N. Sinha, P. Suryanarayana, G.B. Reddy, Inhibition of protein glycation by procyanidin-B2 enriched fraction of cinnamon: delay of diabetic cataract in rats, IUBMB Life 65 (2013) 941–950. Z. Zhang, B.Y. Li, X.L. Li, M. Cheng, F. Yu, W.D. Lu, Q. Cai, J.F. Wang, R.H. Zhou, H.Q. Gao, L. Shen, Proteomic analysis of kidney and protective effects of grape seed procyanidin B2 in db/db mice indicate MFG-E8 as a key molecule in the development of diabetic nephropathy, Biochim. Biophys. Acta (1832) 805–816. M. Saraswat, P. Suryanarayana, P.Y. Reddy, M.A. Patil, N. Balakrishna, G.B. Reddy, Antiglycating potential of Zingiber officinalis and delay of diabetic cataract in rats, Mol. Vis. 16 (2010) 1525–1537. S. Vasan, X. Zhang, A. Kapurniotu, J. Bernhagen, S. Teichberg, J. Basgen, D. Wagle, D. Shih, I. Terlecky, R. Bucala, A. Cerami, J. Egan, P. Ulrich, An agent cleaving glucose-derived protein crosslinks in vitro and in vivo, Nature 382 (1996) 275–278. V. Scivittaro, M.B. Ganz, M.F. Weiss, AGEs induce oxidative stress and activate protein kinase C-beta(II) in neonatal mesangial cells, Am. J. Physiol. Renal Physiol. 278 (2000) F676–683. Y. Ishibashi, T. Matsui, M. Takeuchi, S. Yamagishi, Beneficial effects of metformin and irbesartan on advanced glycation end products (AGEs)RAGE-induced proximal tubular cell injury, Pharmacol. Res. 65 (2012) 297– 302. M. Brownlee, A. Cerami, H. Vlassara, Advanced glycosylation end products in tissue and the biochemical basis of diabetic complications, N. Engl. J. Med. 318 (1988) 1315–1321. M. Brownlee, A. Cerami, H. Vlassara, Advanced products of nonenzymatic glycosylation and the pathogenesis of diabetic vascular disease, Diabetes Metab. Rev. 4 (1988) 437–451. Z. Makita, S. Radoff, E.J. Rayfield, Z. Yang, E. Skolnik, V. Delaney, E.A. Friedman, A. Cerami, H. Vlassara, Advanced glycosylation end products in patients with diabetic nephropathy, N. Engl. J. Med. 325 (1991) 836–842. R. Deng, A review of the hypoglycemic effects of five commonly used herbal food supplements, Recent Pat. Food Nutr. Agric. 4 (2012) 50–60. M. KestilÃ, U. Lenkkeri, M. MÃnnikkÃ, J. Lamerdin, P. McCready, H. Putaala, V. Ruotsalainen, T. Morita, M. Nissinen, R. Herva, C.E. Kashtan, L. Peltonen, C. Holmberg, A. Olsen, K. Tryggvason, Positionally cloned gene for a novel glomerular protein nephrin is mutated in congenital nephrotic syndrome, Mol. Cell 1 (1998) 575–582. P.S. Topham, H. Kawachi, S.A. Haydar, S. Chugh, T.A. Addona, K.B. Charron, L.B. Holzman, M. Shia, F. Shimizu, D.J. Salant, Nephritogenic mAb 5-1-6 is directed at the extracellular domain of rat nephrin, J. Clin. Invest. 104 (1999) 1559– 1566. M. Hosoyamada, K. Yan, Y. Nishibori, Y. Takiue, A. Kudo, H. Kawakami, T. Shibasaki, H. Endou, Nephrin and podocin expression around the onset of puromycin aminonucleoside nephrosis, J. Pharm. Sci. 97 (2005) 234–241. F. Bonnet, M.E. Cooper, H. Kawachi, T.J. Allen, G. Boner, Z. Cao, Irbesartan normalises the deficiency in glomerular nephrin expression in a model of diabetes and hypertension, Diabetologia 44 (2001) 874–877. J.M. Forbes, F. Bonnet, L.M. Russo, W.C. Burns, Z. Cao, R. Candido, H. Kawachi, T.J. Allen, M.E. Cooper, G. Jerums, T.M. Osicka, Modulation of nephrin in the diabetic kidney: association with systemic hypertension and increasing albuminuria, J. Hypertens. 20 (2002) 985–992. S. Doublier, G. Salvidio, E. Lupia, V. Ruotsalainen, D. Verzola, G. Deferrari, G. Camussi, Nephrin expression is reduced in human diabetic nephropathy: evidence for a distinct role for glycated albumin and angiotensin II, Diabetes 52 (2003) 1023–1030. Y. Wu, J. Dong, L. Yuan, C. Liang, K. Ren, W. Zhang, F. Fang, J. Shen, Nephrin and podocin loss is prevented by mycophenolate mofetil in early experimental diabetic nephropathy, Cytokine 44 (2008) 85–91.
76
P. Muthenna et al. / Chemico-Biological Interactions 222 (2014) 68–76
[30] R.G. Langham, D.J. Kelly, A.J. Cox, N.M. Thomson, H. Holthofer, P. Zaoui, N. Pinel, D.J. Cordonnier, R.E. Gilbert, Proteinuria and the expression of the podocyte slit diaphragm protein, nephrin, in diabetic nephropathy: effects of angiotensin converting enzyme inhibition, Diabetologia 45 (2002) 1572–1576. [31] D.J. Kelly, P. Aaltonen, A.J. Cox, J.R. Rumble, R. Langham, S. Panagiotopoulos, G. Jerums, H. Holthofer, R.E. Gilbert, Expression of the slit-diaphragm protein, nephrin, in experimental diabetic nephropathy: differing effects of antiproteinuric therapies, Nephrol. Dial. Transplant. 17 (2002) 1327–1332. [32] S. Yamagishi, Y. Inagaki, T. Okamoto, S. Amano, K. Koga, M. Takeuchi, Z. Makita, Advanced glycation end product-induced apoptosis and overexpression of vascular endothelial growth factor and monocyte chemoattractant protein-1 in human-cultured mesangial cells, J. Biol. Chem. 277 (2002) 20309–20315. [33] N. Banba, T. Nakamura, M. Matsumura, H. Kuroda, Y. Hattori, K. Kasai, Possible relationship of monocyte chemoattractant protein-1 with diabetic nephropathy, Kidney Int. 58 (2000) 684–690. [34] F.Y. Chow, D.J. Nikolic-Paterson, E. Ozols, R.C. Atkins, B.J. Rollin, G.H. Tesch, Monocyte chemoattractant protein-1 promotes the development of diabetic renal injury in streptozotocin-treated mice, Kidney Int. 69 (2006) 73–80. [35] E. Tarabra, S. Giunti, F. Barutta, G. Salvidio, D. Burt, G. Deferrari, R. Gambino, D. Vergola, S. Pinach, P.C. Perin, G. Camussi, G. Gruden, Effect of the monocyte chemoattractant protein-1/CC chemokine receptor 2 system on nephrin expression in streptozotocin-treated mice and human cultured podocytes, Diabetes 58 (2009) 2109–2118. [36] G.H. Tesch, MCP-1/CCL2: a new diagnostic marker and therapeutic target for progressive renal injury in diabetic nephropathy, Am. J. Physiol. Renal Physiol. 294 (2008) F697–701.
[37] T.S. Ha, High glucose and advanced glycosylated end-products affect the expression of alpha-actinin-4 in glomerular epithelial cells, Nephrology (Carlton) 11 (2006) 435–441. [38] I. Quack, M. Woznowski, S.A. Potthoff, R. Palmer, E. Konigshausen, S. Sivritas, M. Schiffer, J. Stegbauer, O. Vonend, L.C. Rump, L. Sellin, PKC alpha mediates beta-arrestin2-dependent nephrin endocytosis in hyperglycemia, J. Biol. Chem. 286 (2011) 12959–12970. [39] J. Menne, M. Meier, J.K. Park, M. Boehne, T. Kirsch, C. Lindschau, R. Ociepka, M. Leitges, J. Rinta-Valkama, H. Holthofer, H. Haller, Nephrin loss in experimental diabetic nephropathy is prevented by deletion of protein kinase C alpha signaling in-vivo, Kidney Int. 70 (2006) 1456–1462. [40] M. Meier, J.K. Park, D. Overheu, T. Kirsch, C. Lindschau, F. Gueler, M. Leitges, J. Menne, H. Haller, Deletion of protein kinase C-beta isoform in vivo reduces renal hypertrophy but not albuminuria in the streptozotocin-induced diabetic mouse model, Diabetes 56 (2007) 346–354. [41] H. Vlassara, H. Fuh, T. Donnelly, M. Cybulsky, Advanced glycation endproducts promote adhesion molecule (VCAM-1, ICAM-1) expression and atheroma formation in normal rabbits, Mol. Med. 1 (1995) 447–456. [42] I. Tossidou, B. Teng, J. Menne, N. Shushakova, J.K. Park, J.U. Becker, F. Modde, M. Leitges, H. Haller, M. Schiffer, Podocytic PKC-alpha is regulated in murine and human diabetes and mediates nephrin endocytosis, PLoS One 5 (2010) e10185. [43] P.U. Rani, R. Kesavan, R. Ganugula, P.U. Kumar, G.B. Reddy, M. Dixit, Ellagic acid inhibits PDGF-BB-induced vascular smooth muscle cell proliferation and prevents atheroma formation in streptozotocin-induced diabetic rats, J. Nutr. Biochem. 24 (2013) 1830–1839.