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Increased renal semicarbazide-sensitive amine oxidase activity and methylglyoxal levels in aristolochic acid-induced nephrotoxicity
Life Sciences 114 (2014) 4–11
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Increased renal semicarbazide-sensitive amine oxidase activity and methylglyoxal levels in aristolochic acid-induced nephrotoxicity Tzu-Chuan Huang, Shih-Ming Chen, Yi-Chieh Li, Jen-Ai Lee ⁎ School of Pharmacy, College of Pharmacy, Taipei Medical University, No. 250, Wuxing St., Taipei 11031, Taiwan
a r t i c l e
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Article history: Received 28 February 2014 Accepted 25 July 2014 Available online 8 August 2014 Keywords: Aristolochic acid Nephrotoxicity Semicarbazide-sensitive amine oxidase Methylglyoxal Nε-(carboxymethyl)lysine Metformin
a b s t r a c t Aims: Aristolochic acid (AA) nephrotoxicity is related to accumulation of methylglyoxal (MGO) and Nε(carboxymethyl)lysine (CML) in the mouse kidney. We studied the activity of renal semicarbazide-sensitive amine oxidase (SSAO), a key enzyme involved in MGO generation, in AA-treated mice, and investigated nephroprotective effects produced by metformin, a MGO scavenger. Methods: Mice were orally administered water or metformin for 15 days (12 or 24 mg kg−1 day−1), and injected AA (5 mg kg−1 day−1) intraperitoneally for 8 days starting on day 8. Renal function was studied, and histopathological examination, determination of renal SSAO activity, and measurement of MGO levels were performed. Key findings: Compared to control mice, AA-injected mice showed significant renal damage and approximately 2.7-fold greater renal SSAO activity (p b 0.05). Further, compared to control treatment, administration of 12 mg/kg metformin inhibited formation of renal lesions, and significantly decreased renal MGO levels (37.33 ± 9.78 vs. 5.89 ± 2.64 μg/mg of protein, respectively, p b 0.01). In the AA-treated mice, metformin also inhibited the accumulation of CML in renal tubules, but did not affect SSAO activity. Significance: This study is the first to show elevated renal SSAO activity in AA-treated mice, which could be involved in MGO accumulation. Moreover, MGO scavenging by metformin reduces AA nephrotoxicity. These findings suggest that reducing MGO accumulation produces nephroprotection, revealing new therapeutic strategies for the management. SSAO is a key enzyme involved in MGO generation, and consequently, inhibition of renal SSAO activity is worth investigating in AA nephrotoxicity and other renal pathologies further. © 2014 Elsevier Inc. All rights reserved.
Introduction Aristolochic acid (AA), a compound found in plants of the genus Aristolochia, causes nephrotoxicity (AA-induced nephrotoxicity, or AAN) characterized by initial damage to the proximal renal tubule, with later development of interstitial fibrosis and urothelial carcinoma {Debelle et al., 2002}. The U.S. Food and Drug Administration has recommended cessation of the use of botanical products containing AA {Schwetz, 2001}. However, products containing AA remain available online, and some herbal remedies have been found to be contaminated with AA, which has led to cases of AAN {Gold and Slone, 2003; Li et al., 2011}. There is no established optimal clinical recommendation for AAN therapy. Steroid therapy slows the progression of renal failure that characteristically accompanies progressive interstitial fibrosis in AAN {Vanherweghem et al., 1996}. However, steroid therapy is not effective in all cases of AAN, and long-term administration of steroids is associated with major side effects. Currently, the top priorities in the management of AAN are to control the progression of nephropathy ⁎ Corresponding author. Tel.: +886 2 2736 1661x6125; fax: +886 2 2736 1661x6120. E-mail addresses: [email protected] (T.-C. Huang), [email protected] (S.-M. Chen), [email protected] (Y.-C. Li), [email protected] (J.-A. Lee).
http://dx.doi.org/10.1016/j.lfs.2014.07.034 0024-3205/© 2014 Elsevier Inc. All rights reserved.
and to prepare for renal replacement therapy {Gokmen et al., 2013}. Therefore, it is important to develop a clinically effective and safe treatment for AAN. Accumulation of methylglyoxal (MGO) and Nε-(carboxymethyl)lysine (CML) in kidney tissue has been demonstrated in a mouse model of AAN Li et al., 2012. MGO is an endogenous, highly-reactive, and cytotoxic aldehyde compound {Inoue and Kimura, 1995}. It can react with and modify DNA and other biomolecules, which alters their structures and functions, and results in the formation of advanced glycation end-products (AGEs), such as CML {Brownlee, 1994}. We speculated that renal MGO and CML were related to damage produced by AA, but the causes of AA-induced MGO generation and the effect of MGO reduction on AAN have not been investigated. Semicarbazide-sensitive amine oxidase (SSAO; EC 1.4.3.21) is involved in a MGO generation pathway that converts aminoacetone into MGO {Inoue and Kimura, 1995}. SSAO is found in several tissues, with particularly high activity in kidney {Lewinsohn, 1984; Lyles and Singh, 1985}. SSAO activates profibrogenic cytokines {Wong et al., 2013} that are involved in the progression of renal fibrosis that is characteristic of AAN. The activity of renal SSAO may be closely related to the production of MGO and the pathological progression of AAN, but this hypothesis has not been verified.
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The MGO inhibitor metformin was used to investigate whether a decrease in MGO levels mitigates the severity of AAN. Metformin (N,Ndimethylimidodicarbonimidic diamide) is generally accepted as the first-line treatment for type 2 diabetes mellitus. In addition, metformin is a MGO scavenger and an inhibitor of the formation of AGEs. The drug has been shown to reduce systemic levels of MGO and AGEs in patients with type 2 diabetes mellitus, and to prevent diabetic complications {Beisswenger et al., 1999; Yamagishi et al., 2008; Ruggiero-Lopez et al., 1999; Kiho et al., 2005 #6;Rahbar and Figarola, 2003}. Metformin has also been shown to ameliorate gentamicin-induced renal toxicity in rats by decreasing renal MGO levels {Amini et al., 2012; Li et al., 2013}. The safety, widespread clinical use, and proven efficacy of metformin make it an ideal MGO inhibitor for evaluation in animals with AAN. This study investigated the relationship between increased renal MGO levels and SSAO activity in renal tissue by using a mouse model of AAN. Furthermore, the MGO inhibitor metformin was administered to confirm that reduction in MGO levels is nephroprotective in AAN mice, and to investigate the effect of metformin on renal SSAO activity.
Materials and methods Animal experiments All animal experiments were approved by the Animal Care and Use Committee of Taipei Medical University, Taiwan. Thirty-two C57BL/6 mice (7-week-old males) were purchased from the National Laboratory Animal Breeding and Research Center (Taipei, Taiwan). These mice were housed in temperature-controlled (25 ± 2 °C) and humiditycontrolled (65% ± 5%) facility with a 12/12 light/dark photoperiod and access to standardized food pellets (TMI, USA) and tap water ad libitum. After 1 week of acclimatization, the mice were divided randomly into 4 groups (each group consisted of 8 mice). Control group: mice treated with water (P.O) and saline (i.p.) AA group: mice treated with water (P.O) and AA (i.p.) AAML group: mice treated with low dose metformin (P.O) and AA (i.p.) AAMH group: mice treated with high dose metformin (P.O) and AA (i.p.) The control group was orally administered 0.1 mL water for 15 days (days 1–15) and intraperitoneally injected with 0.15 mL saline for 8 days (day 8–15). The AA group was orally administered 0.1 mL water for 15 days and intraperitoneally injected with 0.15 mL AA (days 8–15; 5 mg kg− 1 day− 1). The AAML and AAMH groups were treated with 0.1 mL metformin at doses of 12 and 24 mg kg−1 day−1, respectively, for 15 days, and injected with 0.15 mL AA for 8 days (days 8–15; 5 mg kg− 1 day−1). At the end of the treatment period, the mice were placed in rodent metabolic cages with free access to food for 12-h urine collection. At the end of the experiment, the mice were sacrificed under pentobarbital (50 mg/kg), and a blood sample was collected by intracardiac puncture. A thorough necropsy was performed on all mice, and the kidneys were dissected for histopathological evaluation, immunohistochemistry, determination of SSAO activity, and quantification of MGO levels.
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Serum and urinary creatinine assays Creatinine levels in the serum and urinary samples were measured using high-performance liquid chromatography (HPLC), as described previously {Lee et al., 2005; Marsilio et al., 1999}. The measured concentrations were expressed in milligrams per deciliter, and 0.1 mmol/L cimetidine in 0.01 mol/L HCl aqueous solution was used as the internal standard. A Capcell Pak C18 column (250 mm × 4.6 mm, ID; particle size, 5 μm; Shiseido, Tokyo, Japan) was used, and absorbance was monitored at 234 nm. The mobile phase was composed of acetonitrile and 100 mmol/L of PBS containing 30 mmol/L sodium lauryl sulfate adjusted to pH 3.0 with hydrochloric acid (36/60, v/v). The flow rate was 0.8 mL/min. Measurement of the glomerular filtration rate Creatinine clearance (CCr), an index of the glomerular filtration rate (GFR), was calculated using the following equation adapted from Hsu and co-workers {Hsu et al., 2008}: CCr ¼
Ucr UV 100 1 Scr BW 720
Where CCr is creatinine clearance (mL min−1 kg−1 BW); Ucr, urinary creatinine concentration (mg/dL); UV, is urine volume (mL); Scr, serum creatinine (mg/dL); BW, body weight (g); and 720, the period in which urine was collected (21 h, expressed in min). Determination of urinary protein levels and N-acetyl-β-D-glucosaminidase activity Urinary protein levels were quantified using a Bio-Rad protein assay kit with bovine serum albumin as the standard, according to the manufacturer’s instructions. Urinary N-acetyl-β-D-glucosaminidase (NAG, EC 3.2.1.30) activity was determined using the method described by Leaback and Walker {Leaback and Walker, 1961}. NAG reacted with the 4-methylumbelliferyl-N-acetyl-β-glucosaminide, and fluorescence was measured and used to quantify NAG concentration. Determination of blood urea nitrogen concentration The concentration of blood urea nitrogen (BUN) was determined using a urease assay kit (Sigma-Aldrich, St Louis, MO), according to the manufacturer’s instructions. Urea was hydrolyzed by urease to ammonia and carbonate. In the presence of glutamic dehydrogenase, ammonia reacts with ketoglutaric acid with the concurrent oxidation of NADH to NAD. BUN activity is directly proportional to the rate of conversion of NADH to NAD, and it is monitored at 340 nm {Kaltwasser and Schlegel, 1966}. Urinary microalbumin Microalbumin levels were measured using a commercial kit (Good Biotech Corp., Taiwan) based on immunoturbidimetric methods, with procedures performed according to the manufacturer’s instructions. The samples were conditioned with a Tris buffer before incubation with an anti-albumin antibody for 10 min. The samples were then analyzed at 405 nm.
Preparation of urine, blood, and kidney samples Histopathological examination Sediments was removed from the urine samples by centrifugation (700 × g at 4 °C for 5 min), and the supernatant was stored at −80 °C until analysis. The blood samples were centrifuged (700 × g, 15 min) and the serum was stored at − 20 °C. The kidneys were embedded in paraffin, sectioned (thickness, 3–5 μm), and stained with periodic acid-Schiff stain for immunohistochemistry.
A histopathological evaluation was performed on the kidneys of the mice according to the guidelines proposed previously {Sato et al., 2004}. The histopathological severity of the lesions was rated from one to five (1 = minimal; 2 = slight; 3 = moderate; 4 = moderately severe; 5 = severe/high). The sum of scores for each sample, reflecting tubular
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trophy, cell infiltration, and interstitial fibrosis, was defined as the tubulointerstitial histological score.
one-way analysis of variance (ANOVA) and Tukey's studentized range in the SAS 9.3 package (SAS Institute, Inc., Cary, NC, USA). Differences were considered significant at p b 0.05.
SSAO activity assays Results SSAO activity was determined by fluorimetrical technique, using the Amplex Red Monoamine Oxidase Assay kit (Molecular Probes Leiden, Netherlands) to measure hydrogen peroxide production, according to the manufacturer’s instructions. Briefly, 100 μL kidney homogenates were pre-incubated for 30 min at room temperature with 1 μmol/ L L-deprenyl to inhibit any monoamine oxidase-B contamination. The reaction was started by the addition of 100 μL reaction buffer (400 μmol/L Amplex Red, 2 U/mL horseradish peroxidase, 2 mmol/L benzylamine, 50 mmol/L sodium phosphate, pH 7.4) and the fluorescence (excitation, 530 nm; emission, 590 nm) was monitored during 60 min using a Synergy HT microplate reader (Bio-Tek Instruments, Inc., Winooski, VT). A range of H2O2 concentrations were used for the standard calibration. SSAO activity was expressed as pmol/L H2O2 min mg−1 protein. Measurements of MGO in kidney homogenates The determination of MGO levels in kidney homogenates by HPLC was performed according to a previously published method {EspinosaMansilla et al., 2007}. Briefly, 0.5 mol/L ammonium chloride buffer (pH 10.0) was added to the kidney homogenates to create an alkaline environment. Then, 5,6-diamino-2,4-dihydroxy-pyrimidine was used as a derivatization reagent. After incubation for 30 min at 60 °C, the derivatization reaction was stopped with 0.01 mol/L citric acid (pH 6.0). HPLC analysis was performed using an ODS column (Biosil, 250 mm; ID 4.6 mm; particle size, 5 μm; Biosil Chemical Co. Ltd., Taipei, Taiwan) at 33 °C. The MGO derivative was separated using a mobile phase composed of a mixture of acetonitrile and 0.01 mol/L citric acid buffer adjusted to pH 6.0 with NaOH (3/97, v/v), with a flow rate of 0.7 mL/min. Derivatization reduction products were injected in 50-μL volumes, and eluted fractions were analyzed by monitoring fluorescence at excitation and emission wavelengths of 330 and 500 nm, respectively. Immunohistochemistry for CML in kidney section Paraffin-embedded sections (thickness, 3–5 μm) were used to determine the localization of CML. After blocking endogenous peroxidase activity by incubating the sections in 0.3% H2O2 for 10 min, the sections were incubated overnight with CML antibodies (diluted 50 times). The sections were reacted for 1 h with goat anti-rabbit IgGs conjugated with peroxidase-labeled secondary antibodies, diluted 250 times. Peroxidase activity was visualized using the diaminobenzidine stain. Immunohistochemistry was performed without the primary antibody to obtain the negative control (data not shown). Measurement of renal CML levels by ELISA Renal CML were determined using the OxiSelect CML competitive ELISA kit (Cell Biolabs, San Diego, CA) according to the manufacturer’s instructions. First, a CML conjugate was coated on the ELISA plate. Kidney homogenates (150 μg of protein) are then added to the CML conjugate-coated plate. After a brief incubation, the anti-CML monoclonal antibody is added, followed by an HRP conjugated secondary antibody. The quantity of CML adduct in kidney homogenates was determined by comparing its absorbance with that of a known CML− bovine serum albumin standard curve.
Biochemical indices Urinary microalbumin levels in mice Urinary microalbumin levels were significantly increased in all AA-treated mice (AA, AAML, and AAMH groups), as compared to the levels in the control group (Fig. 1A). While the levels measured in both the AAML and AAMH groups were significantly lower than that in the AA group, no significant difference was observed between the AAML and AAMH groups. Urinary NAG levels Fig. 1B illustrates the effect of metformin administration on urinary NAG levels. AA-treated mice that did not receive metformin (the AA group) exhibited higher NAG levels than the control group. Compared with the AA group, the AAML and AAMH groups showed decreased urinary NAG levels, which were not statistically significant. Blood urea nitrogen (BUN) concentration Fig. 1C shows the BUN concentrations of the experimental groups. BUN was significantly elevated in all AA-treated groups, as compared to that in the control group. However, BUN levels in metformintreated mice (both the AAML and AAMH groups) were lower than that of the AA group. This difference was significant only when comparing the AAML group with the AA group (133.11 ± 39.19 vs. 244.88 ± 37.97 mg/dL, respectively, p b 0.05). Creatinine clearance level Creatinine clearance (CCr) was significantly lower in the AA group (1.29 ± 0.59 mL min−1 kg−1 BW) than in the control group (10.18 ± 1.80 mL min− 1 kg−1 BW; Fig. 1D). CCr for AAML and AAMH groups were 3.55 ± 1.13 and 2.12 ± 1.12 mL min−1 kg−1 BW, respectively. CCr was significantly higher in the AAML group than that in the AA group. Morphological findings and changes in tubulointerstitial histological scores In the control mice, all renal tissue samples appeared histologically normal (Fig. 2A). Renal lesions detected on visual inspection and severe changes observed using microscopy indicate that AA exerted a strong nephrotoxic effect (Fig. 2B\D). Tubular atrophy, cell infiltration, and fibrosis, considered to be the primary indices of AA-induced renal damage, were observed in all AA-treated mice (AA, AAML, and AAMH groups). In addition, we observed hyaline casts, tubular dilatation, and degenerative tubular cells, which are also considered an indications of acute tubular necrosis. The morphology of the glomerular portion did not appear to be affected by AA treatment. Median scores for the renal degeneration evidenced by of tubular atrophy, cell infiltration, and interstitial fibrosis were calculated using data collected from each treatment group (Fig. 2E). Severity scores in the AA group were significantly higher than those in the AAML and AAMH groups, indicating reduced renal damage in metformin-treated mice. Treatment with a lower dose of metformin partially reversed the tubulointerstitial nephritis, and the severity of toxic nephropathy in the AAMH group was slightly higher than that in the AAML group. SSAO activity in mouse kidney
Statistical analysis All data were expressed as mean ± SD for the 8 mice in each group. The significance of differences among groups was analyzed using
Renal SSAO activity levels of each treatment group are shown in Fig. 3. SSAO activity in the control group was 0.46 ± 0.10 pmol/L H2O2 min mg− 1 protein, while in the AA group it was approximately
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Fig. 1. Biochemical indices measured in mice: (A) urinary mcroalbumin levels, (B) urinary N-acetyl-β-D-glucosaminidase (NAG), (C) blood urea nitrogen (BUN), (D) creatinine clearance (CCr). The data are presented as means ± SD for each group (n = 8). *p b 0.05, compared with control group; #p b 0.05, compared with AA group.
2.7 times higher (1.21 ± 0.64 pmol/L H2O2 min mg− 1 protein, p b 0.05). SSAO activities in the AAML and AAMH groups were 1.06 ± 0.77 and 1.39 ± 0.86 pmol/L H2O2 min mg− 1 protein, respectively, with no statistically significant differences observed compared to the AA group. MGO levels in mouse kidney Fig. 4A and B show representative HPLC chromatograms that depict the MGO peak used for the quantification of tissue levels. MGO was derivated and demonstrated a detectable fluorescence peak with a retention time of approximately 27 min, with the peak height normalized for the protein concentration (Fig. 4C). MGO levels in the kidneys were determined to be higher in the AA group than in the control group (37.33 ± 9.78 vs 5.89 ± 2.64 μg/mg protein, p b 0.01). Conversely, MGO levels in metformin-treated rats (AAML and AAMH groups) were lower than that in the AA group (9.81 ± 5.04 and 17.79 ± 7.88 vs. 37.33 ± 9.78 μg/mg protein, respectively, p b 0.01). Immunohistological staining for CML and CML levels in mouse kidney An immunohistochemical analysis was performed to evaluate CML localization in mice kidneys, following AA and metformin treatments (Fig. 5). While faint CML staining was detected in the control group (Fig. 5A), a notably higher signal was observed in the AA, AAML, and AAMH groups, especially in the tubular region and basement membrane (Fig. 5B, C, and D). As shown in Fig. 5E, measurements of renal CML using ELISA revealed non-detection in the control group (below the kit detection limit), and showed an increase in the kidneys of AA, AAML, and AAMH group (15.97 ± 3.25, 7.55 ± 3.98, and 7.56 ± 6.04 рg/μg protein, respectively). Compared with AA group, renal CML levels decreased in AAML and AAMH group significantly (p b 0.05).
Discussion In current experiments, we applied a previously described experimental protocol to induce AAN in mice {Baudoux et al., 2012}. Following 8 days of daily intraperitoneal AA injections at 5 mg kg−1 day−1, proximal renal tubular dysfunction and structural destruction were observed using biochemical tests and renal biopsy, reflecting the primary characteristics of acute AAN. Administration regimen used in this study successfully induced AAN in mice, with all animals surviving the entire treatment period. The pathological effects of AA administration were observed primarily in the proximal tubular tissue, with tubular necrosis, atrophy, and degeneration found using renal biopsy. Treatment with AA did not significantly alter glomerular structure, supporting previously reported findings {Sato et al., 2004; Huang et al., 2013; Debelle et al., 2002; Baudoux et al., 2012}. Impaired renal function in AA-injected mice was manifested through alterations in the biochemical indices, such as increased urinary levels of microalbumin, urinary NAG levels, and BUN levels, as well as decreased CCr. The renal levels of MGO were also found to be elevated in the AA-injected mice, while CML was observed to accumulate in tubular cells and the basement membrane. Our histopathological findings suggest that AA primarily affected the tubular cells, causing a leakage of cells and the protrusion of basement membrane. These results correspond to the findings of the previous study {Li et al., 2012}. Excess kidney levels of MGO and CML, therefore, appear to be related to AA-induced damage. Renal SSAO activity increased in the AA-injected mice. SSAO is involved in protein metabolism, and converts aminoacetone to MGO, while CML is a downstream product of MGO metabolism. Increased SSAO activity in mouse kidney is quite likely a factor contributing to the acceleration of MGO and CML formation in AA-induced damage. We speculated that inhibition of SSAO may decrease MGO and CML formation, and elicit nephroprotective effects against AAN. Several studies also suggested that potent inhibition of SSAO may be of therapeutic
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Fig. 2. Morphological findings (periodic acid – Schiff stain) and changes of tubulointerstitial histological scores. Light micrographs of renal tissue of the (A) control, (B) AA, (C) AAML, and (D) AAMH groups. Quantitative analysis of lesions is shown (E). (*): tubular atrophy, (#): cell infiltration, (▲): fibrosis.
value, particularly in the treatment of pathologies related to inflammatory {Tabi et al., 2013; Martelius et al., 2008; O'Rourke et al., 2008}. However, widespread clinical use of previously identified SSAO
Fig. 3. SSAO activity in mouse kidney. The activity of SSAO in the AA, AAML, and AAMH groups were significantly increased, as compared to the control group (mean ± SD, n=8). *p b 0.05, compared with control group; #p b 0.05, compared with AA group.
inhibitors, including hydralazine and semicarbazide, is not recommended, due to the potential for harmful side-effects. Hydralazine is an antihypertension drug that activates gated potassium channels on the vascular smooth muscle, causing an efflux of potassium and a subsequent hyperpolarization of the cell, resulting in vasodilation. It may increase heart rate and cardiac output, potentially causing angina pectoris or a myocardial infarction. Patients treated with hydralazine for a long period may develop a lupus-like syndrome or other immune-related diseases {Handler, 2012}. Semicarbazide is a metabolite of the veterinary drug nitrofurazone. Due to its potential genotoxic and carcinogenic activity {Vlastos et al., 2010 #294;Hirakawa et al., 2003}, the World Health Organization notes that dietary semicarbazide content should be monitored, and uses semicarbazide levels as an indicator for the use of nitrofurazone in animals used for food. Due to of these recognized risks, hydralazine and semicarbazide were not considered ideal SSAO inhibitors, and are seen as unsuitable for use in treatment of renal injury. In order to investigate whether decreased MGO and CML levels are associated with a slowing of the progression of AAN, we treated AAN mice with a MGO and AGE inhibitor metformin. Our results suggested that the administration of metformin (12 or 24 mg kg−1 day−1)
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Fig. 4. MGO levels in the renal tissues of AA-treated mice. MGO was quantified using HPLC, with representative chromatograms of samples from the (A) AA and (B) AAML groups presented. MGO was eluted at 27 min, and its concentration was normalized to the sample protein concentration. The quantification of MGO is shown in (C). * p b 0.05, compared with control group; # p b 0.05, compared with AA group.
decreased MGO and CML levels in the mouse kidney, and ameliorated AA-induced renal damage, as evidenced by the reversal of both histopathological and biochemical indices of nephropathology. Urinary levels of microalbumin and BUN were decreased, while CCr was increased in metformin-treated mice, indicating that administration of metformin improved renal function. The histopathological analysis showed reduced severity of AA-induced renal lesions in metformintreated mice, compared to the pathological appearance in mice treated with AA alone. These findings suggest that the nephroprotective effects
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of metformin in AAN were associated with its MGO- and CMLscavenging ability. Although metformin is considered to be a scavenger of MGO and inhibitor of AGEs {Beisswenger et al., 1999; Yamagishi et al., 2008; Ruggiero-Lopez et al., 1999; Kiho et al., 2005}, its exact mechanism of action has not been clearly elucidated. We measured renal SSAO activities in metformin-treated mice, but did not observe a significant difference in renal SSAO activity between metformin-treated mice and mice treated with AA alone. This finding suggests that metformin decreases MGO through a pathway that does not involve the inhibition of SSAO activity. Previous studies proposed that metformin can trap the carbonyl group of MGO to form an inactive condensation product in vivo {Ruggiero-Lopez et al., 1999; Beisswenger and Ruggiero-Lopez, 2003}, resulting in diminished toxicity of MGO and reduced production of CML. Based on our observations, we believe that the condensation reaction between metformin and MGO could contribute to the nephroprotective effects of metformin in AA-treated mice. In the current study, a high dose of metformin (24 mg kg−1 day−1) elicited less of a nephroprotective effect compared to a low dose (12 mg kg− 1 day−1). Based on the measurements of urinary microalbumin levels, BUN, and CCr, as well as the tubulointerstitial histological findings, renal damage was less severe in the AAML group than in the AAMH group. Renal MGO and CML levels were also lower in the AAML group than in the AAMH group. These observations provided a rationale for a reassessment of the adjustment of metformin dose in the treatment of renal diseases. According to the U.S. Food and Drug Administration, metformin therapy must be used cautiously in patients with renal insufficiency, due to the potential risk of lactic acidosis. However, reducing metformin dosage and monitoring the physiological indicators will allow metformin to be safely used in chronic kidney disease {Scheen, 2013; Duong et al., 2012; Duong et al., 2012}. Metformin has also been shown to protect rat kidney tissue against gentamicin-induced injury {Amini et al., 2012; Li et al., 2013}. Therefore, in patients with nephropathy, metformin dose could be adjusted instead of discontinuing the therapy outright, because appropriate use of metformin might elicite nephroprotection. According to the Physician's Desk Reference and Micromedex® 2.0, the recommended adult dose of metformin in Type II diabetics should be 500–2000 mg per day orally. We translated the drug dosages from those used in our mouse model (12 or 24 mg kg− 1 day − 1) to human dosages using a published formula {Reagan-Shaw et al., 2008}, finding human equivalent dose for metformin to be 1 or 2 mg/kg, which corresponds to a 60 or 120 mg dose of metformin administered to a 60 kg person. The dose of metformin we used in our experiments is therefore lower than the dose recommended for diabetes therapy. Moreover, we monitored the plasma lactate concentration in the treated mice, and detected no significant change in any of the groups (unpublished data), confirming that the dosages of metformin used in this study were safe. Because metformin is excreted by tubular secretion, we speculated that poor nephroprotection observed with higher metformin doses may be a result of increased renal workload. Despite these issues, the results of our study suggest that metformin is an appropriate potential therapy for AAN or other renal diseases. While these findings provide another therapeutic application for metformin, the relationship between the degree of nephroprotective effects and the dosage of metformin in treatment of nephropathy is yet to be clarified. Conclusion Our study was the first to detect increased renal SSAO activity in AAtreated mice, which might be associated with the accumulation of MGO and CML in the kidney. SSAO inhibitors may reduce MGO, but currently available SSAO inhibitors are not suitable for treatment of AAN because of their harmful side-effects, causing other visible damage. Metformin is an oral antidiabetic drug, as well as a MGO scavenger, that acts in part by condensing with MGO. Metformin administration to AAN mice
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Fig. 5. CML in mouse kidney. Immunohistochemical staining for CML in kidney sections was shown in (A) control, (B) AA, (C) AAML, and (D) AAMH group. Faint CML staining was detected in control mice, and the amount of CML staining (arrow) appeared most evident in tubular region and basement membrane of AA-treated mice. Nuclei were counterstained with hematoxylin. Original magnification: 400×. Detection of the renal CML level by ELISA was shown in (E). The renal CML level in control group was non-detected (below the kit detection limit), and decreased in AAML and AAMH group compared with that in AA group. # p b 0.05, compared with AA group.
decreased renal levels of MGO and CML, and ameliorated AA-induced renal damage. However, metformin reduced renal injury via pathways independent of SSAO activity reduction. Our findings indicate that the activity of renal SSAO, a key enzyme involved in MGO generation, increased in the AA-treated mice, and scavenging MGO elicited effective nephroprotection. Inhibition of SSAO activity might be a promising novel therapeutic approach, offering new therapeutic horizons for management of AAN. Further studies are warranted to identify the ideal SSAO-inhibiting agents and to determine the potential for use of metformin in AAN and other renal pathologies. Conflict of interest statement We all authors have no conflict of interest concerning this work.
Acknowledgements This work was financially supported by the National Science Council of the Republic of China (NSC102-2320-B-038-015).
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Marsilio R, Dall'Amico R, Giordano G, Murer L, Montini G, Ros M, et al. Rapid determination of creatinine in serum and urine by ion-pair high-performance liquid chromatography. Int J Clin Lab Res 1999;29:103–9. Martelius T, Salmi M, Krogerus L, Loginov R, Schoultz M, Karikoski M, et al. Inhibition of semicarbazide-sensitive amine oxidases decreases lymphocyte infiltration in the early phases of rat liver allograft rejection. Int J Immunopathol Pharmacol 2008;21: 911–20. O'Rourke AM, Wang EY, Miller A, Podar EM, Scheyhing K, Huang L, et al. Anti-inflammatory effects of LJP 1586 [Z-3-fluoro-2-(4-methoxybenzyl)allylamine hydrochloride], an amine-based inhibitor of semicarbazide-sensitive amine oxidase activity. J Pharmacol Exp Ther 2008;324:867–75. Rahbar S, Figarola JL. Novel inhibitors of advanced glycation endproducts. Arch Biochem Biophys 2003;419:63–79. Reagan-Shaw S, Nihal M, Ahmad N. Dose translation from animal to human studies revisited. FASEB J 2008;22:659–61. Ruggiero-Lopez D, Lecomte M, Moinet G, Patereau G, Lagarde M, Wiernsperger N. Reaction of metformin with dicarbonyl compounds. Possible implication in the inhibition of advanced glycation end product formation. Biochem Pharmacol 1999;58:1765–73. Sato N, Takahashi D, Chen SM, Tsuchiya R, Mukoyama T, Yamagata S, et al. Acute nephrotoxicity of aristolochic acids in mice. J Pharm Pharmacol 2004;56:221–9. Scheen AJ. How I treat … with metformin a diabetic patient with moderate renal insufficiency. Rev Med Liege 2013;68:190–5. Schwetz BA. From the Food and Drug Administration. JAMA 2001;285:2705. Tabi T, Szoko E, Merey A, Toth V, Matyus P, Gyires K. Study on SSAO enzyme activity and anti-inflammatory effect of SSAO inhibitors in animal model of inflammation. J Neural Transm 2013;120:963–7. Vanherweghem JL, Abramowicz D, Tielemans C, Depierreux M. Effects of steroids on the progression of renal failure in chronic interstitial renal fibrosis: a pilot study in Chinese herbs nephropathy. Am J Kidney Dis 1996;27:209–15. Vlastos D, Moshou H, Epeoglou K. Evaluation of genotoxic effects of semicarbazide on cultured human lymphocytes and rat bone marrow. Food Chem Toxicol 2010;48: 209–14. Wong MY, Saad S, Pollock C, Wong MG. Semicarbazide-sensitive amine oxidase and kidney disease. Am J Physiol Renal Physiol 2013;305:F1637–44. Yamagishi S, Nakamura K, Matsui T, Ueda S, Fukami K, Okuda S. Agents that block advanced glycation end product (AGE)-RAGE (receptor for AGEs)-oxidative stress system: a novel therapeutic strategy for diabetic vascular complications. Expert Opin Investig Drugs 2008;17:983–96.
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Increased renal semicarbazide-sensitive amine oxidase activity and methylglyoxal levels in aristolochic acid-induced nephrotoxicity Tzu-Chuan Huang, Shih-Ming Chen, Yi-Chieh Li, Jen-Ai Lee ⁎ School of Pharmacy, College of Pharmacy, Taipei Medical University, No. 250, Wuxing St., Taipei 11031, Taiwan
a r t i c l e
i n f o
Article history: Received 28 February 2014 Accepted 25 July 2014 Available online 8 August 2014 Keywords: Aristolochic acid Nephrotoxicity Semicarbazide-sensitive amine oxidase Methylglyoxal Nε-(carboxymethyl)lysine Metformin
a b s t r a c t Aims: Aristolochic acid (AA) nephrotoxicity is related to accumulation of methylglyoxal (MGO) and Nε(carboxymethyl)lysine (CML) in the mouse kidney. We studied the activity of renal semicarbazide-sensitive amine oxidase (SSAO), a key enzyme involved in MGO generation, in AA-treated mice, and investigated nephroprotective effects produced by metformin, a MGO scavenger. Methods: Mice were orally administered water or metformin for 15 days (12 or 24 mg kg−1 day−1), and injected AA (5 mg kg−1 day−1) intraperitoneally for 8 days starting on day 8. Renal function was studied, and histopathological examination, determination of renal SSAO activity, and measurement of MGO levels were performed. Key findings: Compared to control mice, AA-injected mice showed significant renal damage and approximately 2.7-fold greater renal SSAO activity (p b 0.05). Further, compared to control treatment, administration of 12 mg/kg metformin inhibited formation of renal lesions, and significantly decreased renal MGO levels (37.33 ± 9.78 vs. 5.89 ± 2.64 μg/mg of protein, respectively, p b 0.01). In the AA-treated mice, metformin also inhibited the accumulation of CML in renal tubules, but did not affect SSAO activity. Significance: This study is the first to show elevated renal SSAO activity in AA-treated mice, which could be involved in MGO accumulation. Moreover, MGO scavenging by metformin reduces AA nephrotoxicity. These findings suggest that reducing MGO accumulation produces nephroprotection, revealing new therapeutic strategies for the management. SSAO is a key enzyme involved in MGO generation, and consequently, inhibition of renal SSAO activity is worth investigating in AA nephrotoxicity and other renal pathologies further. © 2014 Elsevier Inc. All rights reserved.
Introduction Aristolochic acid (AA), a compound found in plants of the genus Aristolochia, causes nephrotoxicity (AA-induced nephrotoxicity, or AAN) characterized by initial damage to the proximal renal tubule, with later development of interstitial fibrosis and urothelial carcinoma {Debelle et al., 2002}. The U.S. Food and Drug Administration has recommended cessation of the use of botanical products containing AA {Schwetz, 2001}. However, products containing AA remain available online, and some herbal remedies have been found to be contaminated with AA, which has led to cases of AAN {Gold and Slone, 2003; Li et al., 2011}. There is no established optimal clinical recommendation for AAN therapy. Steroid therapy slows the progression of renal failure that characteristically accompanies progressive interstitial fibrosis in AAN {Vanherweghem et al., 1996}. However, steroid therapy is not effective in all cases of AAN, and long-term administration of steroids is associated with major side effects. Currently, the top priorities in the management of AAN are to control the progression of nephropathy ⁎ Corresponding author. Tel.: +886 2 2736 1661x6125; fax: +886 2 2736 1661x6120. E-mail addresses: [email protected] (T.-C. Huang), [email protected] (S.-M. Chen), [email protected] (Y.-C. Li), [email protected] (J.-A. Lee).
http://dx.doi.org/10.1016/j.lfs.2014.07.034 0024-3205/© 2014 Elsevier Inc. All rights reserved.
and to prepare for renal replacement therapy {Gokmen et al., 2013}. Therefore, it is important to develop a clinically effective and safe treatment for AAN. Accumulation of methylglyoxal (MGO) and Nε-(carboxymethyl)lysine (CML) in kidney tissue has been demonstrated in a mouse model of AAN Li et al., 2012. MGO is an endogenous, highly-reactive, and cytotoxic aldehyde compound {Inoue and Kimura, 1995}. It can react with and modify DNA and other biomolecules, which alters their structures and functions, and results in the formation of advanced glycation end-products (AGEs), such as CML {Brownlee, 1994}. We speculated that renal MGO and CML were related to damage produced by AA, but the causes of AA-induced MGO generation and the effect of MGO reduction on AAN have not been investigated. Semicarbazide-sensitive amine oxidase (SSAO; EC 1.4.3.21) is involved in a MGO generation pathway that converts aminoacetone into MGO {Inoue and Kimura, 1995}. SSAO is found in several tissues, with particularly high activity in kidney {Lewinsohn, 1984; Lyles and Singh, 1985}. SSAO activates profibrogenic cytokines {Wong et al., 2013} that are involved in the progression of renal fibrosis that is characteristic of AAN. The activity of renal SSAO may be closely related to the production of MGO and the pathological progression of AAN, but this hypothesis has not been verified.
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The MGO inhibitor metformin was used to investigate whether a decrease in MGO levels mitigates the severity of AAN. Metformin (N,Ndimethylimidodicarbonimidic diamide) is generally accepted as the first-line treatment for type 2 diabetes mellitus. In addition, metformin is a MGO scavenger and an inhibitor of the formation of AGEs. The drug has been shown to reduce systemic levels of MGO and AGEs in patients with type 2 diabetes mellitus, and to prevent diabetic complications {Beisswenger et al., 1999; Yamagishi et al., 2008; Ruggiero-Lopez et al., 1999; Kiho et al., 2005 #6;Rahbar and Figarola, 2003}. Metformin has also been shown to ameliorate gentamicin-induced renal toxicity in rats by decreasing renal MGO levels {Amini et al., 2012; Li et al., 2013}. The safety, widespread clinical use, and proven efficacy of metformin make it an ideal MGO inhibitor for evaluation in animals with AAN. This study investigated the relationship between increased renal MGO levels and SSAO activity in renal tissue by using a mouse model of AAN. Furthermore, the MGO inhibitor metformin was administered to confirm that reduction in MGO levels is nephroprotective in AAN mice, and to investigate the effect of metformin on renal SSAO activity.
Materials and methods Animal experiments All animal experiments were approved by the Animal Care and Use Committee of Taipei Medical University, Taiwan. Thirty-two C57BL/6 mice (7-week-old males) were purchased from the National Laboratory Animal Breeding and Research Center (Taipei, Taiwan). These mice were housed in temperature-controlled (25 ± 2 °C) and humiditycontrolled (65% ± 5%) facility with a 12/12 light/dark photoperiod and access to standardized food pellets (TMI, USA) and tap water ad libitum. After 1 week of acclimatization, the mice were divided randomly into 4 groups (each group consisted of 8 mice). Control group: mice treated with water (P.O) and saline (i.p.) AA group: mice treated with water (P.O) and AA (i.p.) AAML group: mice treated with low dose metformin (P.O) and AA (i.p.) AAMH group: mice treated with high dose metformin (P.O) and AA (i.p.) The control group was orally administered 0.1 mL water for 15 days (days 1–15) and intraperitoneally injected with 0.15 mL saline for 8 days (day 8–15). The AA group was orally administered 0.1 mL water for 15 days and intraperitoneally injected with 0.15 mL AA (days 8–15; 5 mg kg− 1 day− 1). The AAML and AAMH groups were treated with 0.1 mL metformin at doses of 12 and 24 mg kg−1 day−1, respectively, for 15 days, and injected with 0.15 mL AA for 8 days (days 8–15; 5 mg kg− 1 day−1). At the end of the treatment period, the mice were placed in rodent metabolic cages with free access to food for 12-h urine collection. At the end of the experiment, the mice were sacrificed under pentobarbital (50 mg/kg), and a blood sample was collected by intracardiac puncture. A thorough necropsy was performed on all mice, and the kidneys were dissected for histopathological evaluation, immunohistochemistry, determination of SSAO activity, and quantification of MGO levels.
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Serum and urinary creatinine assays Creatinine levels in the serum and urinary samples were measured using high-performance liquid chromatography (HPLC), as described previously {Lee et al., 2005; Marsilio et al., 1999}. The measured concentrations were expressed in milligrams per deciliter, and 0.1 mmol/L cimetidine in 0.01 mol/L HCl aqueous solution was used as the internal standard. A Capcell Pak C18 column (250 mm × 4.6 mm, ID; particle size, 5 μm; Shiseido, Tokyo, Japan) was used, and absorbance was monitored at 234 nm. The mobile phase was composed of acetonitrile and 100 mmol/L of PBS containing 30 mmol/L sodium lauryl sulfate adjusted to pH 3.0 with hydrochloric acid (36/60, v/v). The flow rate was 0.8 mL/min. Measurement of the glomerular filtration rate Creatinine clearance (CCr), an index of the glomerular filtration rate (GFR), was calculated using the following equation adapted from Hsu and co-workers {Hsu et al., 2008}: CCr ¼
Ucr UV 100 1 Scr BW 720
Where CCr is creatinine clearance (mL min−1 kg−1 BW); Ucr, urinary creatinine concentration (mg/dL); UV, is urine volume (mL); Scr, serum creatinine (mg/dL); BW, body weight (g); and 720, the period in which urine was collected (21 h, expressed in min). Determination of urinary protein levels and N-acetyl-β-D-glucosaminidase activity Urinary protein levels were quantified using a Bio-Rad protein assay kit with bovine serum albumin as the standard, according to the manufacturer’s instructions. Urinary N-acetyl-β-D-glucosaminidase (NAG, EC 3.2.1.30) activity was determined using the method described by Leaback and Walker {Leaback and Walker, 1961}. NAG reacted with the 4-methylumbelliferyl-N-acetyl-β-glucosaminide, and fluorescence was measured and used to quantify NAG concentration. Determination of blood urea nitrogen concentration The concentration of blood urea nitrogen (BUN) was determined using a urease assay kit (Sigma-Aldrich, St Louis, MO), according to the manufacturer’s instructions. Urea was hydrolyzed by urease to ammonia and carbonate. In the presence of glutamic dehydrogenase, ammonia reacts with ketoglutaric acid with the concurrent oxidation of NADH to NAD. BUN activity is directly proportional to the rate of conversion of NADH to NAD, and it is monitored at 340 nm {Kaltwasser and Schlegel, 1966}. Urinary microalbumin Microalbumin levels were measured using a commercial kit (Good Biotech Corp., Taiwan) based on immunoturbidimetric methods, with procedures performed according to the manufacturer’s instructions. The samples were conditioned with a Tris buffer before incubation with an anti-albumin antibody for 10 min. The samples were then analyzed at 405 nm.
Preparation of urine, blood, and kidney samples Histopathological examination Sediments was removed from the urine samples by centrifugation (700 × g at 4 °C for 5 min), and the supernatant was stored at −80 °C until analysis. The blood samples were centrifuged (700 × g, 15 min) and the serum was stored at − 20 °C. The kidneys were embedded in paraffin, sectioned (thickness, 3–5 μm), and stained with periodic acid-Schiff stain for immunohistochemistry.
A histopathological evaluation was performed on the kidneys of the mice according to the guidelines proposed previously {Sato et al., 2004}. The histopathological severity of the lesions was rated from one to five (1 = minimal; 2 = slight; 3 = moderate; 4 = moderately severe; 5 = severe/high). The sum of scores for each sample, reflecting tubular
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trophy, cell infiltration, and interstitial fibrosis, was defined as the tubulointerstitial histological score.
one-way analysis of variance (ANOVA) and Tukey's studentized range in the SAS 9.3 package (SAS Institute, Inc., Cary, NC, USA). Differences were considered significant at p b 0.05.
SSAO activity assays Results SSAO activity was determined by fluorimetrical technique, using the Amplex Red Monoamine Oxidase Assay kit (Molecular Probes Leiden, Netherlands) to measure hydrogen peroxide production, according to the manufacturer’s instructions. Briefly, 100 μL kidney homogenates were pre-incubated for 30 min at room temperature with 1 μmol/ L L-deprenyl to inhibit any monoamine oxidase-B contamination. The reaction was started by the addition of 100 μL reaction buffer (400 μmol/L Amplex Red, 2 U/mL horseradish peroxidase, 2 mmol/L benzylamine, 50 mmol/L sodium phosphate, pH 7.4) and the fluorescence (excitation, 530 nm; emission, 590 nm) was monitored during 60 min using a Synergy HT microplate reader (Bio-Tek Instruments, Inc., Winooski, VT). A range of H2O2 concentrations were used for the standard calibration. SSAO activity was expressed as pmol/L H2O2 min mg−1 protein. Measurements of MGO in kidney homogenates The determination of MGO levels in kidney homogenates by HPLC was performed according to a previously published method {EspinosaMansilla et al., 2007}. Briefly, 0.5 mol/L ammonium chloride buffer (pH 10.0) was added to the kidney homogenates to create an alkaline environment. Then, 5,6-diamino-2,4-dihydroxy-pyrimidine was used as a derivatization reagent. After incubation for 30 min at 60 °C, the derivatization reaction was stopped with 0.01 mol/L citric acid (pH 6.0). HPLC analysis was performed using an ODS column (Biosil, 250 mm; ID 4.6 mm; particle size, 5 μm; Biosil Chemical Co. Ltd., Taipei, Taiwan) at 33 °C. The MGO derivative was separated using a mobile phase composed of a mixture of acetonitrile and 0.01 mol/L citric acid buffer adjusted to pH 6.0 with NaOH (3/97, v/v), with a flow rate of 0.7 mL/min. Derivatization reduction products were injected in 50-μL volumes, and eluted fractions were analyzed by monitoring fluorescence at excitation and emission wavelengths of 330 and 500 nm, respectively. Immunohistochemistry for CML in kidney section Paraffin-embedded sections (thickness, 3–5 μm) were used to determine the localization of CML. After blocking endogenous peroxidase activity by incubating the sections in 0.3% H2O2 for 10 min, the sections were incubated overnight with CML antibodies (diluted 50 times). The sections were reacted for 1 h with goat anti-rabbit IgGs conjugated with peroxidase-labeled secondary antibodies, diluted 250 times. Peroxidase activity was visualized using the diaminobenzidine stain. Immunohistochemistry was performed without the primary antibody to obtain the negative control (data not shown). Measurement of renal CML levels by ELISA Renal CML were determined using the OxiSelect CML competitive ELISA kit (Cell Biolabs, San Diego, CA) according to the manufacturer’s instructions. First, a CML conjugate was coated on the ELISA plate. Kidney homogenates (150 μg of protein) are then added to the CML conjugate-coated plate. After a brief incubation, the anti-CML monoclonal antibody is added, followed by an HRP conjugated secondary antibody. The quantity of CML adduct in kidney homogenates was determined by comparing its absorbance with that of a known CML− bovine serum albumin standard curve.
Biochemical indices Urinary microalbumin levels in mice Urinary microalbumin levels were significantly increased in all AA-treated mice (AA, AAML, and AAMH groups), as compared to the levels in the control group (Fig. 1A). While the levels measured in both the AAML and AAMH groups were significantly lower than that in the AA group, no significant difference was observed between the AAML and AAMH groups. Urinary NAG levels Fig. 1B illustrates the effect of metformin administration on urinary NAG levels. AA-treated mice that did not receive metformin (the AA group) exhibited higher NAG levels than the control group. Compared with the AA group, the AAML and AAMH groups showed decreased urinary NAG levels, which were not statistically significant. Blood urea nitrogen (BUN) concentration Fig. 1C shows the BUN concentrations of the experimental groups. BUN was significantly elevated in all AA-treated groups, as compared to that in the control group. However, BUN levels in metformintreated mice (both the AAML and AAMH groups) were lower than that of the AA group. This difference was significant only when comparing the AAML group with the AA group (133.11 ± 39.19 vs. 244.88 ± 37.97 mg/dL, respectively, p b 0.05). Creatinine clearance level Creatinine clearance (CCr) was significantly lower in the AA group (1.29 ± 0.59 mL min−1 kg−1 BW) than in the control group (10.18 ± 1.80 mL min− 1 kg−1 BW; Fig. 1D). CCr for AAML and AAMH groups were 3.55 ± 1.13 and 2.12 ± 1.12 mL min−1 kg−1 BW, respectively. CCr was significantly higher in the AAML group than that in the AA group. Morphological findings and changes in tubulointerstitial histological scores In the control mice, all renal tissue samples appeared histologically normal (Fig. 2A). Renal lesions detected on visual inspection and severe changes observed using microscopy indicate that AA exerted a strong nephrotoxic effect (Fig. 2B\D). Tubular atrophy, cell infiltration, and fibrosis, considered to be the primary indices of AA-induced renal damage, were observed in all AA-treated mice (AA, AAML, and AAMH groups). In addition, we observed hyaline casts, tubular dilatation, and degenerative tubular cells, which are also considered an indications of acute tubular necrosis. The morphology of the glomerular portion did not appear to be affected by AA treatment. Median scores for the renal degeneration evidenced by of tubular atrophy, cell infiltration, and interstitial fibrosis were calculated using data collected from each treatment group (Fig. 2E). Severity scores in the AA group were significantly higher than those in the AAML and AAMH groups, indicating reduced renal damage in metformin-treated mice. Treatment with a lower dose of metformin partially reversed the tubulointerstitial nephritis, and the severity of toxic nephropathy in the AAMH group was slightly higher than that in the AAML group. SSAO activity in mouse kidney
Statistical analysis All data were expressed as mean ± SD for the 8 mice in each group. The significance of differences among groups was analyzed using
Renal SSAO activity levels of each treatment group are shown in Fig. 3. SSAO activity in the control group was 0.46 ± 0.10 pmol/L H2O2 min mg− 1 protein, while in the AA group it was approximately
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Fig. 1. Biochemical indices measured in mice: (A) urinary mcroalbumin levels, (B) urinary N-acetyl-β-D-glucosaminidase (NAG), (C) blood urea nitrogen (BUN), (D) creatinine clearance (CCr). The data are presented as means ± SD for each group (n = 8). *p b 0.05, compared with control group; #p b 0.05, compared with AA group.
2.7 times higher (1.21 ± 0.64 pmol/L H2O2 min mg− 1 protein, p b 0.05). SSAO activities in the AAML and AAMH groups were 1.06 ± 0.77 and 1.39 ± 0.86 pmol/L H2O2 min mg− 1 protein, respectively, with no statistically significant differences observed compared to the AA group. MGO levels in mouse kidney Fig. 4A and B show representative HPLC chromatograms that depict the MGO peak used for the quantification of tissue levels. MGO was derivated and demonstrated a detectable fluorescence peak with a retention time of approximately 27 min, with the peak height normalized for the protein concentration (Fig. 4C). MGO levels in the kidneys were determined to be higher in the AA group than in the control group (37.33 ± 9.78 vs 5.89 ± 2.64 μg/mg protein, p b 0.01). Conversely, MGO levels in metformin-treated rats (AAML and AAMH groups) were lower than that in the AA group (9.81 ± 5.04 and 17.79 ± 7.88 vs. 37.33 ± 9.78 μg/mg protein, respectively, p b 0.01). Immunohistological staining for CML and CML levels in mouse kidney An immunohistochemical analysis was performed to evaluate CML localization in mice kidneys, following AA and metformin treatments (Fig. 5). While faint CML staining was detected in the control group (Fig. 5A), a notably higher signal was observed in the AA, AAML, and AAMH groups, especially in the tubular region and basement membrane (Fig. 5B, C, and D). As shown in Fig. 5E, measurements of renal CML using ELISA revealed non-detection in the control group (below the kit detection limit), and showed an increase in the kidneys of AA, AAML, and AAMH group (15.97 ± 3.25, 7.55 ± 3.98, and 7.56 ± 6.04 рg/μg protein, respectively). Compared with AA group, renal CML levels decreased in AAML and AAMH group significantly (p b 0.05).
Discussion In current experiments, we applied a previously described experimental protocol to induce AAN in mice {Baudoux et al., 2012}. Following 8 days of daily intraperitoneal AA injections at 5 mg kg−1 day−1, proximal renal tubular dysfunction and structural destruction were observed using biochemical tests and renal biopsy, reflecting the primary characteristics of acute AAN. Administration regimen used in this study successfully induced AAN in mice, with all animals surviving the entire treatment period. The pathological effects of AA administration were observed primarily in the proximal tubular tissue, with tubular necrosis, atrophy, and degeneration found using renal biopsy. Treatment with AA did not significantly alter glomerular structure, supporting previously reported findings {Sato et al., 2004; Huang et al., 2013; Debelle et al., 2002; Baudoux et al., 2012}. Impaired renal function in AA-injected mice was manifested through alterations in the biochemical indices, such as increased urinary levels of microalbumin, urinary NAG levels, and BUN levels, as well as decreased CCr. The renal levels of MGO were also found to be elevated in the AA-injected mice, while CML was observed to accumulate in tubular cells and the basement membrane. Our histopathological findings suggest that AA primarily affected the tubular cells, causing a leakage of cells and the protrusion of basement membrane. These results correspond to the findings of the previous study {Li et al., 2012}. Excess kidney levels of MGO and CML, therefore, appear to be related to AA-induced damage. Renal SSAO activity increased in the AA-injected mice. SSAO is involved in protein metabolism, and converts aminoacetone to MGO, while CML is a downstream product of MGO metabolism. Increased SSAO activity in mouse kidney is quite likely a factor contributing to the acceleration of MGO and CML formation in AA-induced damage. We speculated that inhibition of SSAO may decrease MGO and CML formation, and elicit nephroprotective effects against AAN. Several studies also suggested that potent inhibition of SSAO may be of therapeutic
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Fig. 2. Morphological findings (periodic acid – Schiff stain) and changes of tubulointerstitial histological scores. Light micrographs of renal tissue of the (A) control, (B) AA, (C) AAML, and (D) AAMH groups. Quantitative analysis of lesions is shown (E). (*): tubular atrophy, (#): cell infiltration, (▲): fibrosis.
value, particularly in the treatment of pathologies related to inflammatory {Tabi et al., 2013; Martelius et al., 2008; O'Rourke et al., 2008}. However, widespread clinical use of previously identified SSAO
Fig. 3. SSAO activity in mouse kidney. The activity of SSAO in the AA, AAML, and AAMH groups were significantly increased, as compared to the control group (mean ± SD, n=8). *p b 0.05, compared with control group; #p b 0.05, compared with AA group.
inhibitors, including hydralazine and semicarbazide, is not recommended, due to the potential for harmful side-effects. Hydralazine is an antihypertension drug that activates gated potassium channels on the vascular smooth muscle, causing an efflux of potassium and a subsequent hyperpolarization of the cell, resulting in vasodilation. It may increase heart rate and cardiac output, potentially causing angina pectoris or a myocardial infarction. Patients treated with hydralazine for a long period may develop a lupus-like syndrome or other immune-related diseases {Handler, 2012}. Semicarbazide is a metabolite of the veterinary drug nitrofurazone. Due to its potential genotoxic and carcinogenic activity {Vlastos et al., 2010 #294;Hirakawa et al., 2003}, the World Health Organization notes that dietary semicarbazide content should be monitored, and uses semicarbazide levels as an indicator for the use of nitrofurazone in animals used for food. Due to of these recognized risks, hydralazine and semicarbazide were not considered ideal SSAO inhibitors, and are seen as unsuitable for use in treatment of renal injury. In order to investigate whether decreased MGO and CML levels are associated with a slowing of the progression of AAN, we treated AAN mice with a MGO and AGE inhibitor metformin. Our results suggested that the administration of metformin (12 or 24 mg kg−1 day−1)
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Fig. 4. MGO levels in the renal tissues of AA-treated mice. MGO was quantified using HPLC, with representative chromatograms of samples from the (A) AA and (B) AAML groups presented. MGO was eluted at 27 min, and its concentration was normalized to the sample protein concentration. The quantification of MGO is shown in (C). * p b 0.05, compared with control group; # p b 0.05, compared with AA group.
decreased MGO and CML levels in the mouse kidney, and ameliorated AA-induced renal damage, as evidenced by the reversal of both histopathological and biochemical indices of nephropathology. Urinary levels of microalbumin and BUN were decreased, while CCr was increased in metformin-treated mice, indicating that administration of metformin improved renal function. The histopathological analysis showed reduced severity of AA-induced renal lesions in metformintreated mice, compared to the pathological appearance in mice treated with AA alone. These findings suggest that the nephroprotective effects
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of metformin in AAN were associated with its MGO- and CMLscavenging ability. Although metformin is considered to be a scavenger of MGO and inhibitor of AGEs {Beisswenger et al., 1999; Yamagishi et al., 2008; Ruggiero-Lopez et al., 1999; Kiho et al., 2005}, its exact mechanism of action has not been clearly elucidated. We measured renal SSAO activities in metformin-treated mice, but did not observe a significant difference in renal SSAO activity between metformin-treated mice and mice treated with AA alone. This finding suggests that metformin decreases MGO through a pathway that does not involve the inhibition of SSAO activity. Previous studies proposed that metformin can trap the carbonyl group of MGO to form an inactive condensation product in vivo {Ruggiero-Lopez et al., 1999; Beisswenger and Ruggiero-Lopez, 2003}, resulting in diminished toxicity of MGO and reduced production of CML. Based on our observations, we believe that the condensation reaction between metformin and MGO could contribute to the nephroprotective effects of metformin in AA-treated mice. In the current study, a high dose of metformin (24 mg kg−1 day−1) elicited less of a nephroprotective effect compared to a low dose (12 mg kg− 1 day−1). Based on the measurements of urinary microalbumin levels, BUN, and CCr, as well as the tubulointerstitial histological findings, renal damage was less severe in the AAML group than in the AAMH group. Renal MGO and CML levels were also lower in the AAML group than in the AAMH group. These observations provided a rationale for a reassessment of the adjustment of metformin dose in the treatment of renal diseases. According to the U.S. Food and Drug Administration, metformin therapy must be used cautiously in patients with renal insufficiency, due to the potential risk of lactic acidosis. However, reducing metformin dosage and monitoring the physiological indicators will allow metformin to be safely used in chronic kidney disease {Scheen, 2013; Duong et al., 2012; Duong et al., 2012}. Metformin has also been shown to protect rat kidney tissue against gentamicin-induced injury {Amini et al., 2012; Li et al., 2013}. Therefore, in patients with nephropathy, metformin dose could be adjusted instead of discontinuing the therapy outright, because appropriate use of metformin might elicite nephroprotection. According to the Physician's Desk Reference and Micromedex® 2.0, the recommended adult dose of metformin in Type II diabetics should be 500–2000 mg per day orally. We translated the drug dosages from those used in our mouse model (12 or 24 mg kg− 1 day − 1) to human dosages using a published formula {Reagan-Shaw et al., 2008}, finding human equivalent dose for metformin to be 1 or 2 mg/kg, which corresponds to a 60 or 120 mg dose of metformin administered to a 60 kg person. The dose of metformin we used in our experiments is therefore lower than the dose recommended for diabetes therapy. Moreover, we monitored the plasma lactate concentration in the treated mice, and detected no significant change in any of the groups (unpublished data), confirming that the dosages of metformin used in this study were safe. Because metformin is excreted by tubular secretion, we speculated that poor nephroprotection observed with higher metformin doses may be a result of increased renal workload. Despite these issues, the results of our study suggest that metformin is an appropriate potential therapy for AAN or other renal diseases. While these findings provide another therapeutic application for metformin, the relationship between the degree of nephroprotective effects and the dosage of metformin in treatment of nephropathy is yet to be clarified. Conclusion Our study was the first to detect increased renal SSAO activity in AAtreated mice, which might be associated with the accumulation of MGO and CML in the kidney. SSAO inhibitors may reduce MGO, but currently available SSAO inhibitors are not suitable for treatment of AAN because of their harmful side-effects, causing other visible damage. Metformin is an oral antidiabetic drug, as well as a MGO scavenger, that acts in part by condensing with MGO. Metformin administration to AAN mice
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Fig. 5. CML in mouse kidney. Immunohistochemical staining for CML in kidney sections was shown in (A) control, (B) AA, (C) AAML, and (D) AAMH group. Faint CML staining was detected in control mice, and the amount of CML staining (arrow) appeared most evident in tubular region and basement membrane of AA-treated mice. Nuclei were counterstained with hematoxylin. Original magnification: 400×. Detection of the renal CML level by ELISA was shown in (E). The renal CML level in control group was non-detected (below the kit detection limit), and decreased in AAML and AAMH group compared with that in AA group. # p b 0.05, compared with AA group.
decreased renal levels of MGO and CML, and ameliorated AA-induced renal damage. However, metformin reduced renal injury via pathways independent of SSAO activity reduction. Our findings indicate that the activity of renal SSAO, a key enzyme involved in MGO generation, increased in the AA-treated mice, and scavenging MGO elicited effective nephroprotection. Inhibition of SSAO activity might be a promising novel therapeutic approach, offering new therapeutic horizons for management of AAN. Further studies are warranted to identify the ideal SSAO-inhibiting agents and to determine the potential for use of metformin in AAN and other renal pathologies. Conflict of interest statement We all authors have no conflict of interest concerning this work.
Acknowledgements This work was financially supported by the National Science Council of the Republic of China (NSC102-2320-B-038-015).
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