Original Paper Nephron Exp Nephrol 2014;126:148–156 DOI: 10.1159/000362556
Received: October 24, 2013 Accepted: March 25, 2014 Published online: May 16, 2014
Synergistic Effects of Leflunomide and Benazepril in Streptozotocin-Induced Diabetic Nephropathy Hua Jin a Shang Guo Piao a, b Ji Zhe Jin a Ying Shun Jin a Zhen Hua Cui a Hai Feng Jin a Hai Lan Zheng a Jin Ji Li a Yu Ji Jiang a Chul Woo Yang b Can Li a a
Nephrology and Dialysis Unit, Department of Internal Medicine, Yanbian University Hospital, Yanji, Jilin Province, PR China; b Transplant Research Center, Convergent Research Consortium in Immunologic Disease, The Catholic University of Korea, Seoul, Korea
Key Words Leflunomide · Benazepril · Diabetic nephropathy · Toll-like receptor-2 · Connective tissue growth factor · 8-Hydroxy-2’-deoxyguanosine
Abstract Background: Leflunomide (LEF) and benazepril have renoprotective effects on diabetic nephropathy (DN) through their anti-inflammatory and anti-fibrotic activities. This study investigated whether combined treatment using LEF and benazepril affords superior protection compared with the respective monotherapies. Methods: Diabetes was induced with streptozotocin (STZ, 65 mg/kg) by intraperitoneal injection in male Wistar rats. Two weeks after STZ injection, diabetic rats were treated daily for 12 weeks with LEF (10 mg/kg), benazepril (10 mg/kg), or a combination of both. Basic parameters (body weight, fasting blood glucose level, and 24 h urinary protein excretion), histopathology, inflammatory [inflammatory cell infiltration (ED-1), monocyte chemoattractant protein-1 (MCP-1), and Tolllike receptor-2 (TLR-2)] and glomerulosclerotic factors
© 2014 S. Karger AG, Basel 1660–2129/14/1263–0148$39.50/0 E-Mail [email protected] www.karger.com/nee
[transforming growth factor-β1 (TGF-β1) and connective tissue growth factor (CTGF)], and oxidative stress (8-hydroxy-2’-deoxyguanosine, 8-OHdG) were studied. Results: Benazepril or LEF treatment significantly prevented body weight loss and 24 h urinary protein excretion induced by diabetes; combined treatment with LEF and benazepril further improved these parameters compared with giving each drug alone (all p < 0.01). Increased expression of inflammatory (MCP-1 and TLR-2) and glomerulosclerotic (TGF-β1 and CTGF) factors in diabetic rat kidney was reduced by treatment with either LEF or benazepril and was further reduced by the combined administration of the two drugs (p < 0.01). These effects were accompanied by suppression of urinary 8-OHdG excretion. There was no significant between-group difference in blood glucose level. Conclusions: LEF treatment lessens DN, and combined treatment with LEF and benazepril provides synergistic effects in preventing DN. © 2014 S. Karger AG, Basel
Can Li, MD Nephrology and Dialysis Unit, Department of Internal Medicine Yanbian University Hospital, #1327 Juzi Street Yanji, Jilin Province 133000 (PR China) E-Mail lican @ ybu.edu.cn Chul Woo Yang, MD Transplant Research Center, Convergent Research Consortium in Immunologic Disease The Catholic University of Korea Seoul 137-040 (Korea) E-Mail yangch @ catholic.ac.kr
Introduction
Leflunomide (LEF) is a prodrug that is rapidly converted to its active metabolite A77 1726, which inhibits de novo pyrimidine nucleotide biosynthesis by reducing the activity of the enzyme dihydroorotate dehydrogenase. Because of this activity, the clinical use of LEF has produced a significant reduction in rheumatoid arthritis. However, LEF possesses both immunosuppressive and antiviral properties. LEF may thus exert pleiotropic effects in the management of cytomegalovirus infection, BK virus nephropathy, and chronic allograft dysfunction in renal transplant recipients. The renoprotective effects of LEF have also been reported in lupus nephritis, IgA nephropathy, and diabetic nephropathy (DN) [1]. Nevertheless, the precise mechanism of the renoprotective effects of LEF remains to be elucidated. Diabetes mellitus (DM) is the leading cause of endstage renal disease worldwide. The prevalence of DN caused by type 2 DM is significantly increasing not only in developed countries but also in developing countries. Despite strict control of blood glucose, hypertension, and metabolic abnormalities, the number of diabetic patients entering end-stage renal disease because of DM remains extremely high. DN is characterized by a progressive accumulation of extracellular matrix components in the glomerular mesangium and tubular interstitium, which eventually leads to proteinuria and renal insufficiency [2]. It is well recognized that glomerulosclerosis, along with tubulointerstitial injury, is a major feature and an important predictor of renal dysfunction in DN. The mechanism responsible for DN is multifactorial, and there is overwhelming evidence implicating the renin-angiotensin system (RAS), inflammatory cytokines, and transforming growth factor-β1 (TGF-β1) as important players [3, 4]. Of these, RAS plays a critical role in the mediation of inflammation and fibrotic process in DN. Blockade of RAS by angiotensin II blockers or angiotensin-converting enzyme inhibitors (ACE-I) confers renoprotection. We hypothesized that the combined treatment with LEF and ACE-I (benazepril) may prevent the progression of DN. To test the hypothesis, LEF and benazepril were administered separately or combined to rats with streptozotocin (STZ)-induced DM. Our study clearly demonstrates that combined treatment using two drugs has synergistic effects on preventing inflammatory and sclerotic processes in DN.
Leflunomide and Benazepril on DN
Materials and Methods Experiment Schedule The experimental protocol was approved by the Animal Care Committee of the Catholic University of Korea. Male Wistar rats (Charles River Technologies, Korea) weighing 220–250 g, were housed in individual cages in a temperature- and light-controlled environment and allowed free access to tap water and standard laboratory chow. Rats were randomized divided into five groups: (1) controls (Con, n = 6): non-diabetic normal animals; (2) diabetic controls (DN, n = 8): diabetic animals treated with saline used as diabetic controls; (3) benazepril treatment (DN+B, n = 8): diabetic animals treated with benazepril at a dose of 10 mg/kg body weight; (4) LEF treatment (DN+L, n = 8): diabetic animals treated with LEF at a dose of 10 mg/kg body weight, and (5) combined treatment (DN+B+L, n = 8): diabetic animals treated with both LEF and benazepril. Diabetes was induced by a single intraperitoneal injection of STZ (65 mg/kg), and the induction of diabetes was defined when blood glucose level was ≥16.7 mmol/l (300 mg/dl). Benazepril and LEF treatment were started 2 weeks after STZ injection and were administered via oral gavage for 12 weeks. At the end of the study, animals were euthanized under ketamine anesthesia and kidney tissue was rapidly removed for morphological examination. The dosage and method of benazepril [5] and LEF [6] administration were chosen based on previous reports. Biochemical Analysis Body weight was recorded periodically for each rat and blood glucose levels were measured. Urine samples were collected over a 24-hour period in individual metabolic cages (Tecniplast Gazzada Srl, Buguggiate, Italy) for the measurement of urinary protein excretion (UPE) at 0, 4, 8, and 12 weeks, and were determined by the Bradford method. Systolic blood pressure (SBP) was measured in conscious rats every 4 months using the tail-cuff method with a plethysmography by a tail manometer-tachometer system (BP2000; Visitech System, Apex, N.C., USA). Histopathology Kidney tissues were fixed in periodate-lysine-paraformaldehyde solution and embedded in wax. After dewaxing, 4-μm sections were processed and stained with periodic acid-Schiff (PAS). Semiquantitative analysis was performed by counting the percentage of the stained areas per field of glomerulus at 400× magnification on a color image autoanalyzer (TDI Scope Eye Version 3.5 for Windows; Olympus). A minimum of 40 glomeruli per section were counted and averaged. Immunohistochemistry After dewaxing, sections were incubated with a 0.5% Triton X100-PBS solution for 30 min and washed with phosphate-buffered saline (PBS) three times. Non-specific binding sites were blocked for 1 h with normal donkey serum diluted 1:10 in PBS, and then incubated overnight at 4 ° C with ED-1 (Serotec, Inc., Indianapolis, Ind., USA) diluted in 1:1,000 in a humid environment. After rinsing in Tris-buffered saline (TBS), sections were incubated in peroxidase-conjugated donkey anti-mouse or rabbit IgG Fab fragment (Jackson ImmunoResearch Laboratories, West Grove, Pa., USA) for 30 min. For staining, sections were incubated with a mixture of 0.05% 3,3-diaminobenzidine containing 0.01% H2O2 at room temperature until a brown color was visible, washed with
Nephron Exp Nephrol 2014;126:148–156 DOI: 10.1159/000362556
149
Con
35
DN DN+B
Body weight (g)
400
DN+L DN+B+L
350
†
†
300 250
*
200 0
a
4
* 8
*
Fasting glucose levels (mmol/l)
450
*
4
8
*
25 20 15 10 5 0
b
12
Time (months)
160
*
60
150
* *
30
†
* † †, ‡
†, ‡
†, ‡
*
140 SBP (mm Hg)
50 UPE (mg/dl)
*
0
70
10
100 0 4
8
12
Time (months)
†
†
120 110
0
*
*
*
130
20
0
c
*
12
Time (months)
40
30
0
4
d
8
12
Time (months)
7
*
KW/BW ratio × 1,000
6
†
†
5
‡
4 3 2 1
Fig. 1. Basic parameters during the experiment period: (a) body weight, (b) fasting blood glucose, (c) UPE, (d) SBP, and (e) kid-
ney weight to body weight ratio (KW/BW) ×1,000. * p < 0.01 vs. Con; † p < 0.01 vs. DN; ‡ p < 0.01 vs. DN+B or DN+L.
150
Nephron Exp Nephrol 2014;126:148–156 DOI: 10.1159/000362556
e
0
Con
DN
DN+B
DN+L DN+B+L
Jin /Piao /Jin /Jin /Cui /Jin /Zheng /Li /Jiang / Yang /Li
c
DN
Control
DN+B
DN+B+L
b
0.6
†
0.4
†
0.3 0.2
‡
0.1 0
Con
DN
DN+B+L
15
*
0.5
DN
DN+B
DN+B
DN+L DN+B+L
ED-1-positive cells (%/0.5 mm2)
Glomerular sclerosis index (%/0.5 mm2)
a
Control
d
*
12
†
†
9 6
‡ 3 0
Con
DN
DN+B
DN+L DN+B+L
Fig. 2. Representative photomicrographs of PAS staining (a) and immunohistochemistry for ED-1 (b) and semiquantitative analyses (c, d). ×400. * p < 0.01 vs. Con; † p < 0.01 vs. DN; ‡ p < 0.01 vs. DN±B or DN±L.
TBS, counterstained with hematoxylin, and examined under light microscopy. The procedure of immunohistochemistry for monocyte chemoattractant protein-1 (MCP-1), Toll-like receptor-2 (TLR-2), TGF-β1, and connective tissue growth factor (CTGF) was similar to that for ED-1. The number of ED-1-positive cells was counted at least 40 glomeruli per section under 400× magnification. Semiquantitative analyses were performed by counting the percentage of the stained areas per field of glomerulus at 400× magnification on a color image autoanalyzer (TDI Scope Eye Version 3.5 for Windows; Olympus). A minimum of 40 glomeruli per section were counted and averaged. Immunoblotting Renal cortex tissue was homogenized in lysis buffer (20 mM/l Tris-Cl, pH 7.6, 150 mM/l NaCl, 1% (w/v) sodium deoxycholate,
Leflunomide and Benazepril on DN
1% (v/v) Triton X-100, 0.1% SDS, 2 mM/l NaVO3, and freshly added 1% (v/v) aprotinin, leupeptin (1 μg/ml), pepstatin (1 μg/ ml), and 1 mM/l phenylmethyl-sulfonyl-fluoride). Homogenates were centrifuged at 3,000 rpm for 15 min at 4 ° C, and the protein concentration of the lysate was determined using a protein microassay of the Bradford method (Bio-Rad, Hercules, Calif., USA). Protein samples were resolved on 15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then electroblotted onto Bio-Blot nitrocellulose membrane (Bio-Rad). An equal amount of protein loading (50 μg) was verified by Ponceau S staining. The membrane was blocked for 1 h in TBS-added Tween-20 (TBS-T, 10 mM Tris-Cl, 150 mM NaCl, pH 8.0, 0.05% Tween-20) containing 5% non-fat powdered milk. MCP-1 or CTGF (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif., USA) was detected by incubating for 1 h with anti-MCP-1 or anti-CT
Nephron Exp Nephrol 2014;126:148–156 DOI: 10.1159/000362556
151
Control
DN
DN+B
DN+B+L
a
Control
b
TLR-2 expression (%/0.5 mm2)
MCP-1 expression (%/0.5 mm2)
*
0.5
† †
0.4 0.3
‡
0.2 0.1 0
DN+B+L
DN+B
0.25
0.6
c
DN
Con
DN
DN+B DN+L DN+B+L
d
*
0.20
†
0.15
†
0.10 ‡ 0.05 0
Con
DN
DN+B DN+L DN+B+L
Fig. 3. Representative photomicrographs of immunohistochemistry for MCP-1 (a) and TLR-2 (b) and semiquantitative analyses (c, d). ×400. * p < 0.01 vs. Con; † p < 0.01 vs. DN; ‡ p < 0.01 vs. DN+B or DN+L.
GF antibody diluted 1: 2,000. Primary antibody incubation was followed by six washes of TBS-T. The blot was then incubated with secondary antibody (anti-rabbit Ig, horseradish peroxidase) conjugate at 1: 1,000 (Amersham Biosciences, Little Chalfont, UK) for 1 h. Antibody-reactive protein was detected using enhanced chemiluminescence (Amersham Biosciences). Optical densities were obtained using the Con group as 100% reference and normalized with β-actin.
Statistical Analysis Data are expressed as mean ± SEM. Multiple comparisons among groups were performed by one-way ANOVA with the post hoc Bonferroni test (SPSS software version 19.0; IBM, Armonk, N.Y., USA). Statistical significance was accepted when p < 0.05.
Results Enzyme-Linked Immunosorbent Assay 24-Hour urinary concentrations of the DNA adduct 8-hydroxy2′-deoxyguanosine (8-OHdG) were determined using a competitive enzyme-linked immunosorbent assay (Japan Institute for the Control of Aging, Shizuoka, Japan) according to the manufacturer’s protocol. All samples were assayed in triplicate.
152
Nephron Exp Nephrol 2014;126:148–156 DOI: 10.1159/000362556
Synergistic Effect of LEF and Benazepril on Basic Parameters As shown in figure 1, DN rats displayed loss of body weight, increase in the ratio of kidney weight to body Jin /Piao /Jin /Jin /Cui /Jin /Zheng /Li /Jiang / Yang /Li
Control
DN
DN+B
DN+B+L
a
b
† †
0.4 0.3
‡
0.2 0.1
Con
DN
DN+B
DN+L DN+B+L
TLR-2 expression (%/0.5 mm2)
MCP-1 expression (%/0.5 mm2)
*
0.5
0
DN
DN+B
DN+B+L
0.25
0.6
c
Control
d
*
0.20
†
0.15
†
0.10 ‡ 0.05 0
Con
DN
DN+B
DN+L DN+B+L
Fig. 4. Representative photomicrographs of immunohistochemistry for TGF-β1 (a) and CTGF (b) and semiquantitative analyses (c, d). ×400. * p < 0.01 vs. Con; † p < 0.01 vs. DN; ‡ p < 0.01 vs. DN+B or DN+L.
weight (a marker for the development of DN), and enhanced UPE. The changes in all of these parameters were reversed by either LEF or benazepril, and the effects were more pronounced with combined treatment using two drugs. There was no significant difference in blood glucose levels between treatment groups. Diabetic rats had higher SBP levels than control rats at the start of the treatment (128 ± 5 vs. 116 ± 4 mm Hg, p < 0.05; fig.1d). Blockade of RAS with benazepril markedly lowered the SBP of diabetic rats throughout treatment versus the untreated diabetic rats. However, LEF treatment did not influence SBP in either treatment groups (p < 0.05).
Synergistic Effect of LEF and Benazepril on Histology Rats with STZ-induced diabetes showed histological features typical of glomerulopathy, characterized by glomerular hypertrophy, mesangial expansion, thickening of the basement membrane, arteriolar hyalinosis, and nodular or sclerosis (fig. 2a). The glomeruli sclerosis index was increased ninefold in the DN group compared with the Con group (0.492 ± 0.057 vs. 0.054 ± 0.014%/mm2, p < 0.01). This pathological abnormality was significantly improved in the DN±B (0.375 ± 0.027%/mm2 vs. DN, p < 0.01) and DN±L (0.406 ± 0.024%/mm2 vs. DN, p < 0.01) groups, and additional improvement was observed following co-administra-
Leflunomide and Benazepril on DN
Nephron Exp Nephrol 2014;126:148–156 DOI: 10.1159/000362556
153
Con
DN
DN+B
DN+L
DN+B+L
Con
MCP-1
130 kDa
DŽ$FWLQ
42 kDa
DN+B+L 130 kDa
CTGF
*
42 kDa
400 †
300
&7*)DŽDFWLQ
0&3DŽDFWLQ
DN+L
500
400
†
‡
200
* †
†
300
‡
100 50
100 0
DN+B
DŽ$FWLQ
500
a
DN
Con
DN
DN+B DN+L DN+B+L
b
0
Con
DN
DN+B DN+L DN+B+L
Fig. 5. Immunoblotting of MCP-1 (a) and CTGF (b). * p < 0.01 vs. Con; † p < 0.01 vs. DN; ‡ p < 0.01 vs. DN±B
or DN±L.
tion of LEF and benazepril (DN±B±L 0.153 ± 0.028%/ mm2 vs. DN, p < 0.01; fig. 2c). Synergistic Effect of LEF and Benazepril on Macrophage Infiltration Macrophages, as detected by immunohistochemical staining for ED-1, were rarely observed within the glomerulus of the control rats, but their numbers were markedly higher in the diabetic controls (10.3 ± 2.6 vs. 2.7 ± 0.6, p < 0.01; fig. 2b). Concomitant administration of LEF or benazepril significantly decreased the number of ED1-positive cells (DN±B 7.3 ± 2.7 vs. DN, DN±L 8.2 ± 2.6 vs. DN, p < 0.01, respectively) and this decrease was more pronounced with combined treatment with LEF ± benazepril (4.1 ± 1.3 vs. DN, p < 0.01; fig. 2d). Synergistic Effect of LEF and Benazepril on MCP-1 and TLR-2 In the DN group, glomerular MCP-1 (0.460 ± 0.050 vs. 0.020 ± 0.010%/mm2, p < 0.01) and TLR-2 (0.180 ± 0.029 vs. 0.011 ± 0.001%/mm2, p < 0.01) expression were much higher than in the Con group, whereas their expression decreased in the DN+B (MCP-1 0.350 ± 0.030%/mm2 vs. DN, p < 0.01; TLR-2 0.140 ± 0.018%/mm2 vs. DN, p < 0.01) and DN+L groups (MCP-1 0.400 ± 0.060%/mm2 vs. DN, p < 0.01; TLR2 0.130 ± 0.015%/mm2 vs. DN, p < 0.01), and a further decrease was observed in the DN+B+L group (MCP-1 0.230 ± 154
Nephron Exp Nephrol 2014;126:148–156 DOI: 10.1159/000362556
0.030%/mm2 vs. DN+B or DN+L; TLR-2 0.067 ± 0.003%/ mm2, p < 0.01, respectively; fig. 3). Immunoblotting of MCP-1 further conformed that a combination of LEF and benazepril significantly decreased MCP-1 protein expression compared with giving each drug alone (see fig. 5a). Synergistic Effect of LEF and Benazepril on TGF-β1 and CTGF Figure 4 shows a significant upregulation of TGF-β1 (0.184 ± 0.025 vs. 0.02 ± 0.003%/mm2, p < 0.01) and CTGF (0.190 ± 0.023 vs. 0.014 ± 0.003%/mm2, p < 0.01) in the glomeruli of DN rats, and a downregulation in both the DN+B (TGF-β1 0.137 ± 0.017%/mm2 vs. DN; CTGF 0.142 ± 0.020%/mm2, p < 0.01, respectively) and DN+L groups (TGF-β1 0.146 ± 0.019%/mm2 vs. DN; CTGF 0.151 ± 0.023%/mm2, p < 0.01, respectively). The greatest effect was observed in the DN+B+L group (TGF-β1 0.070 ± 0.003%/mm2 vs. DN+B or DN+L; CTGF 0.008 ± 0.009%/mm2, p < 0.01, respectively). Immunoblotting of CTGF further conformed that a combination of LEF and benazepril significantly decreased CTGF protein expression compared with giving each drug alone (fig. 5b). Synergistic Effect of LEF and Benazepril on Oxidative Stress The present study analyzed oxidative stress by evaluating urinary 8-OHdG excretion in the treatment groups. Jin /Piao /Jin /Jin /Cui /Jin /Zheng /Li /Jiang / Yang /Li
Urinary 8-OHdG excretion (ng/day)
400
* 300
† † ‡
200
100
0
Con
DN
DN+B
DN+L DN+B+L
Fig. 6. Urinary 8-OHdG excretion in the treatment groups. * p
Received: October 24, 2013 Accepted: March 25, 2014 Published online: May 16, 2014
Synergistic Effects of Leflunomide and Benazepril in Streptozotocin-Induced Diabetic Nephropathy Hua Jin a Shang Guo Piao a, b Ji Zhe Jin a Ying Shun Jin a Zhen Hua Cui a Hai Feng Jin a Hai Lan Zheng a Jin Ji Li a Yu Ji Jiang a Chul Woo Yang b Can Li a a
Nephrology and Dialysis Unit, Department of Internal Medicine, Yanbian University Hospital, Yanji, Jilin Province, PR China; b Transplant Research Center, Convergent Research Consortium in Immunologic Disease, The Catholic University of Korea, Seoul, Korea
Key Words Leflunomide · Benazepril · Diabetic nephropathy · Toll-like receptor-2 · Connective tissue growth factor · 8-Hydroxy-2’-deoxyguanosine
Abstract Background: Leflunomide (LEF) and benazepril have renoprotective effects on diabetic nephropathy (DN) through their anti-inflammatory and anti-fibrotic activities. This study investigated whether combined treatment using LEF and benazepril affords superior protection compared with the respective monotherapies. Methods: Diabetes was induced with streptozotocin (STZ, 65 mg/kg) by intraperitoneal injection in male Wistar rats. Two weeks after STZ injection, diabetic rats were treated daily for 12 weeks with LEF (10 mg/kg), benazepril (10 mg/kg), or a combination of both. Basic parameters (body weight, fasting blood glucose level, and 24 h urinary protein excretion), histopathology, inflammatory [inflammatory cell infiltration (ED-1), monocyte chemoattractant protein-1 (MCP-1), and Tolllike receptor-2 (TLR-2)] and glomerulosclerotic factors
© 2014 S. Karger AG, Basel 1660–2129/14/1263–0148$39.50/0 E-Mail [email protected] www.karger.com/nee
[transforming growth factor-β1 (TGF-β1) and connective tissue growth factor (CTGF)], and oxidative stress (8-hydroxy-2’-deoxyguanosine, 8-OHdG) were studied. Results: Benazepril or LEF treatment significantly prevented body weight loss and 24 h urinary protein excretion induced by diabetes; combined treatment with LEF and benazepril further improved these parameters compared with giving each drug alone (all p < 0.01). Increased expression of inflammatory (MCP-1 and TLR-2) and glomerulosclerotic (TGF-β1 and CTGF) factors in diabetic rat kidney was reduced by treatment with either LEF or benazepril and was further reduced by the combined administration of the two drugs (p < 0.01). These effects were accompanied by suppression of urinary 8-OHdG excretion. There was no significant between-group difference in blood glucose level. Conclusions: LEF treatment lessens DN, and combined treatment with LEF and benazepril provides synergistic effects in preventing DN. © 2014 S. Karger AG, Basel
Can Li, MD Nephrology and Dialysis Unit, Department of Internal Medicine Yanbian University Hospital, #1327 Juzi Street Yanji, Jilin Province 133000 (PR China) E-Mail lican @ ybu.edu.cn Chul Woo Yang, MD Transplant Research Center, Convergent Research Consortium in Immunologic Disease The Catholic University of Korea Seoul 137-040 (Korea) E-Mail yangch @ catholic.ac.kr
Introduction
Leflunomide (LEF) is a prodrug that is rapidly converted to its active metabolite A77 1726, which inhibits de novo pyrimidine nucleotide biosynthesis by reducing the activity of the enzyme dihydroorotate dehydrogenase. Because of this activity, the clinical use of LEF has produced a significant reduction in rheumatoid arthritis. However, LEF possesses both immunosuppressive and antiviral properties. LEF may thus exert pleiotropic effects in the management of cytomegalovirus infection, BK virus nephropathy, and chronic allograft dysfunction in renal transplant recipients. The renoprotective effects of LEF have also been reported in lupus nephritis, IgA nephropathy, and diabetic nephropathy (DN) [1]. Nevertheless, the precise mechanism of the renoprotective effects of LEF remains to be elucidated. Diabetes mellitus (DM) is the leading cause of endstage renal disease worldwide. The prevalence of DN caused by type 2 DM is significantly increasing not only in developed countries but also in developing countries. Despite strict control of blood glucose, hypertension, and metabolic abnormalities, the number of diabetic patients entering end-stage renal disease because of DM remains extremely high. DN is characterized by a progressive accumulation of extracellular matrix components in the glomerular mesangium and tubular interstitium, which eventually leads to proteinuria and renal insufficiency [2]. It is well recognized that glomerulosclerosis, along with tubulointerstitial injury, is a major feature and an important predictor of renal dysfunction in DN. The mechanism responsible for DN is multifactorial, and there is overwhelming evidence implicating the renin-angiotensin system (RAS), inflammatory cytokines, and transforming growth factor-β1 (TGF-β1) as important players [3, 4]. Of these, RAS plays a critical role in the mediation of inflammation and fibrotic process in DN. Blockade of RAS by angiotensin II blockers or angiotensin-converting enzyme inhibitors (ACE-I) confers renoprotection. We hypothesized that the combined treatment with LEF and ACE-I (benazepril) may prevent the progression of DN. To test the hypothesis, LEF and benazepril were administered separately or combined to rats with streptozotocin (STZ)-induced DM. Our study clearly demonstrates that combined treatment using two drugs has synergistic effects on preventing inflammatory and sclerotic processes in DN.
Leflunomide and Benazepril on DN
Materials and Methods Experiment Schedule The experimental protocol was approved by the Animal Care Committee of the Catholic University of Korea. Male Wistar rats (Charles River Technologies, Korea) weighing 220–250 g, were housed in individual cages in a temperature- and light-controlled environment and allowed free access to tap water and standard laboratory chow. Rats were randomized divided into five groups: (1) controls (Con, n = 6): non-diabetic normal animals; (2) diabetic controls (DN, n = 8): diabetic animals treated with saline used as diabetic controls; (3) benazepril treatment (DN+B, n = 8): diabetic animals treated with benazepril at a dose of 10 mg/kg body weight; (4) LEF treatment (DN+L, n = 8): diabetic animals treated with LEF at a dose of 10 mg/kg body weight, and (5) combined treatment (DN+B+L, n = 8): diabetic animals treated with both LEF and benazepril. Diabetes was induced by a single intraperitoneal injection of STZ (65 mg/kg), and the induction of diabetes was defined when blood glucose level was ≥16.7 mmol/l (300 mg/dl). Benazepril and LEF treatment were started 2 weeks after STZ injection and were administered via oral gavage for 12 weeks. At the end of the study, animals were euthanized under ketamine anesthesia and kidney tissue was rapidly removed for morphological examination. The dosage and method of benazepril [5] and LEF [6] administration were chosen based on previous reports. Biochemical Analysis Body weight was recorded periodically for each rat and blood glucose levels were measured. Urine samples were collected over a 24-hour period in individual metabolic cages (Tecniplast Gazzada Srl, Buguggiate, Italy) for the measurement of urinary protein excretion (UPE) at 0, 4, 8, and 12 weeks, and were determined by the Bradford method. Systolic blood pressure (SBP) was measured in conscious rats every 4 months using the tail-cuff method with a plethysmography by a tail manometer-tachometer system (BP2000; Visitech System, Apex, N.C., USA). Histopathology Kidney tissues were fixed in periodate-lysine-paraformaldehyde solution and embedded in wax. After dewaxing, 4-μm sections were processed and stained with periodic acid-Schiff (PAS). Semiquantitative analysis was performed by counting the percentage of the stained areas per field of glomerulus at 400× magnification on a color image autoanalyzer (TDI Scope Eye Version 3.5 for Windows; Olympus). A minimum of 40 glomeruli per section were counted and averaged. Immunohistochemistry After dewaxing, sections were incubated with a 0.5% Triton X100-PBS solution for 30 min and washed with phosphate-buffered saline (PBS) three times. Non-specific binding sites were blocked for 1 h with normal donkey serum diluted 1:10 in PBS, and then incubated overnight at 4 ° C with ED-1 (Serotec, Inc., Indianapolis, Ind., USA) diluted in 1:1,000 in a humid environment. After rinsing in Tris-buffered saline (TBS), sections were incubated in peroxidase-conjugated donkey anti-mouse or rabbit IgG Fab fragment (Jackson ImmunoResearch Laboratories, West Grove, Pa., USA) for 30 min. For staining, sections were incubated with a mixture of 0.05% 3,3-diaminobenzidine containing 0.01% H2O2 at room temperature until a brown color was visible, washed with
Nephron Exp Nephrol 2014;126:148–156 DOI: 10.1159/000362556
149
Con
35
DN DN+B
Body weight (g)
400
DN+L DN+B+L
350
†
†
300 250
*
200 0
a
4
* 8
*
Fasting glucose levels (mmol/l)
450
*
4
8
*
25 20 15 10 5 0
b
12
Time (months)
160
*
60
150
* *
30
†
* † †, ‡
†, ‡
†, ‡
*
140 SBP (mm Hg)
50 UPE (mg/dl)
*
0
70
10
100 0 4
8
12
Time (months)
†
†
120 110
0
*
*
*
130
20
0
c
*
12
Time (months)
40
30
0
4
d
8
12
Time (months)
7
*
KW/BW ratio × 1,000
6
†
†
5
‡
4 3 2 1
Fig. 1. Basic parameters during the experiment period: (a) body weight, (b) fasting blood glucose, (c) UPE, (d) SBP, and (e) kid-
ney weight to body weight ratio (KW/BW) ×1,000. * p < 0.01 vs. Con; † p < 0.01 vs. DN; ‡ p < 0.01 vs. DN+B or DN+L.
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Nephron Exp Nephrol 2014;126:148–156 DOI: 10.1159/000362556
e
0
Con
DN
DN+B
DN+L DN+B+L
Jin /Piao /Jin /Jin /Cui /Jin /Zheng /Li /Jiang / Yang /Li
c
DN
Control
DN+B
DN+B+L
b
0.6
†
0.4
†
0.3 0.2
‡
0.1 0
Con
DN
DN+B+L
15
*
0.5
DN
DN+B
DN+B
DN+L DN+B+L
ED-1-positive cells (%/0.5 mm2)
Glomerular sclerosis index (%/0.5 mm2)
a
Control
d
*
12
†
†
9 6
‡ 3 0
Con
DN
DN+B
DN+L DN+B+L
Fig. 2. Representative photomicrographs of PAS staining (a) and immunohistochemistry for ED-1 (b) and semiquantitative analyses (c, d). ×400. * p < 0.01 vs. Con; † p < 0.01 vs. DN; ‡ p < 0.01 vs. DN±B or DN±L.
TBS, counterstained with hematoxylin, and examined under light microscopy. The procedure of immunohistochemistry for monocyte chemoattractant protein-1 (MCP-1), Toll-like receptor-2 (TLR-2), TGF-β1, and connective tissue growth factor (CTGF) was similar to that for ED-1. The number of ED-1-positive cells was counted at least 40 glomeruli per section under 400× magnification. Semiquantitative analyses were performed by counting the percentage of the stained areas per field of glomerulus at 400× magnification on a color image autoanalyzer (TDI Scope Eye Version 3.5 for Windows; Olympus). A minimum of 40 glomeruli per section were counted and averaged. Immunoblotting Renal cortex tissue was homogenized in lysis buffer (20 mM/l Tris-Cl, pH 7.6, 150 mM/l NaCl, 1% (w/v) sodium deoxycholate,
Leflunomide and Benazepril on DN
1% (v/v) Triton X-100, 0.1% SDS, 2 mM/l NaVO3, and freshly added 1% (v/v) aprotinin, leupeptin (1 μg/ml), pepstatin (1 μg/ ml), and 1 mM/l phenylmethyl-sulfonyl-fluoride). Homogenates were centrifuged at 3,000 rpm for 15 min at 4 ° C, and the protein concentration of the lysate was determined using a protein microassay of the Bradford method (Bio-Rad, Hercules, Calif., USA). Protein samples were resolved on 15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then electroblotted onto Bio-Blot nitrocellulose membrane (Bio-Rad). An equal amount of protein loading (50 μg) was verified by Ponceau S staining. The membrane was blocked for 1 h in TBS-added Tween-20 (TBS-T, 10 mM Tris-Cl, 150 mM NaCl, pH 8.0, 0.05% Tween-20) containing 5% non-fat powdered milk. MCP-1 or CTGF (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif., USA) was detected by incubating for 1 h with anti-MCP-1 or anti-CT
Nephron Exp Nephrol 2014;126:148–156 DOI: 10.1159/000362556
151
Control
DN
DN+B
DN+B+L
a
Control
b
TLR-2 expression (%/0.5 mm2)
MCP-1 expression (%/0.5 mm2)
*
0.5
† †
0.4 0.3
‡
0.2 0.1 0
DN+B+L
DN+B
0.25
0.6
c
DN
Con
DN
DN+B DN+L DN+B+L
d
*
0.20
†
0.15
†
0.10 ‡ 0.05 0
Con
DN
DN+B DN+L DN+B+L
Fig. 3. Representative photomicrographs of immunohistochemistry for MCP-1 (a) and TLR-2 (b) and semiquantitative analyses (c, d). ×400. * p < 0.01 vs. Con; † p < 0.01 vs. DN; ‡ p < 0.01 vs. DN+B or DN+L.
GF antibody diluted 1: 2,000. Primary antibody incubation was followed by six washes of TBS-T. The blot was then incubated with secondary antibody (anti-rabbit Ig, horseradish peroxidase) conjugate at 1: 1,000 (Amersham Biosciences, Little Chalfont, UK) for 1 h. Antibody-reactive protein was detected using enhanced chemiluminescence (Amersham Biosciences). Optical densities were obtained using the Con group as 100% reference and normalized with β-actin.
Statistical Analysis Data are expressed as mean ± SEM. Multiple comparisons among groups were performed by one-way ANOVA with the post hoc Bonferroni test (SPSS software version 19.0; IBM, Armonk, N.Y., USA). Statistical significance was accepted when p < 0.05.
Results Enzyme-Linked Immunosorbent Assay 24-Hour urinary concentrations of the DNA adduct 8-hydroxy2′-deoxyguanosine (8-OHdG) were determined using a competitive enzyme-linked immunosorbent assay (Japan Institute for the Control of Aging, Shizuoka, Japan) according to the manufacturer’s protocol. All samples were assayed in triplicate.
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Synergistic Effect of LEF and Benazepril on Basic Parameters As shown in figure 1, DN rats displayed loss of body weight, increase in the ratio of kidney weight to body Jin /Piao /Jin /Jin /Cui /Jin /Zheng /Li /Jiang / Yang /Li
Control
DN
DN+B
DN+B+L
a
b
† †
0.4 0.3
‡
0.2 0.1
Con
DN
DN+B
DN+L DN+B+L
TLR-2 expression (%/0.5 mm2)
MCP-1 expression (%/0.5 mm2)
*
0.5
0
DN
DN+B
DN+B+L
0.25
0.6
c
Control
d
*
0.20
†
0.15
†
0.10 ‡ 0.05 0
Con
DN
DN+B
DN+L DN+B+L
Fig. 4. Representative photomicrographs of immunohistochemistry for TGF-β1 (a) and CTGF (b) and semiquantitative analyses (c, d). ×400. * p < 0.01 vs. Con; † p < 0.01 vs. DN; ‡ p < 0.01 vs. DN+B or DN+L.
weight (a marker for the development of DN), and enhanced UPE. The changes in all of these parameters were reversed by either LEF or benazepril, and the effects were more pronounced with combined treatment using two drugs. There was no significant difference in blood glucose levels between treatment groups. Diabetic rats had higher SBP levels than control rats at the start of the treatment (128 ± 5 vs. 116 ± 4 mm Hg, p < 0.05; fig.1d). Blockade of RAS with benazepril markedly lowered the SBP of diabetic rats throughout treatment versus the untreated diabetic rats. However, LEF treatment did not influence SBP in either treatment groups (p < 0.05).
Synergistic Effect of LEF and Benazepril on Histology Rats with STZ-induced diabetes showed histological features typical of glomerulopathy, characterized by glomerular hypertrophy, mesangial expansion, thickening of the basement membrane, arteriolar hyalinosis, and nodular or sclerosis (fig. 2a). The glomeruli sclerosis index was increased ninefold in the DN group compared with the Con group (0.492 ± 0.057 vs. 0.054 ± 0.014%/mm2, p < 0.01). This pathological abnormality was significantly improved in the DN±B (0.375 ± 0.027%/mm2 vs. DN, p < 0.01) and DN±L (0.406 ± 0.024%/mm2 vs. DN, p < 0.01) groups, and additional improvement was observed following co-administra-
Leflunomide and Benazepril on DN
Nephron Exp Nephrol 2014;126:148–156 DOI: 10.1159/000362556
153
Con
DN
DN+B
DN+L
DN+B+L
Con
MCP-1
130 kDa
DŽ$FWLQ
42 kDa
DN+B+L 130 kDa
CTGF
*
42 kDa
400 †
300
&7*)DŽDFWLQ
0&3DŽDFWLQ
DN+L
500
400
†
‡
200
* †
†
300
‡
100 50
100 0
DN+B
DŽ$FWLQ
500
a
DN
Con
DN
DN+B DN+L DN+B+L
b
0
Con
DN
DN+B DN+L DN+B+L
Fig. 5. Immunoblotting of MCP-1 (a) and CTGF (b). * p < 0.01 vs. Con; † p < 0.01 vs. DN; ‡ p < 0.01 vs. DN±B
or DN±L.
tion of LEF and benazepril (DN±B±L 0.153 ± 0.028%/ mm2 vs. DN, p < 0.01; fig. 2c). Synergistic Effect of LEF and Benazepril on Macrophage Infiltration Macrophages, as detected by immunohistochemical staining for ED-1, were rarely observed within the glomerulus of the control rats, but their numbers were markedly higher in the diabetic controls (10.3 ± 2.6 vs. 2.7 ± 0.6, p < 0.01; fig. 2b). Concomitant administration of LEF or benazepril significantly decreased the number of ED1-positive cells (DN±B 7.3 ± 2.7 vs. DN, DN±L 8.2 ± 2.6 vs. DN, p < 0.01, respectively) and this decrease was more pronounced with combined treatment with LEF ± benazepril (4.1 ± 1.3 vs. DN, p < 0.01; fig. 2d). Synergistic Effect of LEF and Benazepril on MCP-1 and TLR-2 In the DN group, glomerular MCP-1 (0.460 ± 0.050 vs. 0.020 ± 0.010%/mm2, p < 0.01) and TLR-2 (0.180 ± 0.029 vs. 0.011 ± 0.001%/mm2, p < 0.01) expression were much higher than in the Con group, whereas their expression decreased in the DN+B (MCP-1 0.350 ± 0.030%/mm2 vs. DN, p < 0.01; TLR-2 0.140 ± 0.018%/mm2 vs. DN, p < 0.01) and DN+L groups (MCP-1 0.400 ± 0.060%/mm2 vs. DN, p < 0.01; TLR2 0.130 ± 0.015%/mm2 vs. DN, p < 0.01), and a further decrease was observed in the DN+B+L group (MCP-1 0.230 ± 154
Nephron Exp Nephrol 2014;126:148–156 DOI: 10.1159/000362556
0.030%/mm2 vs. DN+B or DN+L; TLR-2 0.067 ± 0.003%/ mm2, p < 0.01, respectively; fig. 3). Immunoblotting of MCP-1 further conformed that a combination of LEF and benazepril significantly decreased MCP-1 protein expression compared with giving each drug alone (see fig. 5a). Synergistic Effect of LEF and Benazepril on TGF-β1 and CTGF Figure 4 shows a significant upregulation of TGF-β1 (0.184 ± 0.025 vs. 0.02 ± 0.003%/mm2, p < 0.01) and CTGF (0.190 ± 0.023 vs. 0.014 ± 0.003%/mm2, p < 0.01) in the glomeruli of DN rats, and a downregulation in both the DN+B (TGF-β1 0.137 ± 0.017%/mm2 vs. DN; CTGF 0.142 ± 0.020%/mm2, p < 0.01, respectively) and DN+L groups (TGF-β1 0.146 ± 0.019%/mm2 vs. DN; CTGF 0.151 ± 0.023%/mm2, p < 0.01, respectively). The greatest effect was observed in the DN+B+L group (TGF-β1 0.070 ± 0.003%/mm2 vs. DN+B or DN+L; CTGF 0.008 ± 0.009%/mm2, p < 0.01, respectively). Immunoblotting of CTGF further conformed that a combination of LEF and benazepril significantly decreased CTGF protein expression compared with giving each drug alone (fig. 5b). Synergistic Effect of LEF and Benazepril on Oxidative Stress The present study analyzed oxidative stress by evaluating urinary 8-OHdG excretion in the treatment groups. Jin /Piao /Jin /Jin /Cui /Jin /Zheng /Li /Jiang / Yang /Li
Urinary 8-OHdG excretion (ng/day)
400
* 300
† † ‡
200
100
0
Con
DN
DN+B
DN+L DN+B+L
Fig. 6. Urinary 8-OHdG excretion in the treatment groups. * p
