- Email: [email protected]
L-Carnitine Protects Against Cyclosporine-Induced Pancreatic and Renal Injury in Rats
L-Carnitine Protects Against Cyclosporine-Induced Pancreatic and Renal Injury in Rats Y. Xiang, S.G. Piao, H.B. Zou, J. Jin, M.R. Fang, D.M. Lei, B.H. Gao, C.W. Yang, and C. Li ABSTRACT Background. L-carnitine has protective effects against various types of injury. This study was designed to evaluate the beneficial effects of L-carnitine on pancreatic and renal injuries caused by cyclosporine (CsA). Methods. Rats maintained on a low sodium diet were given vehicle (olive oil, 1 mL/kg/d), CsA (15 mg/kg/d), L-carnitine (50 or 200 mg/kg/d), or a combination of CsA and L-carnitine for 4 weeks. The impact of L-carnitine on pancreatic injury was assessed by blood glucose levels, plasma insulin concentrations, and hemoglobulin A1c (HbA1c). In addition, the protective effects of L-carnitine against CsA-induced kidney injury were evaluated in terms of renal function, histopathology (inflammatory cell influx and tubulointerstitial fibrosis), oxidative stress (8-hydroxy 20 -deoxyguanosine, 8-OHdG), transforming growth factor-betal (TGF-b1), apoptosis (caspase-3), and autophagy (LC3-II). Results. CsA treatment caused diabetes, renal dysfunction, tubulointerstitial inflammation (ED-1-positive cells), and fibrosis, which were accompanied by an increase in 8-OHdG production and upregulation of TGF-b1, caspase-3, and LC3-II. Concomitant administration of L-carnitine increased plasma insulin concentrations, decreasing plasma glucose and HbA1c levels. In the kidney, L-carnitine induced dose-dependent improvement of renal function, inflammation, and fibrosis in parallel with suppression of the expression of TGF-b1 and 8-OHdG. Furthermore, the administration of L-carnitine at a high dose inhibited the expression of caspase-3 and LC3-II. Conclusion. These findings suggest that L-carnitine has a protective effect against CsA-induced pancreatic and renal injuries.
D
ESPITE THE DEVELOPMENT of newer immunosuppressants, cyclosporine (CsA) remains a potent immunosuppressive agent that is used for treating multiorgan transplantation and autoimmune diseases. However, the clinical usage of CsA is frequently impeded by its
nephrotoxic effects in terms of acute and chronic nephrotoxicity. Acute CsA nephrotoxicity is regarded as being reversible and seems to be caused by a reduction in renal blood flow related to afferent arteriolar vasoconstriction. However, the long-term administration of CsA causes
From the Nephrology & Dialysis Unit, Department of Internal Medicine, (Y.X., S.G.P., J.J., M.R.F., D.M.L., C.L.), YanBian University Hospital, YanJi, JiLin, PR China; Convergent Research Consortium in Immunologic Disease (S.G.P., C.W.Y.), The Catholic University of Korea, Seoul, Korea; Transplant Research Center (S.G.P., C.W.Y.), Seoul St Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea; Division of Nephrology (H.B.Z.), The First Hospital of JiLin University, ChangChun, JiLin, PR China; and Division of Nephrology (B.H.G.), ZhongShan Hospital of DaLian University, DaLian, LiaoNing, PR China.
This work was supported by the National Natural Science Foundation of China (No. 81160092) and a grant of the Korea Healthcare technology R&D Project, Ministry for Health, Welfare & Family Affairs of Korea (A092258). Address reprint requests to Can Li, MD, PhD, Nephrology & Dialysis Unit, Department of Internal Medicine, YanBian University Hospital, YanJi, JiLin, PR China, or Chul Woo Yang, MD, PhD, Transplant Research Center, Seoul, St Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul Korea. E-mail: [email protected] or [email protected]
Crown Copyright ª 2013 Published by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 45, 3127e3134 (2013)
0041-1345/13/$esee front matter http://dx.doi.org/10.1016/j.transproceed.2013.08.041 3127
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progressive renal insufficiency with striped tubulointerstitial fibrosis (TIF), tubular atrophy, inflammatory cell influx, and hyalinosis of the afferent arterioles.1,2 The mechanism underlying chronic CsA nephropathy is multifactorial, and several factors, including the intrarenal reninangiotensin system, inflammatory mediators, the transforming growth factor-beta 1 (TGF-b1), oxidative stress, apoptotic cell death, and excessive autophagy, have been implicated in this condition.2e5 Posttransplant diabetes mellitus (PTDM) is a common and serious complication after kidney transplantation that is associated with decreased graft and patient survival.6 A recent meta-analysis of observational studies and randomized controlled trials reported that the incidence of PTDM in the first year after transplantation varied from 2% to 50%. The incidence of PTDM is higher in the first 6 months after transplantation.7 Although its etiopathogenesis is multifactorial, PTDM is thought to be related to the increased insulin resistance that results from glucocorticoids8 and to the decreased insulin secretion caused by calcineurin inhibitors.9,10 PTDM results in adverse outcomes, such as decreased graft survival, increased cardiovascular mortality, and increased risk of postoperative infections.11 L-carnitine (b-hydroxy-g-N-trimethyl ammonium-butyrate) is a component of lipid metabolism that is important for the production of adenosine triphospate (ATP) through the boxidation of long-chain fatty acids. In this manner, L-carnitine functions as an indirect antioxidant and facilitates the repair of oxidized membranes or lipid bilayers.12 In addition, Lcarnitine activity has also been reported to be a direct scavenger of O2 and H2O2.13 Thus, L-carnitine may possess antioxidant properties beyond its action on lipid metabolism. It is well known that L-carnitine is necessary for the efflux of acyl groups from mitochondrial. Therefore, L-carnitine supplementation is known to play a beneficial role in insulin resistance or diabetic podocyte injury,14,15 as intracellular accumulation of acyl-CoA derivatives has been implicated in the development of insulin resistance. Recently, the renoprotective effects of L-carnitine have also been reported in ischemia-reperfusion injury,16 cisplatininduced nephrotoxicit,17 doxorubicin-induced nephritic syndrome,18 and chronic CsA nephropathy.19 Nevertheless, the molecular mechanism underlying the benefits of Lcarnitine on CsA-induced pancreatic and renal injuries remains unexplored. In view of the findings described above, the present study was undertaken to evaluate the protective effect of Lcarnitine on CsA-induced pancreatic and renal injuries using a well-described rat model of chronic CsA nephropathy.
XIANG, PIAO, ZOU ET AL 180 to 200 g, were housed in cages (Nalge Co, Rochester, NY, USA) in a controlled-temperature and controlled-light environment and allowed free access to a low salt diet (0.05% sodium, Teklad Premier, Madison, Wis, USA) throughout the experimental period. A total of 45 rats were randomized into 6 subgroups and treated daily for 4 weeks as follows: (1) vehicle group (VH, n ¼ 7): rats received olive oil (1 mL/kg/d subcutaneous) (2) VH þ L-carnitine group (VH þ L50, n ¼ 7): rats received olive oil and L-carnitine (50 mg/kg intravenously); (3) VH þ L-carnitine group (VH þ 200, n ¼ 7): rats received olive oil and L-carnitine (200 mg/kg intravenously); (4) CsA group (C, n ¼ 8): rats received CsA (15 mg/kg/ d subcutaneously); (5) CsA þ L-carnitine group (C þ L50, n ¼ 8): rats received CsA and L-carnitine (50 mg/kg intravenously); (6) CsA þ L-carnitine group (C þ L200, n ¼ 8): rats received CsA and L-carnitine (200 mg/kg intravenously). The dosage and method of CsA2 and L-carnitine administration20,21 were chosen based on previous reports. Animals were euthanized under ketamine anesthesia, and blood and the kidney samples were collected for biochemical and morphologic or molecular examinations.
Basic Parameters Rats were pair-fed and daily body weight (BW) was monitored. Before sacrifice, rats were individually housed in metabolic cages (Tecniplast Gazzada S. a r.l., Italy) for 24 hour-urine collection, and blood samples were withdrawn to evaluate serum creatinine (SCr), blood urea nitrogen (BUN), blood glucose levels, and plasma insulin level (Coulter-STKS; Coulter Electronics). An intraperitoneal glucose tolerance test (IPGTT) was performed at the end of the 4-week treatment period as previously described,22 and the area under the curve of glucose (AUCg) was calculated by trapezoidal estimation from the values obtained in the IPGTT. Whole blood CsA level was measured by a monoclonal radioimmunoassay (Incstar Co, Stillwater, Minn, USA).
Histopathology Harvested rat kidney tissues were fixed in periodate-lysineparaformaldehyde solution and embedded in wax. After dewaxing, 4 mm sections were processed and stained with Masson trichrome and hematoxylin. A finding of TIF was defined as a matrix-rich expansion of the interstitium with tubular dilatation, tubular atrophy, tubular cast formation, sloughing of tubular epithelial cells, or thickening of the tubular basement membrane. A minimum of 20 fields per section was assessed using a color image analyzer (TDI Scope Eye Version 3.0 for Windows, Olympus, Japan). Briefly, the images were captured, and the extent of TIF was quantified using the Polygon program by counting the percentage of areas injured per field of cortex under 100 magnification. Histopathologic analysis was performed in randomly selected cortical fields of sections by a pathologist blinded to the identity of the treatment groups.
Enzyme-Linked Immunosorbent assay MATERIALS AND METHODS Experimental design The experimental protocol was approved by the Animal Care Committee of the Catholic University of Korea. Male SpragueDawley rats (Charles River Technology, Korea), initially weighing
Twenty-four-hour urinary concentrations of the DNA adduct 8hydroxy-20 -deoxyguanosine (8-OHdG) were determined using a competitive enzyme-linked immunesorbent assay (Japan Institute for the Control of Aging, Shizuoka, Japan), according to the manufacturer’s protocol. All samples were assayed in triplicate.
L-CARNITINE PROTECTS AGAINST CSA-INDUCED INJURY
3129 detected using enhanced chemiluminescence (Amersham Biosciences, UK). Optical densities were obtained using the VH group as 100% reference and normalized with b-actin.
Immunohistochemistry After dewaxing, sections were incubated with a 0.5% Triton X100phosphate-buffered saline (PBS) solution for 30 minutes and washed with PBS three times. Nonspecific binding sites were blocked for 1 hour with normal donkey serum diluted 1:10 in PBS, and then incubated overnight at 4 C with anti-8-OHdG monoclonal antibody (JaICA; Shizuoka, Japan) diluted in 1:1000 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 Immuno Research Laboratories, West Grove, Pa, USA) for 30 minutes. 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 TBS, counterstained with hematoxylin, and examined under light microscopy. The procedure of immunostaining for TGFb1 (1:500, Santa Cruz Biotechnology, Inc) and ED-1 (1:500, Serotec Inc, UK) was similar to that for 8-OHdG. Semiquantitative analyses of TGF-b1 and ED-1 expression was performed using a color image autoanalyzer (TDI Scope Eye Version 3.5 for Windows; Olympus, Japan) by counting the percentage of stained areas per field of cortex at 100 magnification. A minimum of 20 fields per section were counted and averaged.
Statistical Analysis Data are expressed as mean standard error of the mean unless otherwise specified. Multiple comparisons among groups were performed by one-way analysis of variance with the post hoc Bonferroni test (SPSS software version 19.0, Microsoft Corp, Chicago, Ill, USA). Statistical significance was assumed as P < .05.
RESULTS Effect of L-Carnitine on Basic Parameters in Chronic CsA Nephropathy
CsA-treated rats exhibited weight loss (weight gain: 75.6 2.6 vs 89.9 2.4, P < .01) and decreased renal function (SCr, 0.55 0.02 mg/dL vs 0.42 0.03 mg/dL, P < .01; BUN, 29.4 0.94 mg/dL vs 11.3 0.48 mg/dL, P < .01) compared with VH-treated rats. However, BW was increased (weight gain: 85.3 3.0 vs CsA, P < .05) and renal function was improved after L-carnitine coadministration compared with the CsA group (SCr, 0.48 0.02 mg/ dL vs CsA, P < .05; BUN, 27.4 1.02 mg/dL vs CsA, P < .05). Whole blood CsA concentration was unaffected by the combination of L-carnitine and CsA, as shown in Table 1.
Western Botting Kidney cortex tissue was homogenized in lysis buffer (20 mmol/L Tris-Cl [pH 7.6], 150 mmol/L NaCl, 1% [wt/vol] sodium deoxycholate, 1% [vol/vol] Triton X-100, 0.1% sodium dodecyl sulfate, 2 mmol/L NaVO3, and freshly added 1% [vol/vol] aprotinin, leupeptin [l mg/mL], pepstatin [l mg/mL], and 1 mmol/L phenylmethyl sulfonyl-fluoride. Homogenates were centrifuged at 3000 rpm for 15 minutes at 4 C, and the protein concentration of the lysate was determined using a protein microassay of the Bradford method (Bio-RAD, Hercules, California, USA). Protein samples were resolved on 15% SDS-polyacrylamide gel electrophoresis and then electroblotted onto Bio-Blot nitrocellulose membrane (Bio-RAD). An equal amount of protein loading (80 mg) was verified by Ponceau S staining. The membrane was blocked for 1 hour in TBS added Tween-20 (TBS-T, 10 mmol/Tri-CL, 150 mmol/L NaCl [pH 8.0], 0.05% Tween-20) containing 5% nonfat powdered milk. Anticaspase-3, active form (1:200; Millipore Corporation, Calif, USA) or microtubule-associated protein 1 light chain 3 phospholipidconjugated form (LC3- II, 1:1000; Sigma-Aldrich, USA) was detected by incubating for 1 hour with specific primary antibodies. Primary antibody incubation was followed by 6 washes of TBS-T. The blot was then incubated with secondary antibody (Donkey anti-rat IgG, horseradish peroxidase) conjugate at 1:2000 (Amersham Biosciences, UK) for 1 hour. Antibody-reactive protein was
Effect of L-Carnitine on CsA-Induced Diabetes
Baseline blood glucose concentration did not differ between the 6 groups (Fig 1A). IPGTT was significantly increased in the CsA group compared with the VH group, whereas the CsA þ L group showed a significant decrease in IPGTT compared with the CsA group (Fig 1A). The calculated AUCg did not differ significantly between the VH and VH þ L groups and was lower in the CsA þ L group than it was in the CsA group (L50: 476.2 14.6 mg/dL minutes vs 44 11.2 mg/dL minutes, P < .05; L200: 476.2 14.6 mg/ dL minutes vs 396 9.6 mg/dL minutes, P < .05; Fig 1B). Serum insulin level did not differ significantly between the VH and VH þ L groups. However, serum insulin concentration in the CsA group was significantly lower than that observed in the VH (5.1 0.8 mg/dL minutes vs and 1.1 0.3 mg/dL minutes, P < .05) and VH þ L group (L50: 5.1 1.0 mg/dL minutes vs CsA, P < .05; L200: 4.8 0.4 mg/dL minutes vs CsA, P < .05), and was significantly higher in the
Table 1. Basic Parameters
WG UV SCr BUN CsA con TIF
89.9 11.6 0.42 11.3 0 0
VH
VF þ L50
VH þ L200
CsA
2.4 1.3 0.03 0.48 0 0
88.1 16.0 0.35 10.7 0 0
1.7 1.7 0.03 0.90 0 0
90.1 13.7 0.34 11.0 0 0
3.0 1.9 0.02 0.63 0 0
75.6 18.6 0.55 29.4 2207 34.1
CsA þ L50 a
2.6 2.7a 0.02a 0.94a 205 3.2a
85.4 13.4 0.48 27.4 2345 30.2
b
3.5 2.0 0.02b 1.02b 161 3.6b
CsA þ L200
85.3 17.3 0.41 23.8 2409 28.7
3.0b 4.0 0.03c 1.32b 225 2.5c
WG, weight gain (g); UV, urine volume (mL/d); SCr, serum creatinine (mg/dL); BUN, blood urea nitrogen (mg/dL); CsA con, CsA concentration (ng/mL); TIF, tubulointerstitial fibrosis (%0.5/mm2) CsA, Cyclosporine; VH, vehicle group; L, L-carnitine. a P < .05 vs VH. b P < .05 vs CsA. c P < .05 vs CsA þ L50.
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Fig 1. Effect of L-carnitine (L) on diabetic biomarkers. (A) Blood glucose levels (intraperitoneal glucose tolerance test [IPGTT]). (B) Calculation of area under the curve of glucose (AUCg). (C) Plasma insulin levels. Note that cyclosporine (CsA) induced an elevation in blood glucose and HbA1c and a decrease in plasma insulin levels, whereas coadministration of L reversed all of these parameters. # P < .01 vs vehicle group of (VH); ##P < .05 vs CsA.
CsA þ L group compared with the CsA group (L50: 2.7 0.5 mg/dL minutes vs CsA, P < .05; L200: 2.8 0.2 mg/dL minutes vs CsA, respectively; Fig 1C).
a further decrease was observed in the CsA þ L200 group (23.8 2.5%/0.5 mm2 vs CsA þ L50, P < .05).
Effect of L-Carnitine on Macrophage Infiltration and Fibrosis in Chronic CsA Nephropathy
Immunohistochemistry revealed that the majority of VHtreated rat kidneys were negative for TGF-b1. In contrast, the expression of the TGF-b1 protein was dramatically increased in the tubulointerstitium in CsA-treated rat kidneys (46.0% 2.3%/0.5 mm2 vs 17.1% 0.5%/0.5 mm2, P < .01). It is notable that the sites of strong TGF-b1 protein expression were located in areas of inflammatory cell infiltration and severe TIF (Fig 3A). L-carnitine suppressed TGF-b1 protein expression in a dose-dependent manner (CsA þ L50: 40.0% 1.4%/0.5 mm2, P < .05 vs CsA; CsA þ L200: 30.2% 3.0%/0.5 mm2, P < .05 vs CsA þ L50, respectively; Fig. 3B).
ED-1-positive cells were detected only rarely in the kidneys of VH-treated animals. CsA treatment increased the number of ED-1-positive cells (29 1.0/0.5 mm2 vs 5 0.7/ 0.5 mm2, P < .01). Concomitant administration of L-carnitine significantly decreased the number of ED-1-positive cells (23 1.2/0.5 mm2 vs CsA, P < .01), which was more pronounced after the administration of 200 mg/kg L-carnitine compared with the CsA þ L50 group (Fig 2, 19 2.0/ 0.5 mm2 vs CsA þ L50, P < .05). CsA-treated rats exhibited the typical striped TIF of chronic CsA nephropathy (Table 1). A significant increase in the percentage of TIF was found in the CsA group compared with the VH group (34.1% 3.2%/0.5 mm2 vs 0% 0%/0.5 mm2, P < .01). In contrast, the percentage of TIF decreased markedly when L-carnitine was coadministered (28.7% 3.6%/0.5 mm2 vs CsA, P < .05) and
Effect of L-Carnitine on TGF-b Expression in Chronic CsA Nephropathy
Effect of L-Carnitine on Oxidative Stress in Chronic CsA Nephropathy
Chronic CsA nephropathy is closely associated with oxidative stress. As shown in Fig 4, immunohistochemistry showed a significant increase in 8-OHdG expression and
L-CARNITINE PROTECTS AGAINST CSA-INDUCED INJURY
Fig 2. Representative photomicrographs of immunohistochemistry (A) and semiquantitative analysis (B) of macrophage infiltration. Cyclosporine (CsA) treatment significantly increased the number of ED-1-positive cells, whereas this number was decreased by cotreatment with L-carnitine (L). Immunohistochemical staining: original magnification, 200. #P < .01 vs vehicle group (VH); ##P < .05 vs CsA; *P < .05 vs CsA þ L50.
immunoreactivity in CsA-treated rat kidneys; these signals were confined to cortical tubular epithelial cells. Interestingly, the sites of strong 8-OHdG expression were located in areas of injured or atrophied tubules (Fig 4A). Concomitant administration of L-carnitine significantly decreased intrarenal 8-OHdG expression compared with the CsA treatment alone. Moreover, this decrease was more pronounced in the presence of a higher dose of L-carnitine compared with the CsA þ L50 group. In parallel, urinary 8-OHdG excretion in the CsA-treated group was higher than that observed in the VH-treated group (241.0 37.2 ng/d vs 130.7 17.0 ng/d, P < .01), whereas its level was
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Fig 3. Representative photomicrographs of immunohistochemistry (A) and semiquantitative analysis (B) of transforming growth factor (TGF)-b1 expression. Cyclosporine (CsA) significantly increased intrarenal TGF-b1 immunoreactivity, whereas this was significantly decreased by cotreatment with L-carnitine (L). Immunohistochemical staining: original magnification, 200. # P < 05 vs vehicle group (VH); #P < .05 vs CsA; *P < .05 vs CsA þ L50.
significantly decreased after concomitant administration of L-carnitine, in a dose-dependent manner (Fig 4B, CsA þ L50: 208.3 19.7 ng/d vs CsA, P < .05; CsA þ L200: 177.2 12.7 ng/d vs CsA þ L50, P < .05, respectively). Effect of L-Carnitine on LC3-II and Caspase-3 in Chronic CsA Nephropathy
The presence of autophagy was measured based on LC3-II, whereas apoptosis was assessed based on the detection of the apoptosis-related caspase-3 protein, using Western blotting. The results showed that CsA treatment upregulated LC3-II (183% 11% vs 102% 6%, P < .01) and
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Fig 4. Representative photomicrographs of immunohistochemistry (A) and enzyme-linked immunosorbent assay analysis of the urinary output (B) of 8 hydroxy 20 - deoxygulosine (8-OHdG) CsA treatment significantly increased intrarenal 8-OHdG immunoreactivity and urinary 8-OHdG production, whereas these phenomena were significantly decreased by cotreatment with L-carnitine (L). Immunohistochemical staining: original magnification, 200. # P < .05 vs vehicle group (VH); ##P < .05 vs CsA; *P < .05 vs CsA þ L50.
caspase-3 (169% 13% vs 103% 2%, P < .01) compared with the VH group. Concomitant administration of Lcarnitine at a dose of 200 mg/kg inhibited the expression of LC3-II (Fig 5A and Fig 5B, 133% 10% vs CsA, P < .05 and caspase-3 (Fig 5A and Fig 5C, 124% 12% vs CsA, P < .05) expression. DISCUSSION
The results of the present study demonstrate clearly that L-carnitine offers a protective effect against CsA-induced
XIANG, PIAO, ZOU ET AL
Fig 5. Western blotting of light chain 3 phospholipid conjugated form (LC3-Il) and activated caspase-3. The increased expression of LC3-II and caspase-3 observed in the cyclosporine (CsA) group was significantly suppressed after concomitant treatment with L-carnitine (L). #P < .05 vs vehicle group (VH); ##P <.05 vs CsA.
pancreatic and renal injuries in a rat model of chronic CsA nephropathy. The renoprotective effect of L-carnitine against CsA-induced renal injury was illustrated by its inhibition of tubulointerstitial inflammation and fibrosis, apoptosis, and autophagy. In the pancreas, L-carnitine treatment decreased CsA-induced diabetic control values. Our findings may provide a potential rationale for the clinical use of L-carnitine to reduce CsA-induced nephropathy and diabetes. The main pathogenesis of CsA-induced diabetes is caused by the direct toxicity of CsA on pancreatic b cells, which leads to a decrease in insulin secretion and synthesis23; in contrast, insulin resistance plays a minor role in this phenomenon.24 We demonstrated previously that CsA treatment induces a dramatic reduction in pancreatic islet size, insulin immunoreactivity, and vacuolization of islet cells22 As a result, the present study measured blood diabetic controls. The results of this study revealed that CsA
L-CARNITINE PROTECTS AGAINST CSA-INDUCED INJURY
treatment led to a significant elevation in blood glucose, which was accompanied by a decrease in plasma insulin and increase in hemoglobulin A1c (HbA1c). The concomitant administration of L-carnitine reversed all of the biomarkers. These findings suggest that L-carnitine may counteract CsAinduced toxicity in the pancreas. Our results are consistent with those reported by studies performed using human25 and animal20 samples, which revealed that L-carnitine reverses blood glucose levels, insulin resistance, and HbA1c levels. The mechanism via which L-carnitine improved pancreatic function in this model is unknown; however, 2 possibilities should be considered. First, chronic CsA treatment induces oxidative stress injury, which ultimately leads to the apoptosis of pancreatic b cells induced by low-density lipoprotein.26 L-carnitine ameliorates pancreatic myeloperoxidase and glutathione-S-transferase enzymatic activities in caerulin-induced acute pancreatitis via an antioxidant effect.21 Second, mitochondrial dysfunction plays a central role in ATP synthesis and content in b cells, which regulates glucose-stimulated insulin secretion. Studies performed in vivo27 and in vitro28 have confirmed that L-carnitine improves the mitochondrial membrane potential and upregulates uncoupling protein-2, thus preventing mitochondrial dysfunction in b cells.29 Based on our data regarding blood glucose levels and previous reports, we propose that the benefits of L-carnitine on pancreatic b cells may be associated with its action on mitochondrial and oxidative stress. The most notable finding of our study was that L-carnitine treatment suppressed inflammation and TIF in chronic CsA nephropathy. As reported previously by our group2,30 and others,31 inflammation and TIF induced by CsA are the typical pathologic features of chronic CsA nephropathy. Moreover, the anti-inflammatory and antifibrotic effects of L-carnitine have been observed in isolated rat kidneys from a rat model of this condition.32 However, the molecular mechanism underlying the renoprotection afforded by Lcarnitine remains unknown. Therefore, this study assessed the effect of L-carnitine on macrophage influx and profibrotic TGF-b1. We found that L-carnitine significantly decreased the number of ED-1-positive cells and TGF-b1 expression in a dose-dependent manner, which was accompanied by a marked attenuation of TIF. Our results suggest that L-carnitine inhibits the development of tubulointerstitial inflammation and fibrosis in chronic CsA nephropathy via a mechanism involving suppressed macrophage influx and TGF-b1 expression. It is well accepted that oxidative stress plays a pivotal role in the pathogenesis of chronic CsA nephropathy. In addition, overwhelming evidence suggests that CsA treatment induces oxidative stress injury via the production of reactive oxygen species and malondialdheyde and downregulation of antioxidant enzymes, such as superoxide dismutase and glutathione peroxidase.33 This concept is supported by studies demonstrating that antioxidant treatment significantly ameliorates chronic CsA-induced renal injury.34 L-carnitine, which is a potential antioxidant agent, has been shown to protect
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against various types of nephrotoxicity in vivo17,35 and in vitro36 via its antioxidant properties. In this study, we found that L-carnitine decreased intrarenal 8-OHdG expression and urinary production. The suppression of oxidative stress occurred in parallel with a significant improvement of renal function, as well as TIF. Our findings are supported by the results of previous studies32,35 and suggest that the renoprotective role of L-carnitine observed in this study may be related, in part, to the inhibition of 8-OHdG. L-carnitine protects against renal16 and myocardial37 ischemia-reperfusion injuries; in addition, its antiapoptotic effect is regarded as a protective mechanism against ischemic injury. More recently, we have shown that renal cell apoptosis and excessive autophagy are closely associated with the development of tubulointerstitial injury in chronic CsA nephropathy.5 In the present study, we chose to compare 2 doses of L-carnitine (50 and 200 mg/kg). Interestingly, the lower dose of L-carnitine did not influence apoptosis (active caspase-3) and autophagy (LC3-II), whereas the higher dose of L-carnitine improved not only pathologic damage, but also apoptosis and autophagy. These observations suggest that, although a low dose of L-carnitine (50 mk/kg) failed to overcome the hemodynamic changes caused by CsA, this compound was effective in preventing the development of chronic CsA-induced nephropathy. In summary, CsA-induced diabetes and renal injury are common clinical dilemma in the transplantation field. Our results provide a rationale for the use of the nonnephrotoxic agent L-carnitine in the management of diabetes in transplant recipients taking CsA. REFERENCES 1. Shihab FS, Andoh TF, Tanner AM, et al. Role of transforming growth factor-beta 1 in experimental chronic cyclosporine nephropathy. Kidney Int. 1996;49:1141e1151. 2. Li C, Yang CW, Park JH, et al. Pravastatin treatment attenuates interstitial inflammation and fibrosis in a rat model of chronic cyclosporine-induced nephropathy. Am J Physiol Renal Physiol. 2004;286:F46eF57. 3. Sun BK, Li C, Lim SW, et al. Expression of transforming growth factor-beta-inducible gene-h3 in normal and cyclosporinetreated rat kidney. J Lab Clin Med. 2004;143:175e183. 4. Li C, Lim SW, Sun BK, et al. Expression of apoptosis-related factors in chronic cyclosporine nephrotoxicity after cyclosporine withdrawal. Acta Pharmacol Sin. 2004;25:401e411. 5. Lim SW, Hyoung BJ, Piao SG, et al. Chronic cyclosporine nephropathy is characterized by excessive autophagosome formation and decreased autophagic clearance. Transplantation. 2012;94: 218e225. 6. First MR, Gerber DA, Hariharan S, et al. Posttransplant diabetes mellitus in kidney allograft recipients: incidence, risk factors, and management. Transplantation. 2002;73:379e386. 7. Razeghi E, Heydarian P, Amerian M, et al. The risk factors for diabetes mellitus after kidney transplantation. Saudi J Kidney Dis Transpl. 2010;21:1038e1043. 8. Hjelmesaeth J, Hartmann A, Kofstad J, et al. Glucose intolerance after renal transplantation depends upon prednisolone dose and recipient age. Transplantation. 1997;64:979e983. 9. Ishizuka J, Gugliuzza KK, Wassmuth Z, et al. Effects of FK506 and cyclosporine on dynamic insulin secretion from isolated dog pancreatic islets. Transplantation. 1993;56:1486e1490.
3134 10. Drachenberg CB, Klassen DK, Weir MR, et al. Islet cell damage associated with tacrolimus and cyclosporine: morphological features in pancreas allograft biopsies and clinical correlation. Transplantation. 1999;68:396e402. 11. Cosio FG, Pesavento TE, Kim S, et al. Patient survival after renal transplantation: IV. Impact of posttransplant diabetes. Kidney Int. 2002;62:1440e1446. 12. Arduini A. Carnitine and its acyl esters as secondary antioxidants? Am Heart J. 1992;123:1726e1727. 13. Gülcin I. Antioxidant and antiradical activities of L-carnitine. Life Sci. 2006;78:803e811. 14. Fan JP, Kim D, Kawachi H, et al. Ameliorating effects of Lcarnitine on diabetic podocyte injury. J Med Food. 2010;13:1324e1330. 15. Ringseis R, Keller J, Eder K. Role of carnitine in the regulation of glucose homeostasis and insulin sensitivity: evidence from in vivo and in vitro studies with carnitine supplementation and carnitine deficiency. Eur J Nutr. 2012;51:1e18. 16. Liu Y, Yan S, Ji C, et al. Metabolomic changes and protective effect of (L)-carnitine in rat kidney ischemia/reperfusion injury. Kidney Blood Press Res. 2012;35:373e381. 17. Tufekci O, Gunes D, Ozogul C, et al. Evaluation of the effect of acetyl L-carnitine on experimental cisplatin nephrotoxicity. Chemotherapy. 2009;55:451e459. 18. Boonsanit D, Kanchanapangka S, Buranakarl C. L-carnitine ameliorates doxorubicin-induced nephrotic syndrome in rats. Nephrology (Carlton). 2006;11:313e320. 19. Bertelli A, Giovannini L, Palla R, et al. Protective effect of Lpropionylcarnitine on cyclosporine-induced nephrotoxicity. Drugs Exp Clin Res. 1995;21:221e228. 20. Cresto JC, Fabiano de Bruno LE, Cao GF, et al. The association of acetyl-l-carnitine and nicotinamide remits the experimental diabetes in mice by multiple low-dose streptozotocin. Pancreas. 2006;33:403e411. 21. Arafa HM, Hemeida RA, Hassan MI, et al. Acetyl-L-carnitine ameliorates caerulein-induced acute pancreatitis in rats. Basic Clin Pharmacol Toxicol. 2009;105:30e36. 22. Chung BH, Li C, Sun BK, et al. Rosiglitazone protects against cyclosporine-induced pancreatic and renal injury in rats. Am J Transplant. 2005;5:1856e1867. 23. Basadonna G, Montorsi F, Kakizaki K, et al. Cyclosporin A and islet function. Am J Surg. 1988;156:191e193. 24. Nam JH, Mun JI, Kim SI, et al. beta-Cell dysfunction rather than insulin resistance is the main contributing factor for the development of postrenal transplantation diabetes mellitus. Transplantation. 2001;71:1417e1423. 25. Derosa G, Maffioli P, Salvadeo SA, et al. Effects of combination of sibutramine and L-carnitine compared with sibutramine
XIANG, PIAO, ZOU ET AL monotherapy on inflammatory parameters in diabetic patients. Metabolism. 2011;60:421e429. 26. Jiao J, Dou L, Li M, et al. NADPH oxidase 2 plays a critical role in dysfunction and apoptosis of pancreatic b-cells induced by very low-density lipoprotein. Mol Cell Biochem. 2012;370:103e113. 27. Xia Y, Li Q, Zhong W, et al. L-carnitine ameliorated fatty liver in high-calorie diet/STZ-induced type 2 diabetic mice by improving mitochondrial function. Diabetol Metab Syndr. 2011;3:31. 28. Wan J, Jiang L, Lü Q, et al. Activation of PPARdelta upregulates fatty acid oxidation and energy uncoupling genes of mitochondria and reduces palmitate-induced apoptosis in pancreatic beta-cells. Biochem Biophys Res Commun. 2010;391: 1567e1572. 29. Shen W, Liu K, Tian C, et al. Protective effects of R-alphalipoic acid and acetyl-L-carnitine in MIN6 and isolated rat islet cells chronically exposed to oleic acid. J Cell Biochem. 2008;104: 1232e1243. 30. Li C, Sun BK, Lim SW, et al. Combined effects of losartan and pravastatin on interstitial inflammation and fibrosis in chronic cyclosporine-induced nephropathy. Transplantation. 2005;79:1522e1529. 31. Khanna A. Tacrolimus and cyclosporinein vitro and in vivo induce osteopontin mRNA and protein expression in renal tissues. Nephron Exp Nephrol. 2005;101:ell9eel26. 32. Giovannini L, Palla R, Bertelli AA, et al. Cyclosporine nephrotoxicity evaluated by tissue calcium deposition and tubular enzymes is prevented by L-propionylcarnitine in isolated perfused rat kidney. Transplant Proc. 1996;28:3122e3125. 33. Anjaneyulu M, Tirkey N, Chopra K. Attenuation of cyclosporine-induced renal dysfunction by catechin: possible antioxidant mechanism. Ren Fail. 2003;25:691e707. 34. Zal F, Mostafavi-Pour Z, Vessal M. Comparison of the effects of vitamin E and/or quercetin in attenuating chronic cyclosporine A-induced nephrotoxicity in male rats. Clin Exp Pharmacol Physiol. 2007;34:720e724. 35. Longoni B, Giovannini L, Migliori M, et al. Cyclosporineinduced lipid peroxidation and propionyl carnitine protective effect. Int J Tissue React. 1999;21:7e11. 36. Ye J, Li J, Yu Y, et al. L-carnitine attenuates oxidant injury in HK-2 cells via ROS-mitochondria pathway. Regul Pept. 2010;161: 58e66. 37. Cui J, Das DK, Bertelli A, et al. Effects of L-carnitine and its derivatives on postischemic cardiac function, ventricular fibrillation and necrotic and apoptotic cardiomyocyte death in isolated rat hearts. Mol Cell Biochem. 2003;254:227e234.
D
ESPITE THE DEVELOPMENT of newer immunosuppressants, cyclosporine (CsA) remains a potent immunosuppressive agent that is used for treating multiorgan transplantation and autoimmune diseases. However, the clinical usage of CsA is frequently impeded by its
nephrotoxic effects in terms of acute and chronic nephrotoxicity. Acute CsA nephrotoxicity is regarded as being reversible and seems to be caused by a reduction in renal blood flow related to afferent arteriolar vasoconstriction. However, the long-term administration of CsA causes
From the Nephrology & Dialysis Unit, Department of Internal Medicine, (Y.X., S.G.P., J.J., M.R.F., D.M.L., C.L.), YanBian University Hospital, YanJi, JiLin, PR China; Convergent Research Consortium in Immunologic Disease (S.G.P., C.W.Y.), The Catholic University of Korea, Seoul, Korea; Transplant Research Center (S.G.P., C.W.Y.), Seoul St Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea; Division of Nephrology (H.B.Z.), The First Hospital of JiLin University, ChangChun, JiLin, PR China; and Division of Nephrology (B.H.G.), ZhongShan Hospital of DaLian University, DaLian, LiaoNing, PR China.
This work was supported by the National Natural Science Foundation of China (No. 81160092) and a grant of the Korea Healthcare technology R&D Project, Ministry for Health, Welfare & Family Affairs of Korea (A092258). Address reprint requests to Can Li, MD, PhD, Nephrology & Dialysis Unit, Department of Internal Medicine, YanBian University Hospital, YanJi, JiLin, PR China, or Chul Woo Yang, MD, PhD, Transplant Research Center, Seoul, St Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul Korea. E-mail: [email protected] or [email protected]
Crown Copyright ª 2013 Published by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 45, 3127e3134 (2013)
0041-1345/13/$esee front matter http://dx.doi.org/10.1016/j.transproceed.2013.08.041 3127
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progressive renal insufficiency with striped tubulointerstitial fibrosis (TIF), tubular atrophy, inflammatory cell influx, and hyalinosis of the afferent arterioles.1,2 The mechanism underlying chronic CsA nephropathy is multifactorial, and several factors, including the intrarenal reninangiotensin system, inflammatory mediators, the transforming growth factor-beta 1 (TGF-b1), oxidative stress, apoptotic cell death, and excessive autophagy, have been implicated in this condition.2e5 Posttransplant diabetes mellitus (PTDM) is a common and serious complication after kidney transplantation that is associated with decreased graft and patient survival.6 A recent meta-analysis of observational studies and randomized controlled trials reported that the incidence of PTDM in the first year after transplantation varied from 2% to 50%. The incidence of PTDM is higher in the first 6 months after transplantation.7 Although its etiopathogenesis is multifactorial, PTDM is thought to be related to the increased insulin resistance that results from glucocorticoids8 and to the decreased insulin secretion caused by calcineurin inhibitors.9,10 PTDM results in adverse outcomes, such as decreased graft survival, increased cardiovascular mortality, and increased risk of postoperative infections.11 L-carnitine (b-hydroxy-g-N-trimethyl ammonium-butyrate) is a component of lipid metabolism that is important for the production of adenosine triphospate (ATP) through the boxidation of long-chain fatty acids. In this manner, L-carnitine functions as an indirect antioxidant and facilitates the repair of oxidized membranes or lipid bilayers.12 In addition, Lcarnitine activity has also been reported to be a direct scavenger of O2 and H2O2.13 Thus, L-carnitine may possess antioxidant properties beyond its action on lipid metabolism. It is well known that L-carnitine is necessary for the efflux of acyl groups from mitochondrial. Therefore, L-carnitine supplementation is known to play a beneficial role in insulin resistance or diabetic podocyte injury,14,15 as intracellular accumulation of acyl-CoA derivatives has been implicated in the development of insulin resistance. Recently, the renoprotective effects of L-carnitine have also been reported in ischemia-reperfusion injury,16 cisplatininduced nephrotoxicit,17 doxorubicin-induced nephritic syndrome,18 and chronic CsA nephropathy.19 Nevertheless, the molecular mechanism underlying the benefits of Lcarnitine on CsA-induced pancreatic and renal injuries remains unexplored. In view of the findings described above, the present study was undertaken to evaluate the protective effect of Lcarnitine on CsA-induced pancreatic and renal injuries using a well-described rat model of chronic CsA nephropathy.
XIANG, PIAO, ZOU ET AL 180 to 200 g, were housed in cages (Nalge Co, Rochester, NY, USA) in a controlled-temperature and controlled-light environment and allowed free access to a low salt diet (0.05% sodium, Teklad Premier, Madison, Wis, USA) throughout the experimental period. A total of 45 rats were randomized into 6 subgroups and treated daily for 4 weeks as follows: (1) vehicle group (VH, n ¼ 7): rats received olive oil (1 mL/kg/d subcutaneous) (2) VH þ L-carnitine group (VH þ L50, n ¼ 7): rats received olive oil and L-carnitine (50 mg/kg intravenously); (3) VH þ L-carnitine group (VH þ 200, n ¼ 7): rats received olive oil and L-carnitine (200 mg/kg intravenously); (4) CsA group (C, n ¼ 8): rats received CsA (15 mg/kg/ d subcutaneously); (5) CsA þ L-carnitine group (C þ L50, n ¼ 8): rats received CsA and L-carnitine (50 mg/kg intravenously); (6) CsA þ L-carnitine group (C þ L200, n ¼ 8): rats received CsA and L-carnitine (200 mg/kg intravenously). The dosage and method of CsA2 and L-carnitine administration20,21 were chosen based on previous reports. Animals were euthanized under ketamine anesthesia, and blood and the kidney samples were collected for biochemical and morphologic or molecular examinations.
Basic Parameters Rats were pair-fed and daily body weight (BW) was monitored. Before sacrifice, rats were individually housed in metabolic cages (Tecniplast Gazzada S. a r.l., Italy) for 24 hour-urine collection, and blood samples were withdrawn to evaluate serum creatinine (SCr), blood urea nitrogen (BUN), blood glucose levels, and plasma insulin level (Coulter-STKS; Coulter Electronics). An intraperitoneal glucose tolerance test (IPGTT) was performed at the end of the 4-week treatment period as previously described,22 and the area under the curve of glucose (AUCg) was calculated by trapezoidal estimation from the values obtained in the IPGTT. Whole blood CsA level was measured by a monoclonal radioimmunoassay (Incstar Co, Stillwater, Minn, USA).
Histopathology Harvested rat kidney tissues were fixed in periodate-lysineparaformaldehyde solution and embedded in wax. After dewaxing, 4 mm sections were processed and stained with Masson trichrome and hematoxylin. A finding of TIF was defined as a matrix-rich expansion of the interstitium with tubular dilatation, tubular atrophy, tubular cast formation, sloughing of tubular epithelial cells, or thickening of the tubular basement membrane. A minimum of 20 fields per section was assessed using a color image analyzer (TDI Scope Eye Version 3.0 for Windows, Olympus, Japan). Briefly, the images were captured, and the extent of TIF was quantified using the Polygon program by counting the percentage of areas injured per field of cortex under 100 magnification. Histopathologic analysis was performed in randomly selected cortical fields of sections by a pathologist blinded to the identity of the treatment groups.
Enzyme-Linked Immunosorbent assay MATERIALS AND METHODS Experimental design The experimental protocol was approved by the Animal Care Committee of the Catholic University of Korea. Male SpragueDawley rats (Charles River Technology, Korea), initially weighing
Twenty-four-hour urinary concentrations of the DNA adduct 8hydroxy-20 -deoxyguanosine (8-OHdG) were determined using a competitive enzyme-linked immunesorbent assay (Japan Institute for the Control of Aging, Shizuoka, Japan), according to the manufacturer’s protocol. All samples were assayed in triplicate.
L-CARNITINE PROTECTS AGAINST CSA-INDUCED INJURY
3129 detected using enhanced chemiluminescence (Amersham Biosciences, UK). Optical densities were obtained using the VH group as 100% reference and normalized with b-actin.
Immunohistochemistry After dewaxing, sections were incubated with a 0.5% Triton X100phosphate-buffered saline (PBS) solution for 30 minutes and washed with PBS three times. Nonspecific binding sites were blocked for 1 hour with normal donkey serum diluted 1:10 in PBS, and then incubated overnight at 4 C with anti-8-OHdG monoclonal antibody (JaICA; Shizuoka, Japan) diluted in 1:1000 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 Immuno Research Laboratories, West Grove, Pa, USA) for 30 minutes. 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 TBS, counterstained with hematoxylin, and examined under light microscopy. The procedure of immunostaining for TGFb1 (1:500, Santa Cruz Biotechnology, Inc) and ED-1 (1:500, Serotec Inc, UK) was similar to that for 8-OHdG. Semiquantitative analyses of TGF-b1 and ED-1 expression was performed using a color image autoanalyzer (TDI Scope Eye Version 3.5 for Windows; Olympus, Japan) by counting the percentage of stained areas per field of cortex at 100 magnification. A minimum of 20 fields per section were counted and averaged.
Statistical Analysis Data are expressed as mean standard error of the mean unless otherwise specified. Multiple comparisons among groups were performed by one-way analysis of variance with the post hoc Bonferroni test (SPSS software version 19.0, Microsoft Corp, Chicago, Ill, USA). Statistical significance was assumed as P < .05.
RESULTS Effect of L-Carnitine on Basic Parameters in Chronic CsA Nephropathy
CsA-treated rats exhibited weight loss (weight gain: 75.6 2.6 vs 89.9 2.4, P < .01) and decreased renal function (SCr, 0.55 0.02 mg/dL vs 0.42 0.03 mg/dL, P < .01; BUN, 29.4 0.94 mg/dL vs 11.3 0.48 mg/dL, P < .01) compared with VH-treated rats. However, BW was increased (weight gain: 85.3 3.0 vs CsA, P < .05) and renal function was improved after L-carnitine coadministration compared with the CsA group (SCr, 0.48 0.02 mg/ dL vs CsA, P < .05; BUN, 27.4 1.02 mg/dL vs CsA, P < .05). Whole blood CsA concentration was unaffected by the combination of L-carnitine and CsA, as shown in Table 1.
Western Botting Kidney cortex tissue was homogenized in lysis buffer (20 mmol/L Tris-Cl [pH 7.6], 150 mmol/L NaCl, 1% [wt/vol] sodium deoxycholate, 1% [vol/vol] Triton X-100, 0.1% sodium dodecyl sulfate, 2 mmol/L NaVO3, and freshly added 1% [vol/vol] aprotinin, leupeptin [l mg/mL], pepstatin [l mg/mL], and 1 mmol/L phenylmethyl sulfonyl-fluoride. Homogenates were centrifuged at 3000 rpm for 15 minutes at 4 C, and the protein concentration of the lysate was determined using a protein microassay of the Bradford method (Bio-RAD, Hercules, California, USA). Protein samples were resolved on 15% SDS-polyacrylamide gel electrophoresis and then electroblotted onto Bio-Blot nitrocellulose membrane (Bio-RAD). An equal amount of protein loading (80 mg) was verified by Ponceau S staining. The membrane was blocked for 1 hour in TBS added Tween-20 (TBS-T, 10 mmol/Tri-CL, 150 mmol/L NaCl [pH 8.0], 0.05% Tween-20) containing 5% nonfat powdered milk. Anticaspase-3, active form (1:200; Millipore Corporation, Calif, USA) or microtubule-associated protein 1 light chain 3 phospholipidconjugated form (LC3- II, 1:1000; Sigma-Aldrich, USA) was detected by incubating for 1 hour with specific primary antibodies. Primary antibody incubation was followed by 6 washes of TBS-T. The blot was then incubated with secondary antibody (Donkey anti-rat IgG, horseradish peroxidase) conjugate at 1:2000 (Amersham Biosciences, UK) for 1 hour. Antibody-reactive protein was
Effect of L-Carnitine on CsA-Induced Diabetes
Baseline blood glucose concentration did not differ between the 6 groups (Fig 1A). IPGTT was significantly increased in the CsA group compared with the VH group, whereas the CsA þ L group showed a significant decrease in IPGTT compared with the CsA group (Fig 1A). The calculated AUCg did not differ significantly between the VH and VH þ L groups and was lower in the CsA þ L group than it was in the CsA group (L50: 476.2 14.6 mg/dL minutes vs 44 11.2 mg/dL minutes, P < .05; L200: 476.2 14.6 mg/ dL minutes vs 396 9.6 mg/dL minutes, P < .05; Fig 1B). Serum insulin level did not differ significantly between the VH and VH þ L groups. However, serum insulin concentration in the CsA group was significantly lower than that observed in the VH (5.1 0.8 mg/dL minutes vs and 1.1 0.3 mg/dL minutes, P < .05) and VH þ L group (L50: 5.1 1.0 mg/dL minutes vs CsA, P < .05; L200: 4.8 0.4 mg/dL minutes vs CsA, P < .05), and was significantly higher in the
Table 1. Basic Parameters
WG UV SCr BUN CsA con TIF
89.9 11.6 0.42 11.3 0 0
VH
VF þ L50
VH þ L200
CsA
2.4 1.3 0.03 0.48 0 0
88.1 16.0 0.35 10.7 0 0
1.7 1.7 0.03 0.90 0 0
90.1 13.7 0.34 11.0 0 0
3.0 1.9 0.02 0.63 0 0
75.6 18.6 0.55 29.4 2207 34.1
CsA þ L50 a
2.6 2.7a 0.02a 0.94a 205 3.2a
85.4 13.4 0.48 27.4 2345 30.2
b
3.5 2.0 0.02b 1.02b 161 3.6b
CsA þ L200
85.3 17.3 0.41 23.8 2409 28.7
3.0b 4.0 0.03c 1.32b 225 2.5c
WG, weight gain (g); UV, urine volume (mL/d); SCr, serum creatinine (mg/dL); BUN, blood urea nitrogen (mg/dL); CsA con, CsA concentration (ng/mL); TIF, tubulointerstitial fibrosis (%0.5/mm2) CsA, Cyclosporine; VH, vehicle group; L, L-carnitine. a P < .05 vs VH. b P < .05 vs CsA. c P < .05 vs CsA þ L50.
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XIANG, PIAO, ZOU ET AL
Fig 1. Effect of L-carnitine (L) on diabetic biomarkers. (A) Blood glucose levels (intraperitoneal glucose tolerance test [IPGTT]). (B) Calculation of area under the curve of glucose (AUCg). (C) Plasma insulin levels. Note that cyclosporine (CsA) induced an elevation in blood glucose and HbA1c and a decrease in plasma insulin levels, whereas coadministration of L reversed all of these parameters. # P < .01 vs vehicle group of (VH); ##P < .05 vs CsA.
CsA þ L group compared with the CsA group (L50: 2.7 0.5 mg/dL minutes vs CsA, P < .05; L200: 2.8 0.2 mg/dL minutes vs CsA, respectively; Fig 1C).
a further decrease was observed in the CsA þ L200 group (23.8 2.5%/0.5 mm2 vs CsA þ L50, P < .05).
Effect of L-Carnitine on Macrophage Infiltration and Fibrosis in Chronic CsA Nephropathy
Immunohistochemistry revealed that the majority of VHtreated rat kidneys were negative for TGF-b1. In contrast, the expression of the TGF-b1 protein was dramatically increased in the tubulointerstitium in CsA-treated rat kidneys (46.0% 2.3%/0.5 mm2 vs 17.1% 0.5%/0.5 mm2, P < .01). It is notable that the sites of strong TGF-b1 protein expression were located in areas of inflammatory cell infiltration and severe TIF (Fig 3A). L-carnitine suppressed TGF-b1 protein expression in a dose-dependent manner (CsA þ L50: 40.0% 1.4%/0.5 mm2, P < .05 vs CsA; CsA þ L200: 30.2% 3.0%/0.5 mm2, P < .05 vs CsA þ L50, respectively; Fig. 3B).
ED-1-positive cells were detected only rarely in the kidneys of VH-treated animals. CsA treatment increased the number of ED-1-positive cells (29 1.0/0.5 mm2 vs 5 0.7/ 0.5 mm2, P < .01). Concomitant administration of L-carnitine significantly decreased the number of ED-1-positive cells (23 1.2/0.5 mm2 vs CsA, P < .01), which was more pronounced after the administration of 200 mg/kg L-carnitine compared with the CsA þ L50 group (Fig 2, 19 2.0/ 0.5 mm2 vs CsA þ L50, P < .05). CsA-treated rats exhibited the typical striped TIF of chronic CsA nephropathy (Table 1). A significant increase in the percentage of TIF was found in the CsA group compared with the VH group (34.1% 3.2%/0.5 mm2 vs 0% 0%/0.5 mm2, P < .01). In contrast, the percentage of TIF decreased markedly when L-carnitine was coadministered (28.7% 3.6%/0.5 mm2 vs CsA, P < .05) and
Effect of L-Carnitine on TGF-b Expression in Chronic CsA Nephropathy
Effect of L-Carnitine on Oxidative Stress in Chronic CsA Nephropathy
Chronic CsA nephropathy is closely associated with oxidative stress. As shown in Fig 4, immunohistochemistry showed a significant increase in 8-OHdG expression and
L-CARNITINE PROTECTS AGAINST CSA-INDUCED INJURY
Fig 2. Representative photomicrographs of immunohistochemistry (A) and semiquantitative analysis (B) of macrophage infiltration. Cyclosporine (CsA) treatment significantly increased the number of ED-1-positive cells, whereas this number was decreased by cotreatment with L-carnitine (L). Immunohistochemical staining: original magnification, 200. #P < .01 vs vehicle group (VH); ##P < .05 vs CsA; *P < .05 vs CsA þ L50.
immunoreactivity in CsA-treated rat kidneys; these signals were confined to cortical tubular epithelial cells. Interestingly, the sites of strong 8-OHdG expression were located in areas of injured or atrophied tubules (Fig 4A). Concomitant administration of L-carnitine significantly decreased intrarenal 8-OHdG expression compared with the CsA treatment alone. Moreover, this decrease was more pronounced in the presence of a higher dose of L-carnitine compared with the CsA þ L50 group. In parallel, urinary 8-OHdG excretion in the CsA-treated group was higher than that observed in the VH-treated group (241.0 37.2 ng/d vs 130.7 17.0 ng/d, P < .01), whereas its level was
3131
Fig 3. Representative photomicrographs of immunohistochemistry (A) and semiquantitative analysis (B) of transforming growth factor (TGF)-b1 expression. Cyclosporine (CsA) significantly increased intrarenal TGF-b1 immunoreactivity, whereas this was significantly decreased by cotreatment with L-carnitine (L). Immunohistochemical staining: original magnification, 200. # P < 05 vs vehicle group (VH); #P < .05 vs CsA; *P < .05 vs CsA þ L50.
significantly decreased after concomitant administration of L-carnitine, in a dose-dependent manner (Fig 4B, CsA þ L50: 208.3 19.7 ng/d vs CsA, P < .05; CsA þ L200: 177.2 12.7 ng/d vs CsA þ L50, P < .05, respectively). Effect of L-Carnitine on LC3-II and Caspase-3 in Chronic CsA Nephropathy
The presence of autophagy was measured based on LC3-II, whereas apoptosis was assessed based on the detection of the apoptosis-related caspase-3 protein, using Western blotting. The results showed that CsA treatment upregulated LC3-II (183% 11% vs 102% 6%, P < .01) and
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Fig 4. Representative photomicrographs of immunohistochemistry (A) and enzyme-linked immunosorbent assay analysis of the urinary output (B) of 8 hydroxy 20 - deoxygulosine (8-OHdG) CsA treatment significantly increased intrarenal 8-OHdG immunoreactivity and urinary 8-OHdG production, whereas these phenomena were significantly decreased by cotreatment with L-carnitine (L). Immunohistochemical staining: original magnification, 200. # P < .05 vs vehicle group (VH); ##P < .05 vs CsA; *P < .05 vs CsA þ L50.
caspase-3 (169% 13% vs 103% 2%, P < .01) compared with the VH group. Concomitant administration of Lcarnitine at a dose of 200 mg/kg inhibited the expression of LC3-II (Fig 5A and Fig 5B, 133% 10% vs CsA, P < .05 and caspase-3 (Fig 5A and Fig 5C, 124% 12% vs CsA, P < .05) expression. DISCUSSION
The results of the present study demonstrate clearly that L-carnitine offers a protective effect against CsA-induced
XIANG, PIAO, ZOU ET AL
Fig 5. Western blotting of light chain 3 phospholipid conjugated form (LC3-Il) and activated caspase-3. The increased expression of LC3-II and caspase-3 observed in the cyclosporine (CsA) group was significantly suppressed after concomitant treatment with L-carnitine (L). #P < .05 vs vehicle group (VH); ##P <.05 vs CsA.
pancreatic and renal injuries in a rat model of chronic CsA nephropathy. The renoprotective effect of L-carnitine against CsA-induced renal injury was illustrated by its inhibition of tubulointerstitial inflammation and fibrosis, apoptosis, and autophagy. In the pancreas, L-carnitine treatment decreased CsA-induced diabetic control values. Our findings may provide a potential rationale for the clinical use of L-carnitine to reduce CsA-induced nephropathy and diabetes. The main pathogenesis of CsA-induced diabetes is caused by the direct toxicity of CsA on pancreatic b cells, which leads to a decrease in insulin secretion and synthesis23; in contrast, insulin resistance plays a minor role in this phenomenon.24 We demonstrated previously that CsA treatment induces a dramatic reduction in pancreatic islet size, insulin immunoreactivity, and vacuolization of islet cells22 As a result, the present study measured blood diabetic controls. The results of this study revealed that CsA
L-CARNITINE PROTECTS AGAINST CSA-INDUCED INJURY
treatment led to a significant elevation in blood glucose, which was accompanied by a decrease in plasma insulin and increase in hemoglobulin A1c (HbA1c). The concomitant administration of L-carnitine reversed all of the biomarkers. These findings suggest that L-carnitine may counteract CsAinduced toxicity in the pancreas. Our results are consistent with those reported by studies performed using human25 and animal20 samples, which revealed that L-carnitine reverses blood glucose levels, insulin resistance, and HbA1c levels. The mechanism via which L-carnitine improved pancreatic function in this model is unknown; however, 2 possibilities should be considered. First, chronic CsA treatment induces oxidative stress injury, which ultimately leads to the apoptosis of pancreatic b cells induced by low-density lipoprotein.26 L-carnitine ameliorates pancreatic myeloperoxidase and glutathione-S-transferase enzymatic activities in caerulin-induced acute pancreatitis via an antioxidant effect.21 Second, mitochondrial dysfunction plays a central role in ATP synthesis and content in b cells, which regulates glucose-stimulated insulin secretion. Studies performed in vivo27 and in vitro28 have confirmed that L-carnitine improves the mitochondrial membrane potential and upregulates uncoupling protein-2, thus preventing mitochondrial dysfunction in b cells.29 Based on our data regarding blood glucose levels and previous reports, we propose that the benefits of L-carnitine on pancreatic b cells may be associated with its action on mitochondrial and oxidative stress. The most notable finding of our study was that L-carnitine treatment suppressed inflammation and TIF in chronic CsA nephropathy. As reported previously by our group2,30 and others,31 inflammation and TIF induced by CsA are the typical pathologic features of chronic CsA nephropathy. Moreover, the anti-inflammatory and antifibrotic effects of L-carnitine have been observed in isolated rat kidneys from a rat model of this condition.32 However, the molecular mechanism underlying the renoprotection afforded by Lcarnitine remains unknown. Therefore, this study assessed the effect of L-carnitine on macrophage influx and profibrotic TGF-b1. We found that L-carnitine significantly decreased the number of ED-1-positive cells and TGF-b1 expression in a dose-dependent manner, which was accompanied by a marked attenuation of TIF. Our results suggest that L-carnitine inhibits the development of tubulointerstitial inflammation and fibrosis in chronic CsA nephropathy via a mechanism involving suppressed macrophage influx and TGF-b1 expression. It is well accepted that oxidative stress plays a pivotal role in the pathogenesis of chronic CsA nephropathy. In addition, overwhelming evidence suggests that CsA treatment induces oxidative stress injury via the production of reactive oxygen species and malondialdheyde and downregulation of antioxidant enzymes, such as superoxide dismutase and glutathione peroxidase.33 This concept is supported by studies demonstrating that antioxidant treatment significantly ameliorates chronic CsA-induced renal injury.34 L-carnitine, which is a potential antioxidant agent, has been shown to protect
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against various types of nephrotoxicity in vivo17,35 and in vitro36 via its antioxidant properties. In this study, we found that L-carnitine decreased intrarenal 8-OHdG expression and urinary production. The suppression of oxidative stress occurred in parallel with a significant improvement of renal function, as well as TIF. Our findings are supported by the results of previous studies32,35 and suggest that the renoprotective role of L-carnitine observed in this study may be related, in part, to the inhibition of 8-OHdG. L-carnitine protects against renal16 and myocardial37 ischemia-reperfusion injuries; in addition, its antiapoptotic effect is regarded as a protective mechanism against ischemic injury. More recently, we have shown that renal cell apoptosis and excessive autophagy are closely associated with the development of tubulointerstitial injury in chronic CsA nephropathy.5 In the present study, we chose to compare 2 doses of L-carnitine (50 and 200 mg/kg). Interestingly, the lower dose of L-carnitine did not influence apoptosis (active caspase-3) and autophagy (LC3-II), whereas the higher dose of L-carnitine improved not only pathologic damage, but also apoptosis and autophagy. These observations suggest that, although a low dose of L-carnitine (50 mk/kg) failed to overcome the hemodynamic changes caused by CsA, this compound was effective in preventing the development of chronic CsA-induced nephropathy. In summary, CsA-induced diabetes and renal injury are common clinical dilemma in the transplantation field. Our results provide a rationale for the use of the nonnephrotoxic agent L-carnitine in the management of diabetes in transplant recipients taking CsA. REFERENCES 1. Shihab FS, Andoh TF, Tanner AM, et al. Role of transforming growth factor-beta 1 in experimental chronic cyclosporine nephropathy. Kidney Int. 1996;49:1141e1151. 2. Li C, Yang CW, Park JH, et al. Pravastatin treatment attenuates interstitial inflammation and fibrosis in a rat model of chronic cyclosporine-induced nephropathy. Am J Physiol Renal Physiol. 2004;286:F46eF57. 3. Sun BK, Li C, Lim SW, et al. Expression of transforming growth factor-beta-inducible gene-h3 in normal and cyclosporinetreated rat kidney. J Lab Clin Med. 2004;143:175e183. 4. Li C, Lim SW, Sun BK, et al. Expression of apoptosis-related factors in chronic cyclosporine nephrotoxicity after cyclosporine withdrawal. Acta Pharmacol Sin. 2004;25:401e411. 5. Lim SW, Hyoung BJ, Piao SG, et al. Chronic cyclosporine nephropathy is characterized by excessive autophagosome formation and decreased autophagic clearance. Transplantation. 2012;94: 218e225. 6. First MR, Gerber DA, Hariharan S, et al. Posttransplant diabetes mellitus in kidney allograft recipients: incidence, risk factors, and management. Transplantation. 2002;73:379e386. 7. Razeghi E, Heydarian P, Amerian M, et al. The risk factors for diabetes mellitus after kidney transplantation. Saudi J Kidney Dis Transpl. 2010;21:1038e1043. 8. Hjelmesaeth J, Hartmann A, Kofstad J, et al. Glucose intolerance after renal transplantation depends upon prednisolone dose and recipient age. Transplantation. 1997;64:979e983. 9. Ishizuka J, Gugliuzza KK, Wassmuth Z, et al. Effects of FK506 and cyclosporine on dynamic insulin secretion from isolated dog pancreatic islets. Transplantation. 1993;56:1486e1490.
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