- Email: [email protected]
Simvastatin Down Regulates mRNA Expression of RANTES and CCR5 in Posttransplant Renal Recipients With Hyperlipidemia
Simvastatin Down Regulates mRNA Expression of RANTES and CCR5 in Posttransplant Renal Recipients With Hyperlipidemia Q. Li, D. Wang, Y. Wang, and Q. Xu ABSTRACT Chemokines and hyperlipidemia are involved in the mechanism of chronic allograft nephropathy (CAN). In this study, the mRNA expression of RANTES and its receptor CCR5 on peripheral blood mononuclear cells were measured in renal transplant recipients with hyperlipidemia, and the effect of simvastatin treatment observed to investigate the mechanism and prevention of CAN. Sixty recipients selected from 167 renal transplant recipients were divided into two groups: group A without hyperlipidemia (n ⫽ 30) and group B with hyperlipidemia (n ⫽ 30). The control group consisted of 30 healthy volunteers. The recipients in group B were treated with simvastatin for 3 months. We estimated serum lipid levels and mRNA expressions of RANTES and CCR5. The mRNA expressions of RANTES and CCR5 were significantly higher in renal transplant recipients compared with controls. The expressions were much higher in group B than in group A patients. In group B patients, serum lipid levels decreased dramatically after simvastatin treatment. Meanwhile, the mRNA expressions of RANTES and CCR5 were reduced significantly after 1.5 months of simvastatin treatment to a level significantly lower than that in group A after 3 months of treatment. The increased expressions of RANTES and CCR5 mRNAs in renal transplant recipients with hyperlipidemia might be involved in CAN due to hyperlipidemia. Simvastatin seemed to reduce the chemokine transcripts in renal recipients with hyperlipidemia.
I
N LONG-TERM SURVIVORS of renal transplantation, chronic allograft nephropathy (CAN) and atherosclerosis account for the majority of patient deaths or renal allograft dysfunction.1– 4 Regulated upon activation normal T cell expressed and secreted (RANTES) and its receptor, C-C chemokine receptor 5 (CCR5), are synthesized by arterial smooth muscle cells and endothelial cells exposed to inflammatory stimuli, thus providing a chemotactic stimulus for the recruitment of monocytes into the arterial wall, one of the earliest cellular processes in the pathology of atherosclerosis and CAN.5–11 The expression of RANTES and CCR5 could also be detected in mononuclear cells infiltrating kidney grafts, which suggested that RANTES and CCR5 play important roles in progressive atherosclerosis and CAN in transplant recipients.12–17 In our former study and other studies, hyperlipidemia was documented in more than half of renal transplant recipients. This finding has been considered to be one of the main contributors to CAN and cardiovascular disease.3,4,18 Simvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl CoA
(HMG CoA) reductase is often used to reduce serum lipid levels. Recently studies have shown that HMG CoA inhibitors have many other effects in addition to regulating serum lipid levels.19 –22 HMG CoA inhibitors can inhibit NF-B activation and MCP-1, transforming growth factorbetal mRNA expression, and also ameliorate albuminuria and glomerulosclerosis in five of six nephrectomized rats, in obese rats, and in streptozotocin-induced diabetic uninephrectomized rats.23,24 To date, there have been no reports about its effects on RANTES in renal transplant recipients.
From Department of Nephrology (Q.L., Y.W.), The Second Hospital of Xiamen, Nephrology, Xiamen, China; Department of Urology (D.W.), The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China; and Department of Nephrology (Q.X.), The First People’s Hospital of Shanghai, Shanghai, China. Dr Qingqun Li and Dr Daohu Wang contributed equally to the work and should be considered co-first authors. Address reprint requests to Yuxin Wang, The Department of Nephrology, The Second Hospital of Xiamen, Nephrology, 361021 Xiamen, China. E-mail: [email protected]
© 2006 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/06/$–see front matter doi:10.1016/j.transproceed.2006.08.136
Transplantation Proceedings, 38, 2899 –2904 (2006)
2899
2900
LI, WANG, WANG ET AL ⬍170 mol/L, urine protein was negative, and urine volume was normal in every patient.
In this study, we investigated the mRNA expressions of RANTES and CCR5 in peripheral blood mononuclear cells (PBMCs) among renal, transplant patients with hyperlipidemia and the influence of simvastatin treatment on their expression.
Experimental Design According to the serum lipid levels at 6 months after kidney transplantation, the recipients were divided into two groups; group A without hyperlipidemia, included 16 males and 14 females of mean age 41 ⫾ 9 years; group B with hyperlipidemia total serum cholesterol ⬎ 6.2 mmol/L, serum triglyceride ⬎ 2.3 mmol/L included 17 males and 13 females (mean age 40 ⫾ 10 years). The profile of serum lipids in the renal transplant recipients and controls is shown in Fig 1. Group B patients were treated with simvastatin (Merck, USA; 20 mg/day) to decrease serum lipid levels. To detect serum lipid concentration and the mRNA expression in peripheral mononuclear cells, overnight fasting blood was collected from all patients before and at 6 to 8 months after kidney transplantation and in group B also at 1.5 and 3 months of simvastatin treatment. Thirty healthy volunteers were employed as controls, including 16 males and 14 females of overall mean age of 41 ⫾ 9 years. Informed consent was obtained from each participant.
MATERIALS AND METHODS This study was approved by our institutional ethical committee.
Patients Sixty patients (33 males and 27 females) of mean age 40 ⫾ 9 years were selected from 107 cadaver kidney transplantation recipients. Their primary end-stage renal disease (ESRD) were glomerulonephropathies, and their serum lipid levels were normal before renal transplantation. Every patient was treated with prednisone, mycophenolate mofetil and cyclosporine. Prednisone was dosed at 80 mg/day for the first 2 days, then gradually reduced to 10 mg/day. Cyclosporine was titered according to blood concentrations (100 –200 ng/mL). Serum creatinine level was
A 10
B
9
6
8
4
**
**
5
TG(mmol/L)
TC(mmol/L)
**
6
5
**
7
4 3 2
**
3
**
2
**
**
Group A
Group B
1 1
0 Congtrols
Group A
Group B 0 Congtrols
Before Transplant Before Trans plant
After transplant
C
D
3
5 4. 5 4
**
2 1. 5
3. 5
** **
**
1
LDLC(mmol/L
HDLC(mmol/L)
2. 5
Fig 1. Serum lipid levels before and after renal transplantation versus controls. (A) Serum TC levels in the three groups; (B) Serum TC levels in controls and renal transplant recipients; (C) Serum HDLC levels in controls and renal transplant recipients; (D) Serum LDLC levels of controls and renal transplant recipients. (*P ⬍ .05, **P ⬍ .01 vs group B after renal transplantation).
After transplant
3
**
* **
2. 5
**
2 1. 5 1
0. 5
0. 5
0 Congtrols Before Transplant
Group A
Group B
After transplant
0 Congtrols Before Transplant
Group A
Group B
After transplant
SIMVASTATIN DOWNREGULATES mRNA EXPRESSION
2901
Serum Lipid Serum was collected from peripheral blood samples from each subject after an overnight fast. Serum total cholesterol (TC) and total glycerides (TG) concentrations were measured enzymatically with the Cholesterol-E test and the triglyceride-E test (Wako Pure Chemical Co.). HDL-cholesterol (HDL-C) was measured with the HDL-cholesterol-E test (Wako).
RT-PCR Ten milliliters of blood collected in sodium citrate from the patient was diluted with equal volume of phosphate-buffered saline, overlaid with an equal volume of ficoll-hypaque (Pharmacia), and centrifuged in a table-top centrifuge at 1500g for 30 minutes. The PBMCs band below the plasma was collected and washed three times with phosphate-buffered saline.
RNA Isolation Total RNA was extracted from PBMCs using trizol (Gibco), which is based on the method described by Chomczynski and Sacchi.25 In brief, 10 ⫻ 166 PBMCs from each sample were homogenized in 1 mL trizol before addition of 200 L chloroform. The mixture was
B
10 9
**
**
8
TG(mmol/L)
5 4 3 2
##
3 2
3 months Simvastatin treatment
Before treatment
3 months Simvastatin treatment
1.5 months Simvastatin treatment
Before treatment
D
3
1.5 months Simvastatin treatment
0
0
5 4. 5
2. 5 LDLC(mmol/L)
4
2 1. 5 1
*
3. 5 3 2. 5 2 1. 5 1
0. 5
0. 5
3 months Simvastatin treatment
1.5 months Simvastatin treatment
3 months Simvastatin treatment
0
0 Before treatment
HDLC(mmol/L
**
1
1
C
**
4
1.5 months Simvastatin treatment
TC(mmol/L)
6
Total RNA was transcribed to complementary DNA(cDNA) by reverse transcription (RT) with hexamer random primers (Promega, USA). One microgram of total RNA was added to 0.5 g of primer. A reaction mixture contained 50 ng of random primer, deoxynucleotide triphosphate (0.5 mmol/L), 5⫻buffer 5 L, 0.1 M DTT (2 L), Rnase OUT Rnase Inhib (0.5 L), and 200 units of MMLV reverse transcriptase. All reagents were purchased from Gibco in 20 L of final volume. The reaction was allowed to proceed at 70°C for 5 minutes, 37°C for 60 minutes, and stopped by heating to 95°C for 5 minutes, followed by cooling on ice. The cDNA was stored at ⫺30°C for further procedures.
5
##
7
Reverse Transcription
6
Before treatment
A
centrifuged at 12000g for 15 minutes at 4°C. The supernate was treated with 0.5 mL isopropanol and centrifuged at 12000g for 10 minutes at 4°C. The supernate was discarded and the sediment mixed with 0.75 mL 75% ethanol before centrifugation at 12000g for 10 minutes at 4°C. The precipitate of total RNA was resolved with 20 L DEPC water. The purity of isolated RNA was determined by measuring the ratio of absorbance at 260 and 280 nm (A260/280 ratios), and the concentration estimated by absorbance at 260 nm.
Fig 2. Serum lipid levels before and after renal transplantation among group B patients versus controls. (A) Serum TC levels; (B) Serum TG levels; (C) Serum HDLC levels; (D) Serum LDLC levels. (*P ⬍ .05, **P ⬍ .01 vs controls; #P ⬍ .05, ##P ⬍ .01 vs 1.5 month).
LI, WANG, WANG ET AL
9
7
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Before transplant
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Before transplant
Amplification of Specific Complementary DNA The primer sequences for -actin were: 5=-TGGCACACCTTCTACAATGAGCTGCG-3= (forward) and 5=-CGTCATACTCCTTGCTCCACA TCTGC-3= (reverse), with a 838-bp length of the amplified segments. The primer sequences for RANTES were: 5=-AGCTACTCGGGAGGCTAAGG-3= (forward) and 5=-GAGGCATGCTGACTTCCTTC-3= (reverse), with a 276-bp length of the PCR product. The primer sequences for CCR5 were: 5=-TGTAGGGAGCCCAGAAGAGA-3= (forward) and 5=-CGACATACTCCTTGCTCCCACATCTGC-3= (reverse), with a 168-bp PCR product length. Specific cDNA products corresponding to mRNA for human -actin, RANTES and CCR5 were amplified using PCR. Five microliters of cDNA was taken for PCR, which was performed in PCR buffer, using 0.2 mM/L dNTP, 1 M/L of both primers and 2.0U Taq DNA polymerase. All reagants were purchased from Gibco (USA). GeneAmp2700 Thermal Cycler (USA) was used for amplification with the following sequence profile: one cycle of 5 minutes at 95°C for template denaturation, followed by 35 cycles of 30 seconds of denaturation at 95°C, 45 seconds (at 60°C for RANTES, 57°C for CCR5) for primer annealing, and 60 seconds for polymerase extension at 72°C. All reactions were terminated with a 10-minute extension at 70°C and cooling to
After transplant
4°C. Final products were electrophoresed on 2% agarose gels and analyzed by direct visualization after ethidium bromide staining. Every RT-PCR reaction was repeated at least twice to confirm the results.
Argarose Gel Analysis The amplified PCR products were analyzed on 2.0% agarose gels electrophoresed at 75 V, 25 mA in 40 mmol/L Tris-acetate (pH 8.5), and 2 mmol/L EDTA (TAE buffer) followed by staining with 0.5pg/mL ethidium bromide. Sample products were visualized by UV transillumination for photography of the gel. Specific products were identified by size in relation to a known DNA Marker (Takara) run with each gel. The cDNA of RANTES and CCR5 were semiquantitatively analyzed by densitometric comparison to -actin (internal control) from the same sample after the positive image was digitized by video for computerized densitometry. The results are shown as ratios of intensity of RANTES, CCR5, and -actin.
Statistical Analysis All data are expressed as mean values ⫾ SD. Student’s t test or one-way ANOVA test was used for statistical analysis with the SPSS program.
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Fig 4. The mRNA expression of MCP-1 and CCR2 in group B before and after simvastatin treatment (*P ⬍ .05, **P ⬍ .01 vs before treatment). (A) The mRNA expression of MCP-I in group B before and after simvastatin treatment. (B) The mRNA expression of CCR2 in group B before and after simvastatin treatment (*P ⬍ .05, **P ⬍ .01 vs before treatment).
Group B
Fig 3. The mRNA expression of MCP-1 and CCR2 in controls and the recipients before and after renal transplantation. (A) The mRNA expression of MCP-1 in controls and the recipients before and after renal transplantation (P ⬍ .05 vs group B after renal transplantation). (B) The mRNA expression of CCR2 in controls and the recipients before and after renal transplantation (*P ⬍ .05, **P ⬍ .01 vs comtrols).
3 months Simvastatin treatment
5
5
1.5 months Simvastatin treatment
6
Before Simvastatin treatment
7
CCR5/aBeta-actin
RANTES/Beta-actin
2902
SIMVASTATIN DOWNREGULATES mRNA EXPRESSION
RESULTS
Serum lipid levels were decreased in the renal transplant recipients of group B after treatment with Simvastatin. Simvastatin (20 mg/day) was presented for hyperlipidemic renal transplant recipients with with serum lipid levels determined at 1.5 and 3 months after simvastatin treatment. Serum levels of TC, TG, and LDLC had decreased significantly at 1.5 months compared with before treatment and reduced further at 3 months of simvastatin treatment (Fig 2). The High mRNA Expression of RANTES and CCR5 Were Downregulated by Simvastatin Treatment
The mRNA expressions of RANTES and CCR5 in all recipients were significantly higher compared with those of controls at 6 months after renal transplantation. The mRNA espression of RANTES and CCR5 decreased significantly in all renal transplant recipients, but remained significantly higher than the controls. There was no significant differences in mRNA expression of RANTES on CCR5 between group A and group B before renal transplantation. However, the mRNA expressions of RANTES and CCR5 were significantly higher in group B than group A at 6 months after renal transplantation (Fig 3). For group B, the significantly down-regulation of mRNA expression of RANTES and CCR5 were evident at 1.5 months of simvastatin treatment and reduced to even normal levels in recipients with 3 months of simvastatin treatment (RANTES: 2.3 ⫾ 1.1 vs 2.0 ⫾ 0.8, P ⬎ .05; CCR5: 1.1 ⫾ 0.6 vs 0.8 ⫾ 0.4; P ⬎ .05). These values were significantly lower than those in group A recipients posttransplantation (RANTES: 2.3 ⫾ 1.1 vs 3.3 ⫾ 1.3, P ⬍ .01; CCR5: 1.1 ⫾ 0.6 vs 1.8 ⫾ 0.7, P ⬍ .01; Fig 4). DISCUSSION
Our pervious study suggested that the mRNA expressions of RANTES and CCR5 were up-regulated among posttransplant recipients with hyperlipidemia. RANTES and CCR5 are involved in the pathogenesis of atherosclerosis and CAN by promoting directed migration of inflammatory cells. They play important roles in atherosclerosis and chronic allograft nephropathy. Thus, it was suggested that upregulation of RANTES and CCR5 mRNA that may a main mechanism in cardiovascular disease and CAN caused by hyperlipidemia, and that hyperlipidemia may play an important role in the pathogenesis of atherosclerosis and CAN by inducing the production of RANTES and CCR5. Our results presented here showed that the expression of RANTES and CCR5 were decreased significantly after treatment with simvastatin in renal transplant recipients with hyperlipidemia. After 3 months of treatment the levels were even lower than those among recipients without hyperlipidemia. Simvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl CoA (HMG CoA) reductase, is often used to lower serum lipid levels. Recent studies have shown that it markedly
2903
reduces the incidence of CAN and cardiovascular events besides the action of reducing serum lipid levels. Pretreatment with simvastatin reduces neutrophil adhesion to the venous endothelium in patients undergoing coronary surgery, irrespective of its efficacy at lowering cholesterol concentration.20 –22,24 HMG CoA inhibitors can also reduce activation of RANTES, ameliorating albuminuria and glomerulosclerosis in five of six nephrectomized rats, in obese rats, and in streptozotocin-induced diabetic uninephrectomized rats.22,23 Simvastatin has anti-inflammatory effects through downregulation of cytokines in the endothelium and leukocytes, as well as reducing the expression of the cytokines MCP-1, interleukin-6, and interleukin-8 in circulating monocytes from hypercholesterolemic patients.20 Greenwood et al demonstrated that lovastatin, another HMG CoA inhibitor blocks a key stage in the pathogenesis of neuroinflammation, namely leukocyte migration across the bloodbrain barrier, thus demonstrating a novel effect of statins to modulate the immune response in neuroinflammtory diseases.26 These data support that simvastatin has a role to prevent CAN beyond the plasma cholesterol-lowering activity. Based on the former observations and our results, it may be concluded that simvastatin can be used to reduce the incidence of CAN in renal recipients with hyperlipidemia, even those with normal serum lipid levels. It was suggested by this study that the increased expressions of RANTES and CCR5 mRNA in renal transplant recipients with hyperlipidemia may be involved in CAN due to hyperlipidemia. Thus simvastatin treatment can be used to reduce the incidence of CAN in renal recipients with hyperlipidemia. REFERENCES 1. Nankivell BJ, Borrows RJ, Chir MB, et al: The natural history of chronic allograft nephropathy. N Engl J Med 349:2326, 2003 2. Van der Woude FJ, Hollander AAMJ: Rejection after kidney transplantation: New concepts, new therapeutic options. Transplant Proc 30:2419, 1998 3. Aguirrezabalaga J, Fernandez-Selles C, Otero A, et al: Lipid profiles in liver transplantation in patients receiving tacrolimus or cyclosporin. Transplant Proc 33:1064, 2001 4. Kisielnicka E, Zdrojewski Z, Wroblewska M, et al: Lipid disturbances in a two-year follow-up after successful kidney transplantation. Transplant Proc 32:1358, 2000 5. Moench C, Uhrig A, Lohse AW, et al: Differential diagnosis of cytomegalovirus infection and acute rejection by serum CCchemokine measurement after orthotopic liver transplantation. Transplant Proc 35:2084, 2003 6. Almenar Bonet L, Martinez-Dolz L, Arnau Vives MA, et al: Lipid-lowering effect of atorvastatin in heart transplantation. Transplant Proc 34:179, 2002 7. Berrebi D, Languepin J, Ferkdadji L: Cytokines, chemokine receptors, and homing molecule distribution in the rectum and stomach of pediatric patients with ulcerative colitis. J Pediatr Gastroenterol Nutr 37:300, 2003 8. Anders HJ, Vielhauer V, Schlondorff D: Chemokines and chemokine receptors are involved in the resolution or progression of renal disease. Kidney Int 63:401, 2003 9. Anders HJ, Frink M, Linde Y, et al: CC chemokine ligand 5/RANTES chemokine antagonists aggravate glomerulonephritis despite reduction of glomerular leukocyte infiltration. J Immunol 170:5658, 2003
2904 10. Nelson PJ, Krensky AM: Chemokines, chemokine receptors, and allograft rejection. Immunity 14:377, 2001 11. Handel TM, Lau EK: Chemokine structure and receptor interactions. Ernst Schering Res Found Workshop 45:101, 2004 12. Gibejov A: Chemokine receptors. Acta Univ Palacki Olomuc Fac Med 143:9, 2000 13. Aguirrezabalaga J, Fernandez-Selles C, Fraguela J, et al: Lipid profiles after liver transplantation in patients receiving tacrolimus or cyclosporin. Transplant Proc 34:1551, 2002 14. Aakhus S, Dahi K, Wideroe TE: Hyperlipidemia in renal transplant patients. J Int Med 239:407, 1996 15. Hancock W: Chemokines and transplant immonobiology. J Am Soc Nephrol 13:821, 2002 16. Schwabe RF, Bataller R, Brenner DA: Human hepatic stellate cells express CCR5 and RANTES to induce proliferation and migration. Am J Physiol Gastrointest Liver Physiol 285:G949, 2003 17. Iijima W, Ohtani H, Nakayama T, et al: Infiltrating CD8⫹ T cells in oral lichen planus predominantly express CCR5 and CXCR3 and carry respective chemokine ligands RANTES/CCL5 and IP-10/CXCL10 in their cytolytic granules: a potential selfrecruiting mechanism. Am J Pathol 163:261, 2003 18. Wang Y, Li H, Lu Y, et al: Effect of apolipoprotein E gene polymorphism on serum lipid levels before and after renal transplantation. Chinese J Organ Transpant 25:294, 2004 19. Lopes-Virelia M, Mironina M, Stephen P, et al: Role of Simvastatin as an immunomodulator in type 2 diabetes. Diabetes Care 27:908, 2004
LI, WANG, WANG ET AL 20. Wenke K, Meiser B, Thiery J, et al: Simvastatin initiated early after heart transplantation 8-year prospective experience. Circulation 107:93, 2003 21. Chello M, Mastroroberto P, Patti G, et al: Simvastatin attenuates leucocyte-endothelial interactions after coronary revascularisation with cardiopulmonary bypass. Heart 89:538, 2003 22. Kasiske BL, O’Donnell MP, Garvis WJ, et al: Pharmacologic treatment of hyperlipidemia reduces glomerular injury in the rat 5/6 nephrectomy model of chronic renal failure. Circ Res 62:367, 1988 23. Lee SK, Jin SY, Han DC, et al: Effects of delayed treatment with enalapril and/or lovastatin on the progression of glomerulosclerosis in 5/6 nephrectomized rats. Nephrol Dial Transplant 8:1338, 1993 24. Sparrow CP, Burton CA, Hernande M, et al: Simvastatin has anti-inflammatory and antiatherosclerotic activities independent of plasma cholesterol lowering. Arterioscler Thromb Vasc Biol 21: 115, 2001 25. Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156, 1987 26. Greenwood J, Walters CE, Pryce G, et al: Lovastatin inhibits brain endothelial cell Rho-mediated lymphocyte migration and attenuates experimental autoimmune encephalomyelitis. FASEB J 17:905, 2003
I
N LONG-TERM SURVIVORS of renal transplantation, chronic allograft nephropathy (CAN) and atherosclerosis account for the majority of patient deaths or renal allograft dysfunction.1– 4 Regulated upon activation normal T cell expressed and secreted (RANTES) and its receptor, C-C chemokine receptor 5 (CCR5), are synthesized by arterial smooth muscle cells and endothelial cells exposed to inflammatory stimuli, thus providing a chemotactic stimulus for the recruitment of monocytes into the arterial wall, one of the earliest cellular processes in the pathology of atherosclerosis and CAN.5–11 The expression of RANTES and CCR5 could also be detected in mononuclear cells infiltrating kidney grafts, which suggested that RANTES and CCR5 play important roles in progressive atherosclerosis and CAN in transplant recipients.12–17 In our former study and other studies, hyperlipidemia was documented in more than half of renal transplant recipients. This finding has been considered to be one of the main contributors to CAN and cardiovascular disease.3,4,18 Simvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl CoA
(HMG CoA) reductase is often used to reduce serum lipid levels. Recently studies have shown that HMG CoA inhibitors have many other effects in addition to regulating serum lipid levels.19 –22 HMG CoA inhibitors can inhibit NF-B activation and MCP-1, transforming growth factorbetal mRNA expression, and also ameliorate albuminuria and glomerulosclerosis in five of six nephrectomized rats, in obese rats, and in streptozotocin-induced diabetic uninephrectomized rats.23,24 To date, there have been no reports about its effects on RANTES in renal transplant recipients.
From Department of Nephrology (Q.L., Y.W.), The Second Hospital of Xiamen, Nephrology, Xiamen, China; Department of Urology (D.W.), The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China; and Department of Nephrology (Q.X.), The First People’s Hospital of Shanghai, Shanghai, China. Dr Qingqun Li and Dr Daohu Wang contributed equally to the work and should be considered co-first authors. Address reprint requests to Yuxin Wang, The Department of Nephrology, The Second Hospital of Xiamen, Nephrology, 361021 Xiamen, China. E-mail: [email protected]
© 2006 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/06/$–see front matter doi:10.1016/j.transproceed.2006.08.136
Transplantation Proceedings, 38, 2899 –2904 (2006)
2899
2900
LI, WANG, WANG ET AL ⬍170 mol/L, urine protein was negative, and urine volume was normal in every patient.
In this study, we investigated the mRNA expressions of RANTES and CCR5 in peripheral blood mononuclear cells (PBMCs) among renal, transplant patients with hyperlipidemia and the influence of simvastatin treatment on their expression.
Experimental Design According to the serum lipid levels at 6 months after kidney transplantation, the recipients were divided into two groups; group A without hyperlipidemia, included 16 males and 14 females of mean age 41 ⫾ 9 years; group B with hyperlipidemia total serum cholesterol ⬎ 6.2 mmol/L, serum triglyceride ⬎ 2.3 mmol/L included 17 males and 13 females (mean age 40 ⫾ 10 years). The profile of serum lipids in the renal transplant recipients and controls is shown in Fig 1. Group B patients were treated with simvastatin (Merck, USA; 20 mg/day) to decrease serum lipid levels. To detect serum lipid concentration and the mRNA expression in peripheral mononuclear cells, overnight fasting blood was collected from all patients before and at 6 to 8 months after kidney transplantation and in group B also at 1.5 and 3 months of simvastatin treatment. Thirty healthy volunteers were employed as controls, including 16 males and 14 females of overall mean age of 41 ⫾ 9 years. Informed consent was obtained from each participant.
MATERIALS AND METHODS This study was approved by our institutional ethical committee.
Patients Sixty patients (33 males and 27 females) of mean age 40 ⫾ 9 years were selected from 107 cadaver kidney transplantation recipients. Their primary end-stage renal disease (ESRD) were glomerulonephropathies, and their serum lipid levels were normal before renal transplantation. Every patient was treated with prednisone, mycophenolate mofetil and cyclosporine. Prednisone was dosed at 80 mg/day for the first 2 days, then gradually reduced to 10 mg/day. Cyclosporine was titered according to blood concentrations (100 –200 ng/mL). Serum creatinine level was
A 10
B
9
6
8
4
**
**
5
TG(mmol/L)
TC(mmol/L)
**
6
5
**
7
4 3 2
**
3
**
2
**
**
Group A
Group B
1 1
0 Congtrols
Group A
Group B 0 Congtrols
Before Transplant Before Trans plant
After transplant
C
D
3
5 4. 5 4
**
2 1. 5
3. 5
** **
**
1
LDLC(mmol/L
HDLC(mmol/L)
2. 5
Fig 1. Serum lipid levels before and after renal transplantation versus controls. (A) Serum TC levels in the three groups; (B) Serum TC levels in controls and renal transplant recipients; (C) Serum HDLC levels in controls and renal transplant recipients; (D) Serum LDLC levels of controls and renal transplant recipients. (*P ⬍ .05, **P ⬍ .01 vs group B after renal transplantation).
After transplant
3
**
* **
2. 5
**
2 1. 5 1
0. 5
0. 5
0 Congtrols Before Transplant
Group A
Group B
After transplant
0 Congtrols Before Transplant
Group A
Group B
After transplant
SIMVASTATIN DOWNREGULATES mRNA EXPRESSION
2901
Serum Lipid Serum was collected from peripheral blood samples from each subject after an overnight fast. Serum total cholesterol (TC) and total glycerides (TG) concentrations were measured enzymatically with the Cholesterol-E test and the triglyceride-E test (Wako Pure Chemical Co.). HDL-cholesterol (HDL-C) was measured with the HDL-cholesterol-E test (Wako).
RT-PCR Ten milliliters of blood collected in sodium citrate from the patient was diluted with equal volume of phosphate-buffered saline, overlaid with an equal volume of ficoll-hypaque (Pharmacia), and centrifuged in a table-top centrifuge at 1500g for 30 minutes. The PBMCs band below the plasma was collected and washed three times with phosphate-buffered saline.
RNA Isolation Total RNA was extracted from PBMCs using trizol (Gibco), which is based on the method described by Chomczynski and Sacchi.25 In brief, 10 ⫻ 166 PBMCs from each sample were homogenized in 1 mL trizol before addition of 200 L chloroform. The mixture was
B
10 9
**
**
8
TG(mmol/L)
5 4 3 2
##
3 2
3 months Simvastatin treatment
Before treatment
3 months Simvastatin treatment
1.5 months Simvastatin treatment
Before treatment
D
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0
0
5 4. 5
2. 5 LDLC(mmol/L)
4
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*
3. 5 3 2. 5 2 1. 5 1
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3 months Simvastatin treatment
1.5 months Simvastatin treatment
3 months Simvastatin treatment
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0 Before treatment
HDLC(mmol/L
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1
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1.5 months Simvastatin treatment
TC(mmol/L)
6
Total RNA was transcribed to complementary DNA(cDNA) by reverse transcription (RT) with hexamer random primers (Promega, USA). One microgram of total RNA was added to 0.5 g of primer. A reaction mixture contained 50 ng of random primer, deoxynucleotide triphosphate (0.5 mmol/L), 5⫻buffer 5 L, 0.1 M DTT (2 L), Rnase OUT Rnase Inhib (0.5 L), and 200 units of MMLV reverse transcriptase. All reagents were purchased from Gibco in 20 L of final volume. The reaction was allowed to proceed at 70°C for 5 minutes, 37°C for 60 minutes, and stopped by heating to 95°C for 5 minutes, followed by cooling on ice. The cDNA was stored at ⫺30°C for further procedures.
5
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7
Reverse Transcription
6
Before treatment
A
centrifuged at 12000g for 15 minutes at 4°C. The supernate was treated with 0.5 mL isopropanol and centrifuged at 12000g for 10 minutes at 4°C. The supernate was discarded and the sediment mixed with 0.75 mL 75% ethanol before centrifugation at 12000g for 10 minutes at 4°C. The precipitate of total RNA was resolved with 20 L DEPC water. The purity of isolated RNA was determined by measuring the ratio of absorbance at 260 and 280 nm (A260/280 ratios), and the concentration estimated by absorbance at 260 nm.
Fig 2. Serum lipid levels before and after renal transplantation among group B patients versus controls. (A) Serum TC levels; (B) Serum TG levels; (C) Serum HDLC levels; (D) Serum LDLC levels. (*P ⬍ .05, **P ⬍ .01 vs controls; #P ⬍ .05, ##P ⬍ .01 vs 1.5 month).
LI, WANG, WANG ET AL
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Amplification of Specific Complementary DNA The primer sequences for -actin were: 5=-TGGCACACCTTCTACAATGAGCTGCG-3= (forward) and 5=-CGTCATACTCCTTGCTCCACA TCTGC-3= (reverse), with a 838-bp length of the amplified segments. The primer sequences for RANTES were: 5=-AGCTACTCGGGAGGCTAAGG-3= (forward) and 5=-GAGGCATGCTGACTTCCTTC-3= (reverse), with a 276-bp length of the PCR product. The primer sequences for CCR5 were: 5=-TGTAGGGAGCCCAGAAGAGA-3= (forward) and 5=-CGACATACTCCTTGCTCCCACATCTGC-3= (reverse), with a 168-bp PCR product length. Specific cDNA products corresponding to mRNA for human -actin, RANTES and CCR5 were amplified using PCR. Five microliters of cDNA was taken for PCR, which was performed in PCR buffer, using 0.2 mM/L dNTP, 1 M/L of both primers and 2.0U Taq DNA polymerase. All reagants were purchased from Gibco (USA). GeneAmp2700 Thermal Cycler (USA) was used for amplification with the following sequence profile: one cycle of 5 minutes at 95°C for template denaturation, followed by 35 cycles of 30 seconds of denaturation at 95°C, 45 seconds (at 60°C for RANTES, 57°C for CCR5) for primer annealing, and 60 seconds for polymerase extension at 72°C. All reactions were terminated with a 10-minute extension at 70°C and cooling to
After transplant
4°C. Final products were electrophoresed on 2% agarose gels and analyzed by direct visualization after ethidium bromide staining. Every RT-PCR reaction was repeated at least twice to confirm the results.
Argarose Gel Analysis The amplified PCR products were analyzed on 2.0% agarose gels electrophoresed at 75 V, 25 mA in 40 mmol/L Tris-acetate (pH 8.5), and 2 mmol/L EDTA (TAE buffer) followed by staining with 0.5pg/mL ethidium bromide. Sample products were visualized by UV transillumination for photography of the gel. Specific products were identified by size in relation to a known DNA Marker (Takara) run with each gel. The cDNA of RANTES and CCR5 were semiquantitatively analyzed by densitometric comparison to -actin (internal control) from the same sample after the positive image was digitized by video for computerized densitometry. The results are shown as ratios of intensity of RANTES, CCR5, and -actin.
Statistical Analysis All data are expressed as mean values ⫾ SD. Student’s t test or one-way ANOVA test was used for statistical analysis with the SPSS program.
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Fig 4. The mRNA expression of MCP-1 and CCR2 in group B before and after simvastatin treatment (*P ⬍ .05, **P ⬍ .01 vs before treatment). (A) The mRNA expression of MCP-I in group B before and after simvastatin treatment. (B) The mRNA expression of CCR2 in group B before and after simvastatin treatment (*P ⬍ .05, **P ⬍ .01 vs before treatment).
Group B
Fig 3. The mRNA expression of MCP-1 and CCR2 in controls and the recipients before and after renal transplantation. (A) The mRNA expression of MCP-1 in controls and the recipients before and after renal transplantation (P ⬍ .05 vs group B after renal transplantation). (B) The mRNA expression of CCR2 in controls and the recipients before and after renal transplantation (*P ⬍ .05, **P ⬍ .01 vs comtrols).
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SIMVASTATIN DOWNREGULATES mRNA EXPRESSION
RESULTS
Serum lipid levels were decreased in the renal transplant recipients of group B after treatment with Simvastatin. Simvastatin (20 mg/day) was presented for hyperlipidemic renal transplant recipients with with serum lipid levels determined at 1.5 and 3 months after simvastatin treatment. Serum levels of TC, TG, and LDLC had decreased significantly at 1.5 months compared with before treatment and reduced further at 3 months of simvastatin treatment (Fig 2). The High mRNA Expression of RANTES and CCR5 Were Downregulated by Simvastatin Treatment
The mRNA expressions of RANTES and CCR5 in all recipients were significantly higher compared with those of controls at 6 months after renal transplantation. The mRNA espression of RANTES and CCR5 decreased significantly in all renal transplant recipients, but remained significantly higher than the controls. There was no significant differences in mRNA expression of RANTES on CCR5 between group A and group B before renal transplantation. However, the mRNA expressions of RANTES and CCR5 were significantly higher in group B than group A at 6 months after renal transplantation (Fig 3). For group B, the significantly down-regulation of mRNA expression of RANTES and CCR5 were evident at 1.5 months of simvastatin treatment and reduced to even normal levels in recipients with 3 months of simvastatin treatment (RANTES: 2.3 ⫾ 1.1 vs 2.0 ⫾ 0.8, P ⬎ .05; CCR5: 1.1 ⫾ 0.6 vs 0.8 ⫾ 0.4; P ⬎ .05). These values were significantly lower than those in group A recipients posttransplantation (RANTES: 2.3 ⫾ 1.1 vs 3.3 ⫾ 1.3, P ⬍ .01; CCR5: 1.1 ⫾ 0.6 vs 1.8 ⫾ 0.7, P ⬍ .01; Fig 4). DISCUSSION
Our pervious study suggested that the mRNA expressions of RANTES and CCR5 were up-regulated among posttransplant recipients with hyperlipidemia. RANTES and CCR5 are involved in the pathogenesis of atherosclerosis and CAN by promoting directed migration of inflammatory cells. They play important roles in atherosclerosis and chronic allograft nephropathy. Thus, it was suggested that upregulation of RANTES and CCR5 mRNA that may a main mechanism in cardiovascular disease and CAN caused by hyperlipidemia, and that hyperlipidemia may play an important role in the pathogenesis of atherosclerosis and CAN by inducing the production of RANTES and CCR5. Our results presented here showed that the expression of RANTES and CCR5 were decreased significantly after treatment with simvastatin in renal transplant recipients with hyperlipidemia. After 3 months of treatment the levels were even lower than those among recipients without hyperlipidemia. Simvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl CoA (HMG CoA) reductase, is often used to lower serum lipid levels. Recent studies have shown that it markedly
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reduces the incidence of CAN and cardiovascular events besides the action of reducing serum lipid levels. Pretreatment with simvastatin reduces neutrophil adhesion to the venous endothelium in patients undergoing coronary surgery, irrespective of its efficacy at lowering cholesterol concentration.20 –22,24 HMG CoA inhibitors can also reduce activation of RANTES, ameliorating albuminuria and glomerulosclerosis in five of six nephrectomized rats, in obese rats, and in streptozotocin-induced diabetic uninephrectomized rats.22,23 Simvastatin has anti-inflammatory effects through downregulation of cytokines in the endothelium and leukocytes, as well as reducing the expression of the cytokines MCP-1, interleukin-6, and interleukin-8 in circulating monocytes from hypercholesterolemic patients.20 Greenwood et al demonstrated that lovastatin, another HMG CoA inhibitor blocks a key stage in the pathogenesis of neuroinflammation, namely leukocyte migration across the bloodbrain barrier, thus demonstrating a novel effect of statins to modulate the immune response in neuroinflammtory diseases.26 These data support that simvastatin has a role to prevent CAN beyond the plasma cholesterol-lowering activity. Based on the former observations and our results, it may be concluded that simvastatin can be used to reduce the incidence of CAN in renal recipients with hyperlipidemia, even those with normal serum lipid levels. It was suggested by this study that the increased expressions of RANTES and CCR5 mRNA in renal transplant recipients with hyperlipidemia may be involved in CAN due to hyperlipidemia. Thus simvastatin treatment can be used to reduce the incidence of CAN in renal recipients with hyperlipidemia. REFERENCES 1. Nankivell BJ, Borrows RJ, Chir MB, et al: The natural history of chronic allograft nephropathy. N Engl J Med 349:2326, 2003 2. Van der Woude FJ, Hollander AAMJ: Rejection after kidney transplantation: New concepts, new therapeutic options. Transplant Proc 30:2419, 1998 3. Aguirrezabalaga J, Fernandez-Selles C, Otero A, et al: Lipid profiles in liver transplantation in patients receiving tacrolimus or cyclosporin. Transplant Proc 33:1064, 2001 4. Kisielnicka E, Zdrojewski Z, Wroblewska M, et al: Lipid disturbances in a two-year follow-up after successful kidney transplantation. Transplant Proc 32:1358, 2000 5. Moench C, Uhrig A, Lohse AW, et al: Differential diagnosis of cytomegalovirus infection and acute rejection by serum CCchemokine measurement after orthotopic liver transplantation. Transplant Proc 35:2084, 2003 6. Almenar Bonet L, Martinez-Dolz L, Arnau Vives MA, et al: Lipid-lowering effect of atorvastatin in heart transplantation. Transplant Proc 34:179, 2002 7. Berrebi D, Languepin J, Ferkdadji L: Cytokines, chemokine receptors, and homing molecule distribution in the rectum and stomach of pediatric patients with ulcerative colitis. J Pediatr Gastroenterol Nutr 37:300, 2003 8. Anders HJ, Vielhauer V, Schlondorff D: Chemokines and chemokine receptors are involved in the resolution or progression of renal disease. Kidney Int 63:401, 2003 9. Anders HJ, Frink M, Linde Y, et al: CC chemokine ligand 5/RANTES chemokine antagonists aggravate glomerulonephritis despite reduction of glomerular leukocyte infiltration. J Immunol 170:5658, 2003
2904 10. Nelson PJ, Krensky AM: Chemokines, chemokine receptors, and allograft rejection. Immunity 14:377, 2001 11. Handel TM, Lau EK: Chemokine structure and receptor interactions. Ernst Schering Res Found Workshop 45:101, 2004 12. Gibejov A: Chemokine receptors. Acta Univ Palacki Olomuc Fac Med 143:9, 2000 13. Aguirrezabalaga J, Fernandez-Selles C, Fraguela J, et al: Lipid profiles after liver transplantation in patients receiving tacrolimus or cyclosporin. Transplant Proc 34:1551, 2002 14. Aakhus S, Dahi K, Wideroe TE: Hyperlipidemia in renal transplant patients. J Int Med 239:407, 1996 15. Hancock W: Chemokines and transplant immonobiology. J Am Soc Nephrol 13:821, 2002 16. Schwabe RF, Bataller R, Brenner DA: Human hepatic stellate cells express CCR5 and RANTES to induce proliferation and migration. Am J Physiol Gastrointest Liver Physiol 285:G949, 2003 17. Iijima W, Ohtani H, Nakayama T, et al: Infiltrating CD8⫹ T cells in oral lichen planus predominantly express CCR5 and CXCR3 and carry respective chemokine ligands RANTES/CCL5 and IP-10/CXCL10 in their cytolytic granules: a potential selfrecruiting mechanism. Am J Pathol 163:261, 2003 18. Wang Y, Li H, Lu Y, et al: Effect of apolipoprotein E gene polymorphism on serum lipid levels before and after renal transplantation. Chinese J Organ Transpant 25:294, 2004 19. Lopes-Virelia M, Mironina M, Stephen P, et al: Role of Simvastatin as an immunomodulator in type 2 diabetes. Diabetes Care 27:908, 2004
LI, WANG, WANG ET AL 20. Wenke K, Meiser B, Thiery J, et al: Simvastatin initiated early after heart transplantation 8-year prospective experience. Circulation 107:93, 2003 21. Chello M, Mastroroberto P, Patti G, et al: Simvastatin attenuates leucocyte-endothelial interactions after coronary revascularisation with cardiopulmonary bypass. Heart 89:538, 2003 22. Kasiske BL, O’Donnell MP, Garvis WJ, et al: Pharmacologic treatment of hyperlipidemia reduces glomerular injury in the rat 5/6 nephrectomy model of chronic renal failure. Circ Res 62:367, 1988 23. Lee SK, Jin SY, Han DC, et al: Effects of delayed treatment with enalapril and/or lovastatin on the progression of glomerulosclerosis in 5/6 nephrectomized rats. Nephrol Dial Transplant 8:1338, 1993 24. Sparrow CP, Burton CA, Hernande M, et al: Simvastatin has anti-inflammatory and antiatherosclerotic activities independent of plasma cholesterol lowering. Arterioscler Thromb Vasc Biol 21: 115, 2001 25. Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156, 1987 26. Greenwood J, Walters CE, Pryce G, et al: Lovastatin inhibits brain endothelial cell Rho-mediated lymphocyte migration and attenuates experimental autoimmune encephalomyelitis. FASEB J 17:905, 2003