转化生长因子βⅡ型受体-IgG Fc 嵌合蛋白基因治疗改善环孢霉素A肾毒性
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摘要
目的 转化生长因子β(TGF-β)在环孢霉素A(CsA)所致肾脏毒性的发病过程中可能发挥重要作用。转化生长因子βⅡ型受体—IgG Fc(TGF β RⅡ/IgGFc)是一将人类TGF β RⅡ细胞外区域与IgG1Fc段连接而成的可溶性嵌合蛋白,可在循环中阻断TGF β的活性。本研究旨在探讨TGF β RⅡ/IgG Fc基因转染对小鼠CsA肾毒性的作用。
方法 雌性ICR小鼠予低钠饮食,CsA用药组每日皮下注射CsA 25mg/Kg。在给药的第1天和第7天,对照组在布比卡因预处理的胫骨肌处注射50 μg质粒CAGGS—β半乳糖苷酶(lacZ)(pCAGGS-β galactosidase(lacZ)),治疗组注射等量质粒CAGGS-TGF β RⅡ/IgGFc(pCAGGS-TGF β RⅡ/IgGFc),均联合电穿孔,在CsA给药两周或三周后处死。肾组织中TGF β 1mRNA水平用Northern blot测定,肾组织和血浆中TGFβ1蛋白水平由免疫组化和ELISA法测得。利用石蜡切片PAS和Masson’s trichrom染色半定量评分评估肾小管损害和肾间质纤维化程度,免疫组化方法检测小管间质F4/80阳性的巨噬细胞的表达。
结果 CsA给药2周后,对照组血浆和肾脏TGFβ1表达最高,然后降至基线水平。而TGF β1的mRNA水平直到给药三周仍持续增高。小管间质病变在第二周逐渐显现,第三周病变明显加重。TGF β RⅡ/IgGFc干预组,2周时血浆TGFβ1水平与不用药的低钠饮食对照组相仿,肾组织TGFβ1表达较CsA对照组降低50%(P<0.01),对小管间质病变有保护作用(P<0.01)。第三周时,干预组的肾脏mRNA水平和蛋白水平较对照组没有明显改变,但小管间质病变持续减轻,呈现肾脏保护作用。
结论 TGF β RⅡ/IgG Fc基因转染有效抑制了CsA引起的小管间质病变。在第三周,尽管肾脏mRNA水平和蛋白水平与对照组比没有明显改变,但小管间质病变仍持续缓解。表明早期对TGFβ1的干预治疗对于延缓CsA肾毒性进展具有重要作用。
Background. Transforming growth factor β (TGFβ) is implicated in the pathogenesis of cyclosprine A (CsA) nephrotoxicity. We examined the efficacy of TGFβ RII/IgG Fc, a soluble chimeric protein of the extracellular domain of human TGFβ type II receptor and IgGl Fc, on CsA nephrotoxicity in mice.Methods. Subcutaneous injection of CsA (25 mg/kg/day) was given daily to mice maintained on low sodium diet. On day 1 and 7, an expression vector carrying cDNA for either TGF RII/IgG Fc or β-galactosidase was transfected into the skeletal muscles by electroporation. At 2 or 3 weeks of CsA treatment, and plasma and renal TGF β1 levels, and tubulointerstitial injury and fibrosis were evaluated. Results. By 2 weeks of CsA administration, plasma and renal TGFβ1 levels increased to maximum and, thereafter, declined toward the baseline levels. Renal TGFP lmRNA remained elevated until 3 weeks. Tubulointerstitial alterations became appreciable in 2 weeks and intensified by 3 weeks. At 2 weeks, the TGFβ RII/IgG Fc intervention abolished the increase in plasma TGFβ 1, attenuated the increase in renal TGFβl by 50%, and markedly suppressed the histological alterations. At 3 weeks, the histological alterations remained markedly suppressed by the intervention, with no appreciable effects on the renal TGFβlmRNA and protein.Conclusion. Introduction of TGF β RII/IgG Fc by gene transfer effectively abrogated CsA-induced tubulointerstitial alterations. Suppression of tubulointerstitial changes was evident at 3 weeks when renal TGFP 1 mRNA and protein were comparable to those with CsA alone, suggesting that early
方法 雌性ICR小鼠予低钠饮食,CsA用药组每日皮下注射CsA 25mg/Kg。在给药的第1天和第7天,对照组在布比卡因预处理的胫骨肌处注射50 μg质粒CAGGS—β半乳糖苷酶(lacZ)(pCAGGS-β galactosidase(lacZ)),治疗组注射等量质粒CAGGS-TGF β RⅡ/IgGFc(pCAGGS-TGF β RⅡ/IgGFc),均联合电穿孔,在CsA给药两周或三周后处死。肾组织中TGF β 1mRNA水平用Northern blot测定,肾组织和血浆中TGFβ1蛋白水平由免疫组化和ELISA法测得。利用石蜡切片PAS和Masson’s trichrom染色半定量评分评估肾小管损害和肾间质纤维化程度,免疫组化方法检测小管间质F4/80阳性的巨噬细胞的表达。
结果 CsA给药2周后,对照组血浆和肾脏TGFβ1表达最高,然后降至基线水平。而TGF β1的mRNA水平直到给药三周仍持续增高。小管间质病变在第二周逐渐显现,第三周病变明显加重。TGF β RⅡ/IgGFc干预组,2周时血浆TGFβ1水平与不用药的低钠饮食对照组相仿,肾组织TGFβ1表达较CsA对照组降低50%(P<0.01),对小管间质病变有保护作用(P<0.01)。第三周时,干预组的肾脏mRNA水平和蛋白水平较对照组没有明显改变,但小管间质病变持续减轻,呈现肾脏保护作用。
结论 TGF β RⅡ/IgG Fc基因转染有效抑制了CsA引起的小管间质病变。在第三周,尽管肾脏mRNA水平和蛋白水平与对照组比没有明显改变,但小管间质病变仍持续缓解。表明早期对TGFβ1的干预治疗对于延缓CsA肾毒性进展具有重要作用。
Background. Transforming growth factor β (TGFβ) is implicated in the pathogenesis of cyclosprine A (CsA) nephrotoxicity. We examined the efficacy of TGFβ RII/IgG Fc, a soluble chimeric protein of the extracellular domain of human TGFβ type II receptor and IgGl Fc, on CsA nephrotoxicity in mice.Methods. Subcutaneous injection of CsA (25 mg/kg/day) was given daily to mice maintained on low sodium diet. On day 1 and 7, an expression vector carrying cDNA for either TGF RII/IgG Fc or β-galactosidase was transfected into the skeletal muscles by electroporation. At 2 or 3 weeks of CsA treatment, and plasma and renal TGF β1 levels, and tubulointerstitial injury and fibrosis were evaluated. Results. By 2 weeks of CsA administration, plasma and renal TGFβ1 levels increased to maximum and, thereafter, declined toward the baseline levels. Renal TGFP lmRNA remained elevated until 3 weeks. Tubulointerstitial alterations became appreciable in 2 weeks and intensified by 3 weeks. At 2 weeks, the TGFβ RII/IgG Fc intervention abolished the increase in plasma TGFβ 1, attenuated the increase in renal TGFβl by 50%, and markedly suppressed the histological alterations. At 3 weeks, the histological alterations remained markedly suppressed by the intervention, with no appreciable effects on the renal TGFβlmRNA and protein.Conclusion. Introduction of TGF β RII/IgG Fc by gene transfer effectively abrogated CsA-induced tubulointerstitial alterations. Suppression of tubulointerstitial changes was evident at 3 weeks when renal TGFP 1 mRNA and protein were comparable to those with CsA alone, suggesting that early
引文
1. Mihatsch MJ, Thiel G, Spichtin HP, et al. Morphological findings in kidney transplants after treatment with cyclosporine. Transplant Proc 1983; 15 (Suppl 1): 2821.
2. Rosen S, Greenfeld Z, Brezis M. Chronic cyclosporine-induced nephropathy in rats. Transplantation 1990; 49: 445.
3. Young BA, Burdmann EA, Johnson RJ, et al. Cyclosporine A induced arteriolopathy in a rat model of chronic cyclosporine nephropathy. Kidney Int 1995; 48: 431.
4. Young BA, Burdmann EA, Johnson RJ, et al. Cellular proliferation and macrophage influx precede interstitial fibrosis in cyclosporine nephrotoxicity. Kidney Int 1995; 48: 439.
5. Shihab FS, Andoh TF, Tanner AM, Bennett WM. Sodium depletion enhances fibrosis and the expression of TGF-beta 1 and matrix proteins in experimental chronic cyclosporine nephropathy. Am J Kidney Dis 1997; 30: 71.
6. Border WA, Noble NA. Transforming growth factor β in tissue fibrosis. N Engl J Med 1994; 331: 1286.
7. Pankewycz OG, Miao L, Isaacs R, Guan J, Pruett T, Haussmann G, Sturgill BC. Increased renal tubular expression of transforming growth factor beta in human allografts correlates with cyclosporine toxicity. Kidney Int 1996; 50: 1634.
8. Khanna A, Kapur S, Sharma V, Li B, Suthanthiran M. In vivo hyperexpression of transforming growth factor-β1 in mice: stimulation by cyclosporine. Transplantation 1997; 63: 1037.
9. Wolf G, Thaiss F, Stahl RAK. Cyclosporine stimulates expression of transforming growth factor-β in renal cells. Possible mechanism of cyclosporines antiproliferative effects. Transplantation 1995; 60: 237.
10. Burdmann EL, Andoh TF, Nast CC, et al. Prevention of experimental cyclosporine-induced interstitial fibrosis by losartan and enalapril. Am J Physiol 1995; 269: F491.
11. Shihab FS, Bennett WM, Tanner AM, Andoh TF. Angiotensin Ⅱ blockade decreases TGF-beta 1 and matrix proteins in cyclosporine nephropathy. Kidney Int 1997; 52: 660.
12. Blobe GC, Schiemann WP, Lodish HF. Role of transforming growth factor p in human diseases. N Engl J Med 2000; 342: 1350.
13. Bork P. The modullar architecture of a new family of growth regulators related to connective tissue growth factor. FEBS Lett 1993; 327: 125.
14. Yokoi H, Sugawara A, Mukoyama M, et al. Role of connective tissue growth factor in profibrotic action of transforming growth factor-beta: a potential target for preventing renal fibrosis. Am J Kidney Dis 2001; 38 (Suppl 1): S134.
15. Yokoi H, Mukoyama M, Sugawara A et al. Role of connective tissue growth factor in fibronectin expression and tubulointerstitial fibrosis.
Am J Physiol 2002; 282: F933.
16. Ito Y, Aten J, Bende RJ, et al. Expression of connective tissue growth factor in human renal fibrosis. Kidney Int 1998; 53: 853.
17. Rister BL, Denichilo M, Cortes P, et al. Regulation of connective tissue growth factor activity in cultured rat mesangial cells and its expression in experimental diabetic glomerulosclerosis. J Am Soc Nephrol 2000; 11:25.
18. Finckenberg P, Lassila M, Inkinen K, et al. Cyclosporine induces myocardial connective tissue growth factor in spontaneously hypertensive rats on high-sodium diet. Transplantation 2001; 71: 951.
19. Isaka Y, Akagi Y, Ando Y, et al. Gene therapy by transforming growth factor-β receptor-IgG Fc chimera suppressed extracellular matrix accumulation in experimental glomerulonephritis. Kidney Int 1999; 55: 465.
20. Imai E, Akagi Y, Isaka Y. Towards gene therapy for renal diseases. Nephrologie 1998; 19: 397.
21. Kon V, Hunley T, Fogo A. Combined antagonism of endothelin A/B receptors links endothelin to vasoconstriction whereas angiotensin II effects fibrosis. Studies in chronic cyclosporine nephrotoxicity in rats. Transplantation 1995; 60: 89.
22. Aihara H, Miyazaki J. Gene transfer into muscle by electroporation in vivo. Nature Biotechnol 1998; 16: 867.
23. Ma J, Nishimura H, Fogo A, Kon V, Inagami T, Ichikawa I. Accelerated fibrosis and collagen deposition develop in the renal interstitium of angiotensin type 2 receptor null mutant mice during ureteral obstruction. Kidney Int 1998; 53: 937.
24. Kaneto H, Morrissey J, McCracken R, Reyes A, Klahr S. Enalapril reduces collagen type IV synthesis and expansion of the interstitium in the obstructed kidney. Kidney Int 1994; 45: 1637.
25. Khanna AK, Cairns VR, Becker CG, Hosenpud JD. Transforming growth factor (TGF)-p mimics and anti-TGF-p antibody abrogates the in vivo effects of cyclosporine. Demonstration of a direct role of TGF-β in immunosuppression and nephrotoxicity of cyclosporine. Transplantation 1999; 67: 882.
26. Islam M, Burke JF, Jr, McGowan TA, Zhu Y, Dunn SR, McCue P, Kanalas J, Sharma K. Effect of anti-transforming growth factor-p antibodies in cyclosprine-induced renal dysfunction. Kidney Int 2001; 59: 498.
27. Thomas SE, Andoh TF, Pichler RH, et al. Accelerated apoptosis characterizes cyclosporine-associated interstitial fibrosis. Kidney Int 1998; 53: 897.
28. Wrana JL, Attisano L, Wieser R, Ventura F, Massague J. Mechanism of activation of the TGF-β receptor. Nature 1994; 370: 341.
29. Ling H, Li X, Jha S, Wang W, Karetskaya L, Pratt B, Ledbetter S.
2. Rosen S, Greenfeld Z, Brezis M. Chronic cyclosporine-induced nephropathy in rats. Transplantation 1990; 49: 445.
3. Young BA, Burdmann EA, Johnson RJ, et al. Cyclosporine A induced arteriolopathy in a rat model of chronic cyclosporine nephropathy. Kidney Int 1995; 48: 431.
4. Young BA, Burdmann EA, Johnson RJ, et al. Cellular proliferation and macrophage influx precede interstitial fibrosis in cyclosporine nephrotoxicity. Kidney Int 1995; 48: 439.
5. Shihab FS, Andoh TF, Tanner AM, Bennett WM. Sodium depletion enhances fibrosis and the expression of TGF-beta 1 and matrix proteins in experimental chronic cyclosporine nephropathy. Am J Kidney Dis 1997; 30: 71.
6. Border WA, Noble NA. Transforming growth factor β in tissue fibrosis. N Engl J Med 1994; 331: 1286.
7. Pankewycz OG, Miao L, Isaacs R, Guan J, Pruett T, Haussmann G, Sturgill BC. Increased renal tubular expression of transforming growth factor beta in human allografts correlates with cyclosporine toxicity. Kidney Int 1996; 50: 1634.
8. Khanna A, Kapur S, Sharma V, Li B, Suthanthiran M. In vivo hyperexpression of transforming growth factor-β1 in mice: stimulation by cyclosporine. Transplantation 1997; 63: 1037.
9. Wolf G, Thaiss F, Stahl RAK. Cyclosporine stimulates expression of transforming growth factor-β in renal cells. Possible mechanism of cyclosporines antiproliferative effects. Transplantation 1995; 60: 237.
10. Burdmann EL, Andoh TF, Nast CC, et al. Prevention of experimental cyclosporine-induced interstitial fibrosis by losartan and enalapril. Am J Physiol 1995; 269: F491.
11. Shihab FS, Bennett WM, Tanner AM, Andoh TF. Angiotensin Ⅱ blockade decreases TGF-beta 1 and matrix proteins in cyclosporine nephropathy. Kidney Int 1997; 52: 660.
12. Blobe GC, Schiemann WP, Lodish HF. Role of transforming growth factor p in human diseases. N Engl J Med 2000; 342: 1350.
13. Bork P. The modullar architecture of a new family of growth regulators related to connective tissue growth factor. FEBS Lett 1993; 327: 125.
14. Yokoi H, Sugawara A, Mukoyama M, et al. Role of connective tissue growth factor in profibrotic action of transforming growth factor-beta: a potential target for preventing renal fibrosis. Am J Kidney Dis 2001; 38 (Suppl 1): S134.
15. Yokoi H, Mukoyama M, Sugawara A et al. Role of connective tissue growth factor in fibronectin expression and tubulointerstitial fibrosis.
Am J Physiol 2002; 282: F933.
16. Ito Y, Aten J, Bende RJ, et al. Expression of connective tissue growth factor in human renal fibrosis. Kidney Int 1998; 53: 853.
17. Rister BL, Denichilo M, Cortes P, et al. Regulation of connective tissue growth factor activity in cultured rat mesangial cells and its expression in experimental diabetic glomerulosclerosis. J Am Soc Nephrol 2000; 11:25.
18. Finckenberg P, Lassila M, Inkinen K, et al. Cyclosporine induces myocardial connective tissue growth factor in spontaneously hypertensive rats on high-sodium diet. Transplantation 2001; 71: 951.
19. Isaka Y, Akagi Y, Ando Y, et al. Gene therapy by transforming growth factor-β receptor-IgG Fc chimera suppressed extracellular matrix accumulation in experimental glomerulonephritis. Kidney Int 1999; 55: 465.
20. Imai E, Akagi Y, Isaka Y. Towards gene therapy for renal diseases. Nephrologie 1998; 19: 397.
21. Kon V, Hunley T, Fogo A. Combined antagonism of endothelin A/B receptors links endothelin to vasoconstriction whereas angiotensin II effects fibrosis. Studies in chronic cyclosporine nephrotoxicity in rats. Transplantation 1995; 60: 89.
22. Aihara H, Miyazaki J. Gene transfer into muscle by electroporation in vivo. Nature Biotechnol 1998; 16: 867.
23. Ma J, Nishimura H, Fogo A, Kon V, Inagami T, Ichikawa I. Accelerated fibrosis and collagen deposition develop in the renal interstitium of angiotensin type 2 receptor null mutant mice during ureteral obstruction. Kidney Int 1998; 53: 937.
24. Kaneto H, Morrissey J, McCracken R, Reyes A, Klahr S. Enalapril reduces collagen type IV synthesis and expansion of the interstitium in the obstructed kidney. Kidney Int 1994; 45: 1637.
25. Khanna AK, Cairns VR, Becker CG, Hosenpud JD. Transforming growth factor (TGF)-p mimics and anti-TGF-p antibody abrogates the in vivo effects of cyclosporine. Demonstration of a direct role of TGF-β in immunosuppression and nephrotoxicity of cyclosporine. Transplantation 1999; 67: 882.
26. Islam M, Burke JF, Jr, McGowan TA, Zhu Y, Dunn SR, McCue P, Kanalas J, Sharma K. Effect of anti-transforming growth factor-p antibodies in cyclosprine-induced renal dysfunction. Kidney Int 2001; 59: 498.
27. Thomas SE, Andoh TF, Pichler RH, et al. Accelerated apoptosis characterizes cyclosporine-associated interstitial fibrosis. Kidney Int 1998; 53: 897.
28. Wrana JL, Attisano L, Wieser R, Ventura F, Massague J. Mechanism of activation of the TGF-β receptor. Nature 1994; 370: 341.
29. Ling H, Li X, Jha S, Wang W, Karetskaya L, Pratt B, Ledbetter S.