糖控蛋白78 與環孢素導成之近端腎小管空泡
形成之相關性

研究目的：自從環孢素的使用，可減少移植的排斥，增加器官移植的成功率，使器官移植能廣泛的臨床應用。但環孢素可促成腎毒性導致腎臟移植後的腎病變。在環孢素促成的腎病變中，其中最明顯的是促成近端腎小管空泡的形成。在文獻中有報告，環孢素促成的腎小管空泡是源自內質網。內質網聯繫細胞核及細胞質，是蛋白質合成及摺疊的地方，當細胞受到壓力時蛋白質不能正常的摺疊，出現內質網壓力，內質網壓力影響正常細胞生長，造成細胞死亡。但文獻上對環孢素做成的腎小管空泡並無深入的研究。本研究的目的在探討環孢素做成腎小管空泡的因素，並針對環孢素對內質網壓力的相關研究。研究方法及材料：本研究包括動物活體實驗，及腎小管細胞株之試管實驗。動物活體實驗是利用斯普拉-道來大鼠進行實驗組給予環孢素口服 30mg/kg/day 七天以觀察近端腎小管空泡之形成，在對照組方面給予相對的生理食鹽水做為對照組以觀察是否有腎小管之形成。在解救組方面先給予環孢素後七天後停藥一個月，以觀察停藥後腎小管空泡之現象是否消失。動物活體實驗是利用組織切片及蛋白質學以研究空泡現象之原因。試管內實驗以近端腎小管細胞株NRK52E 進行，觀察環孢素細胞株的空泡之形成，研究環孢素與內質網及內質網壓力之相關伴侶蛋白之表現，並利用基因嵌除及基因敲入方法進行相關蛋白質與環孢素做成空泡因素以及環孢素對內質網壓力的研究。研究結果：動物實驗中，在研究組中環孢素可導致近端腎小管空泡之形成，腎小管空泡在腎臟成條紋形狀。在解救組停藥物後28 天，環孢素做成之空泡消失。利用蛋白學研究，我們發現在實驗組中的腎臟有糖控蛋白78 有超表達的現象。利用特殊染色的組織切片，我們發現，糖控蛋白78 主要是表現在環孢素促成的空泡中。在解救組停藥後 28 天，糖控蛋白78 也隨同空泡的消失而下降。利用腎小管細胞株NRK52E 的實驗，我們發現環孢素可做成細胞株的空泡形成，同時發現空泡中主要成份為糖控蛋白78。利用干擾性小核糖核酸做為糖控蛋白78 的基因剔除可抑制環孢素導致的空泡形成。但抑制糖控蛋白78 的同時，會增加細胞的死亡。如果單獨把糖控蛋白78 的基因敲入NRK52E 細胞株也會造成腎小管細胞株空泡的形成，如果同時插入含有糖控蛋白78 基因的干擾性小核糖核酸可抑制空泡的形成。環孢素與內質網壓力的研究中，我們發現環孢素的腎毒性與內質網壓力之細胞質侶伴蛋白無關包括HSP70、HSP40、HSP90、和HSP60。但環孢素與內質網中摺疊錯誤系統有關，環孢素會增加蛋白摺疊錯誤系統的感測蛋白質包括PTF6、TRE1 和CHOP。其中最重要的是增加控制摺疊錯誤系統的糖控蛋白78 有明顯的表達現象。結論：環孢素促成的腎小管空泡是一種可逆現象。糖控蛋白78 是促成環孢素空泡的主要要素。抑制糖控蛋白78 可抑制環孢素腎小管空泡的形成。但抑制糖控蛋白78 的同時會增加細胞的死亡。糖控蛋白 78 是內質網中控制蛋白摺疊錯誤系統的主要侶伴蛋白。故我們推論，當環孢素做成內質網壓力時腎小管細胞出現糖控蛋白78 會超表達的現象，做成空泡的形成，可能是與維持蛋白摺疊錯誤系統的平衡，及維護細胞的生長有關。

關鍵字

環孢素；糖控蛋白78 ；內質網

並列摘要

Introduction: Since the usage of cyclosporine (CsA) in transplantation, it decrease the incidence of acute rejection and is widely used in organ transplantation. On the other hand, cyclosporine has adverse effect of nephrotoxicity, which could lead to chronic allograft nephropathy in renal transplantation patients. Among the nephrotoxicity, cyclosporine induced vacuolization in proximal renal tubule cell, which was endoplasmic reticulum (ER) in origin. Endoplasmic reticulum is the organelle where protein is produced and folding for maturation. When unfolding protein occurs, it could produce endoplasmic stress, which may lead to cell death. However, the pathogenesis of cyclosporine induced tubular vacuolization (TV) has never been well studied before. The aim of the present study is to study the pathogenesis of cyclosporine induced tubule vacuolization and relation to endoplasmic stress. Method. In the present study, both in vivo and in vitro study was used. In-vivo study, cyclosporine induced acute nephrotoxicity model in Sprague Dawley rats was used. In study the group, cyclosporine 30mg/kg/day was given orally for 7 days and observed for the formation of tubule vacuolization. In control group, same amount of normal saline was given orally for 7 days. In rescue study group, cyclosporine 30mg/kg/day was feed orally for 7 days, and stopped for 28 and observed for the changes of cyclosporine induced vacuolization. We used histochemistry and proteomic method to analyze the cyclosporine induced vacuolization. In-vitro study, proximal renal cell line NRK52E was used in our study. We analyze the effect of cyclosporine induced vacuolization in relation to the chaperones of endoplasmic stress. Using gene insertion and knockout method, we study the importance of chaperones, which is related to cyclosporine induced TV. Results In our acute cyclosporine nephrotoxicity model, cyclosporine induced tubule vacuolization in a stripped pattern in the proximal renal tubule. After stopping the drug for 28 days, rescue therapy group resolution of the tubule vacuolization. In proteomic study, we found that Immunoglobulin binding protein/ glucose regulate protein-78 (Bip/Grp78) was overexpression in our study group. Histochemical stain showed that Bip/Grp78 was overexpression in the cyclosporine induced vacuolization. After cyclosporine stopped for 28 days, Bip/Grp78 was also decrease in association of the tubule vacuolization. In our study, CsA induced vacuolization was found in proximal tubule cell line NRK52E. Using confocal microscope and fluorescence stain, we found that Bip/Grp78 was overexpression in CsA induced vacuolization. Bip/Grp78 specific shRNA was inserted into the NRK52E cell, inhibit cyclosporine induced vacuolization formation. In addition, suppression of Bip/Grp78 enhanced CsA induced cell death. Insertion Bip/Grp78 gene into NRK52E alone could induce vacuolization formation, which was inhibited by Bip/Grp78 specific shRNA. NRK52E cell treated with cyclosporine induced endoplasmic stress involved ER integrated stress response (ISR) chaperones, ATF6, IRE1 and CHOP, but not cytoplasmic ER stress related chaperones-HSP70, HSP 40, HSP27, HSP90 and HSP60. Conclusion In summary, we demonstrated that cyclosporine induced tubule vacuolization was a reversible process in which Bip/Grp78 over-expression is essential for TV formation. Suppression of Bip/Grp78 inhibited cyclosporine induced vacuolization formation in association of cell death. Since Bip/Grp78 was an importance endoplasmic chaperone involving unfolding protein response, it is possible that Bip/Grp78 expression and TV formation may be involved in cellular defense mechanism against CsA nephrotoxicity.

並列關鍵字

Cyclosporine ； Glucose regulation protein 78 ； endoplasma reticulum

參考文獻

1. Naeans M. Kuyper DRJ and Sarwal M. Calcineurin inhibitor nephrotoxicity CJASN 2009;4;481-508.

2. Ponticelli C: De novo thrombotic microangiopathy. An underrated complication of renal transplantation. Clin Nephrol 67: 335–340, 2007

3. Campistol JM , Sacks SH. Mechanisms of nephrotoxicity . Transplanation 2000;69:SS5-SS10.

4. English J, Evan A, Houghton DC, Bennet WM. Cyclosporine induced acute renal dysfunction in rat. Evidence of arteriolar vasoconstriction with preservation of tubular function. Transplantation 1987;44:135-141

5. L’Azou B, Medina J, Frieauff W, Cordier A, Cambar J, Wolf A. In vitro models to study mechanisms involved in cyclosporine A mediated glomerular contraction. Arch Toxicol 1999:73:337-345.

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糖控蛋白78 與環孢素導成之近端腎小管空泡形成之相關性

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