- 學位論文
第四型視黃醇結合蛋白受體訊息傳遞與血脂異常和糖尿病引起腎臟傷害之研究
The Study of Retinol Binding Protein 4 Receptor Signaling in Dyslipidemia and Diabetes-Induced Renal Injury
摘要
糖尿病腎臟病變是我國末期腎衰竭病人類最主要原因，它的病理發生學是多種因素造成。流行病學研究顯示新陳代謝症候群在糖尿病腎臟病變的發生和進行扮演重要的角色。臨床研究指出糖尿病患者血液中視黃醇結合蛋白與脂質異常、高血糖引發粒腺體功能障礙或O-GlcNA醣化轉譯後修飾作用具有相關性。目前糖尿病相關研究多聚焦於視黃醇結合蛋白濃度上升對於糖尿病併發症的影響，但是對於STRA6表現與功能性對於糖尿病併發症的影響之研究依然不完整，特別是在腎臟病變的部分，因此本研究嘗試探討視黃醇結合蛋白受體在糖尿病腎臟病變相關分子機制之影響。 首先本研究發現非視黃醇結合態之視黃醇結合蛋白(apo-RBP4)與STRA6的親合力較視黃醇結合態之視黃醇結合蛋白(holo-RBP4)高，造成apo-RBP4過度刺激STRA6調控之JAK2/STAT5訊息途徑，與活化 cAMP/JNK與p38MAPK訊息途徑最後造成人類腎小管上皮與血管內皮細胞凋亡。而apo-RBP4與STRA6結合，同時抑制維生素A代謝相關蛋白CRBP1 與RAR表現。若透過crbp1基因轉殖可有效逆轉apo-RBP4之效應。再者本研究發現在代謝症候群患者血液陰電性最高的低密度脂蛋白L5可以透過其細胞膜受體LOX1誘發JNK與p38MAPK細胞訊息傳遞途徑抑制STRA6/CRBP1/RARs細胞訊息傳遞途徑與維生素A代謝，造成腎臟細胞細胞凋亡與纖維化。然而透過crbp1基因轉殖可有效逆轉L5之效應。同時本研究在糖尿病小鼠腎臟上發現RBP4與STRA6的結合狀況低下、視黃醇與視黃酸含量下降、CRBP1/RARα途徑弱化與粒腺體功能障礙，同時細胞凋亡與纖維化表現上升。透過抑制ROS生成與crbp1基因轉殖可逆轉上述效應。本研究也發現在糖尿病小鼠(db/db)與肥胖小鼠(ob/ob)腎臟中STRA6與RALDH 發生O-GlcNc轉譯後修飾作用，同時CRBP1/LRAT/CRABP/RARs 途徑也發生異常弱化。透過抑制O-GlcNc轉譯後修飾作用酵素OGT或crbp1基因轉殖能有效抑制上述效應。由上述結果可知非視黃醇結合態之視黃醇結合蛋白、陰電性低密度脂蛋白與高血糖皆透過干擾STRA6調控之維生素A運輸與代謝途徑，造成腎臟細胞傷害。同時crbp1基因轉殖可有效修復STRA6途徑與維生素A代謝，進行細胞保護作用。 本研究透過三種研究模式發現在糖尿病腎臟病變致病機轉中，視黃醇結合蛋白受體途徑是調控細胞維生素A代謝與生物效應 ，維持腎臟細胞抗逆境生理的重要系統。透過了解調控與傷害視黃醇結合蛋白受體途徑機轉，可提供未來開發糖尿病腎臟病變相關治療策略與藥物參考之用。
並列摘要
Daibetic nephropathy has been the leading cause of ESRD in Taiwan. Many studies indicate the pathologensis of diabetic nephropathy are associated with metabolic syndrome. Some studies show serum RBP4 associated with hyperglycemia and dyslipidemia-trigger mitochondria dysfunction and O-GlcNAc modification in nephropathy. In addition, clinic studies focus to investigate the association between elevaton of RBP4 and diabetic complication. However, there have been few studies to investigate role of RBP4 receptor, STRA6 in pathologensis of diabetic nephropathy. Thus, this study first investigate molecular mechanism of STRA6 in pathologensis of diabetic nephropathy This study first showed that the increased apo-/holo-RBP4 ratio delayed the displacement of RBP4 with STRA6, highly enhanced JAK2/STAT5 cascade, up-regulated enhanced cAMP/JNK and p38 cascade, and suppressed CRBP-I/RARα expression, and leaded to apoptosis in vitro. However, crbp1 gene transfection could revese apo-RBP4 effects. Second, this study showed that L5 from metabolic syndrome suppressed STRA6 signaling, and simultaneously induced apoptosis and fibrosis in kidneys through LOX1/JNK and p38 signaling. In additon, crbp1 gene transfection could reverse L5 effects. Third, this study showed that hyperglycemia decreased binding activity of RBP4 with STRA6, impaired STRA6 cascades and retinoid homeostasis, and caused mitochondrial dysfunction, and renal injury. However, ROS inhibition and crbp1 gene transfection contributed to repair STRA6 cascades, retinol metabolism, and mitochondria under high glucose stimulation. Furthermore, this study showed that hyperglycemia decreased binding activity of RBP4 with STRA6, impaired STRA6 cascades and vitamin A metabolism, and induced apoptosis and fibrosis while O-GlcNAc modification of STRA6 and RALDH in kidney of db/db and ob/ob mice. In additon, inhibition of O-GlcNAc modification and crbp1 gene transfection could efficiently inhibit O-GlcNAc modification of STRA6 and RALDH and repair STRA6 cascades and vitamin A metabolism under high glucose condition. In conclusion, this study indicates that apo-RBP4, electronegative LDL, and hyperglycemia-triggered mitochondria dysfunction and O-GlcNAc modification contributes aberrant STRA6 cascade and vitamin A hemostasis to drive the development of diabetic kidney disease. Specially, the crbp1 gene transfections can revesed effects of apo-RBP4, electronegative LDL, and hyperglycemia in vitro through repairing STRA6 cascade and vitamin A metabolism. This study showed three of pathological factor in diabetic nephropathy contributes pathogenesis of kidney through aberrant RBP4/ STRA6 cascades. These results might contribute to understand role of RBP4/ STRA6 cascades in pathogenesis of diabetic nephropathy and provide new direction to develop strategy of therapy or drug for diabetic nephropathy.