背景：糖尿病腎病變是常見的糖尿病發症之一，其特徵在於腎小球硬化和腎小管間質纖維化，病人在高血糖的情況下透過異常調控腎臟中的蛋白質表現及鈣離子恆定進而導致組織病理改變。然而，在病患高血糖的條件下藉由非酶反應產生還原蛋白糖和氨基之複合物稱為糖化終產物，先前研究顯示，糖化終產物會增加Fibronectin和type IV collagen最終導致腎纖維化，且糖化終產物也可以通過CHOP/IRE1訊息傳遞路徑引起的腎臟內質網壓力。Calbindin-D28k參與調節鈣的再吸收，並在細胞內促進鈣離子的擴散且在鈣離子轉運的過程中扮演關鍵的角色。在大鼠的糖尿病模式中，Calbindin-D28k顯著增加於腎小管上皮細胞中的特別在集合管、遠曲小管和近曲小管。另有研究指出，Calbindin-D28k蛋白過度表現可保護環孢菌素造成的腎毒性。然而，Calbindin-D28k在糖尿病腎病或糖化終產物相關的腎臟纖維化中的作用尚不明確，因此在本篇研究中欲探討Calbindin-D28k在糖尿病腎病中扮演的角色，特別是糖化終產物誘導的腎近曲小管細胞腎臟纖維化及內質網壓力的影響。
材料與方法：人類近端腎小管細胞轉染siCalbindin-D28k後接續處理AGEs (100 μg/ml)達48小時後，透過西方墨點法觀察蛋白質的表現情況，並利用MTT試驗測試轉染後的細胞毒性。並利用免疫組織化學染色法觀察Calbindin-D28k在db/db小鼠腎臟的表現情形。
結果：組織病理變化的結果顯示，與對照小鼠相比db / db糖尿病小鼠的腎臟明顯觀察到AGEs以及Calbindin-D28k表現在腎小管的位置。在細胞實驗的部分，AGEs顯著促使Calbindin-D28k、Connective tissue growth factor (CTGF)、Fibronectin及Receptor for AGE (RAGE)表現量上升。然而，腎小管上皮-間質轉分化(EMT)已被確認在腎纖維化的過程中扮演關鍵的角色。AGEs同時也可上調節Vimentin及下調節E-cadherin，最後促使細胞EMT。然而利用抗體中和RAGE的受體，阻斷其下游的訊息傳遞後，Fibronectin、CTGF 、Calbindin-D28k及EMT相關的蛋白表現皆被抑制。然而當細胞轉染siCalbindin-D28k後，會促進AGEs誘導的Fibronectin、CTGF及EMT相關的蛋白表現，綜合上述，Calbindin-D28k在AGEs誘發的腎纖維化中具有保護作用，主要是透過促進RAGE下游相關的訊息傳遞路徑。另一方面，轉染Calbindin-D28k也促進CHOP、GRP78及IRE1的增加的蛋白質表達，因此，Calbindin-D28k同時也在AGEs誘導的內質網壓力下具保護作用。此外，利用4-苯基丁酸（4PBA）處理HK-2細胞以確認腎纖維化和內質網壓力之間的關係，4PBA作為一種化學伴隨蛋白，可以抑制內質網壓力的發生，在實驗中也發現，4PBA可阻斷AGEs造成的內質網壓力產生，同時抑制腎臟的纖維化。
結論：本篇的研究結果顯示，在糖尿病的情況下增加Calbindin-D28k的表現具有保護細胞損害的作用，若將其表現阻斷，則會引發更嚴重的內質網壓力，進而產生更嚴重的腎小管間質纖維化。

Backgrounds：Diabetic nephropathy, characterized by glomerulosclerosis and tubulointerstitial fibrosis, is a common diabetic complication associated with alterations in the expression of some renal proteins and abnormal calcium homeostasis associated with the development of characteristic histopathological features. Advanced glycation end-products (AGEs) are produced from non-enzymatic reactions between reducing sugars and amino groups of proteins under hyperglycemic conditions in diabetes mellitus. Previous studies showed that AGEs increased the synthesis of fibronectin and type IV collagen and finally caused renal fibrosis. AGEs can also induced renal ER-stress via CHOP/IRE1 signaling pathway. Calbindin-D28k is supposed to be involved in the regulation of the reabsorption of calcium and played a pivotal role in the process of calcium transport by facilitating the diffusion of calcium through the cell. Recent animal study demonstrated that Calbindin-D28k expression was markedly increased in tubular epithelial cells of distal convoluted tubules, collecting ducts, and proximal convoluted tubules in diabetic kidney. The present findings demonstrated that Calbindin-D28k protein expression may play a protective role in the kidneys during cyclosporine nephrotoxicity. However, the role of Calbindin-D28k in diabetic nephropathy or AGEs-related renal fibrosis remains unclear. In this study, we would like to investigate whether Calbindin-D28k plays a role in diabetic nephropathy especially in AGEs-related renal fibrosis and ER stress in renal proximal tubule cells.
Materials & Methods：Human renal proximal tubule cell (HK-2) were transfected with scramble or Calbindin-D28k siRNA and then treated with AGEs (100 μg/ml) for 48 hours. The expressions of several critical proteins were determined by Western blotting. Cell viability was evaluated by MTT assay. The expression of Calbindin-D28k in renal tissue of db/db mice were detected by immunohistochemistry.
Results：Histopathological changes, advanced glycation end-products (AGEs), and Calbindin-D28k were obviously observed in the kidneys of db/ db diabetic mice as compared with the control mice. AGEs significantly increased protein expressions Calbindin-D28k, fibrotic factors including fibronectin and connective tissue growth factor (CTGF) and receptor for AGEs (RAGE). Tubular epithelial-to-mesenchymal transition (EMT) is recognized to play a pivotal role in the process of renal fibrosis. AGEs significantly regulated the EMT markers by up-regulation of Vimentin and down-regulation of E-cadherin protein expressions. AGEs-induced protein expressions of fibronectin, CTGF and Calbindin-D28k and promotion of cell EMT by AGEs in HK-2 cells were significantly suppressed by RAGE neutralizing antibody. Transfection with Calbindin-D28k-siRNA augmented the increased protein expressions of fibronectin, CTGF and promote cell EMT in AGEs-treated renal tubular cells. These results suggested that Calbindin-D28k plays a protective role in AGEs-induced renal fibrosis via RAGE-related signal pathway. On the other hand, transfection with Calbindin-D28k-siRNA also promoted the increased protein expressions of CHOP, GRP78 and IRE1. These results suggested that Calbindin-D28k plays a protective role in AGEs-induced ER stress. Further, HK-2 cells were treated with 4-phenylbutyric acid (4PBA) to confirm the role of renal fibrosis and ER stress.4PBA, a chemical chaperone, significantly reduced the ER stress and also block the fibrotic protein expression.
Conclusion：These findings suggest that increased Calbindin-D28k plays an important protective role in decreasing the rate of AGEs-induced renal fibrosis via ER stress during diabetic condition.

口試委員會審定書 I
誌謝 II
Abstract VI
Abbreviation Summary IX
1.1. Diabetes mellitus 1
1.2. Diabetic nephropathy 3
1.2.1 Pathophysiology of diabetic nephropathy 3
1.2.2 Role of proximal tubule cells in diabetic nephropathy 4
1.2.3 Implication of proximal tubule cells fibrosis in diabetic nephropathy 5
1.3. Advanced glycation end-products (AGEs) 6
1.3.1 Role of advanced glycation end products (AGEs) in diabetic nephropathy 6
1.3.2 Involvement of AGEs in proximal tubule cells injury 8
1.4. Calbindin D-28K 9
1.4.1 Physiological role of Calbindin D-28k 9
1.4.2 Relationship between Calbindin-D28k and kidney injury 10
1.4.3 Calbindin-D28k plays a protection role in some disease 11
1.5 Endoplasmic reticulum stress (ER stress) 12
1.5 The interaction between ER stress and fibrosis 13
Part 2: Aims 15
Part 3: Materials and Methods 16
3.1 Cell culture 16
3.1 AGE-BSA preparation 16
3.3 Estimation of cell viability 17
3.4 Protein extraction and Western blot analysis 17
3.5 Blockade of RAGE-ligand interaction assay 18
3.6 RNA interference 19
3.7 Animals 19
3.8 Immunohistochemistry 20
3.9 Statistical analysis 20
Part 4: Results 21
4.1 In vitro and in vivo effects of AGEs on Calbindin-D28k expression. 21
4.2 Calbindin-D28k siRNA in virto delivery enhanced AGEs-induced renal fibrosis 22
4.3 The increases of protein expressions of fibrotic markers and Calbindin-D28k by AGEs in HK-2 cells were significantly suppressed by RAGE neutralizing antibody 23
4.4 Calbindin-D28k siRNA in virto delivery enhanced HK-2 cells ER stress 24
4.5 4-phenylbutyric acid (4PBA) reversed AGE-induced ER stress and fibrosis in proximal tubule cells 25
Part 5: Discussion 27
Part 6: Conclusion 31
Part 7: Figures and figure legends 33
Figure 1. Effects of AGEs on protein expressions of Calbindin-D28K in human renal proximal tubule cell. 33
Figure 2. Immunohistochemical staining for AGEs in the kidneys of diabetic db/db mice. 34
Figure 3. Immunohistochemical staining for Calbindin-D28k in the kidneys of diabetic db/db mice. 35
Figure 4. Effects of AGEs on protein expressions of Calbindin-D28K in mouse mesangial cell and Human Embryonic Kidney cells. 36
Figure 5. Transfection of siCalbindin-D28k downregulated AGEs-induced Calbindin-D28k protein expression. 37
Figure 6. Transfection of siCalbindin-D28k downregulated AGEs-induced Calbindin-D28k protein expression in a dose-dependent manner. 38
Figure 7. Effects of HK-2 cells treated with/without AGEs on siCalbindin-D28k on cell viability. 39
Figure 8. Effects of HK-2 cells treated with AGEs on siCalbindin-D28k and protein expressions of α-SMA, CTGF and Fibronectin in HK-2 cells. 40
Figure 9. Effects of HK-2 cells treated with AGEs on siCalbindin-D28k and protein expressions of E-cadherin, Vimentin and snail in HK-2 cells. 41
Figure 10. Effects of AGEs on protein expressions of RAGE in human renal proximal tubule cell. 42
Figure 11. RAGE blocking suppressed and Calbindin-D28k knockdown enhanced AGEs-induced expression of RAGE and Calbindin-D28K in cultured renal proximal tubular cells. 43
Figure 12. RAGE blocking suppressed and Calbindin-D28k knockdown enhanced AGEs-induced expression of CTGF and Fibronectin in cultured renal proximal tubular cells. 44
Figure 13. Effects of HK-2 cells treated with AGEs on siCalbindin-D28k and protein expressions of p-p38, p38, p-smad2/3 and smad2/3 in HK-2 cells. 45
Figure 14. RAGE blocking suppressed and Calbindin-D28k knockdown enhanced AGEs-induced expression of Vimentin and E-cadherin in cultured renal proximal tubular cells. 46
Figure 15. Effects of HK-2 cells treated with AGEs on siCalbindin-D28k and protein expressions p-p65 and p65 in HK-2 cells. 47
Figure 16. Effects of HK-2 cells treated with AGEs on siCalbindin-D28k and protein expressions of CHOP and GRP78 in HK-2 cells. 48
Figure 17. Effects of HK-2 cells treated with AGEs on siCalbindin-D28k and protein expressions of IRE1, P-eIF2α and eIF2α in HK-2 cells. 49
Figure 18. 4-PBA, a chemical chaperon, reduces ER stress and Calbindin-D28k knockdown enhanced AGEs-induced expression of CHOP and GRP78 in cultured renal proximal tubular cells. 50
Figure 19. 4-PBA, a chemical chaperon, reduces ER stress and Calbindin-D28k knockdown enhanced AGEs-induced expression of PERK and p-JNK in cultured renal proximal tubular cells. 51
Figure 20. 4-PBA, a chemical chaperon, reduces ER stress and Calbindin-D28k knockdown enhanced AGEs-induced expression of α-SMA, Fibronectin and Vimentin in cultured renal proximal tubular cells. 53
Part 8：References 55

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