| 摘要: | 背景：第二型糖尿病產生許多血管性併發症，包括心血管疾病、腦血管疾病、神經病病變、視網膜病變和腎臟病變。其中糖尿病腎臟病變是引起末期腎臟疾病的主要原因。當第二型糖尿病病人產生腎臟病變進而進入末期腎臟疾病時，不僅造成個人生活上的壓力，亦造成國家社會沈重的負擔。但是並非所有糖尿病病人皆會進展成糖尿病腎臟病變，只有一部分糖尿病病人會產生糖尿病腎臟病變，如何早期診斷糖尿病腎臟病變併介入治療是一門很重要的課題。
目前為止，有許多糖尿病腎臟病變的病理機轉被提出，而其中內質網壓力和發炎反應是最常被廣泛討論的。當細胞遭受高糖相關的氧化壓力時，於內質網內摺疊錯誤的蛋白將增多，這時內質網會啟動一連串的反應來消除摺疊錯誤的蛋白。但當摺疊錯誤的蛋白過多超出細胞負荷時，細胞將進行凋亡。當腎膈細胞在持續高糖環境之下，最初腎膈細胞的收縮將能力變差，進而影響腎臟的過濾能力，此時糖尿病腎臟病變會經過一段超過濾率時期。過去有研究發現內質網壓力相關的伴隨蛋白，Chaperonin-containing t-complex polypeptide 1 subunit ?? (TCP-1??)，是形成細胞骨架重要的結構之一，藉由細胞骨架形成進而影響細胞的收縮。在過去的研究中發現腎膈細胞在高糖環境下收縮力轉差扮演相當重要的角色，且於細胞培養液中，TCP-1?猁瑪@度顯著增加，但於體內糖尿病腎病變中的變化仍不清楚。
另外，第二型糖尿病的併發症與動脈粥狀硬化有關。血液中凝血系統與血栓溶解系統是平衡狀態，如果血栓溶解因子缺乏時，就容易引起血栓，而其中纖維溶解蛋白亦扮演此系統中重要的角色。纖維溶解蛋白包括組織型血纖維蛋白溶解酶原活化因子、尿激酶型血纖維蛋白溶解酶原活化因子及其接受體。另外尿激酶型血纖維蛋白溶解酶原活化因子接受體亦會被酵素從細胞膜上切下，形成溶解態尿激酶型血纖維蛋白溶解酶原活化因子接受體。許多研究發現溶解態尿激酶型血纖維蛋白溶解酶原活化因子接受體和發炎與敗血症有關，且參與癌症細胞的轉移。最近更有研究發現溶解態尿激酶型血纖維蛋白溶解酶原活化因子接受體參與局部性腎絲球硬化症的病生理機轉。因此，目前知道溶解態尿激酶型血纖維蛋白溶解酶原活化因子接受體與發炎反應有關，而且與某些腎臟疾病的成因相關，而第二型糖尿病腎病變與發炎反應亦有關係，但目前溶解態尿激酶型血纖維蛋白溶解酶原活化因子接受體在於糖尿病腎臟病變的角色仍不清楚。
最後第二型糖尿病形成除了胰島素阻抗增加外，胰島素分泌開始逐漸缺失，導致血糖升高而形成第二型糖尿病，而且最終必須依賴胰島素治療。在過去實驗中發現胰島細胞仍有再生能力，但在糖尿病病人的胰島細胞凋亡的速度遠大於再生的速度，導致胰島素分泌逐漸變差。有些研究發現尿激酶型血纖維蛋白溶解酶原活化因子不僅參與血栓溶解，也對於免疫調控與組織修復佔了重要的角色，但在胰島細胞的胰島素分泌與再生的功能是否參與其中，並與第二型糖尿病形成是否相關，仍未有研究被探討。
目的：首先，我們先建立第二型糖尿病小鼠的動物模式，並觀察TCP-1?狾b糖尿病小鼠的腎臟表現，以及測量糖尿病小鼠尿液TCP-1?猁瑪@度，另外亦收集第二型糖尿病病人的尿液，並比較有無糖尿病腎臟病變超過濾時期的尿液TCP-1?瓵@度差異。藉此研究是否能用尿液TCP-1?猁瑪@度作為糖尿病腎臟病變超過濾時期的生物標記。第二，我們亦收集各種腎臟疾病，包括糖尿病腎臟病變，的腎臟切片，並比較尿激酶型血纖維蛋白溶解酶原活化因子接受器在各種腎臟疾病的表現，另外測量各種腎臟疾病血液中溶解態尿激酶型血纖維蛋白溶解酶原活化因子接受器的濃度，並觀察並比較糖尿病腎臟病變各時期血液中溶解態尿激酶型血纖維蛋白溶解酶原活化因子接受器的濃度。藉此研究是否能用血液中溶解態尿激酶型血纖維蛋白溶解酶原活化因子接受器的濃度作為糖尿病腎臟病變不同分期的生物標記。藉由上面兩個研究的生物標記，我們便可以早期診斷糖尿病腎臟病變各個分期。最後，我們並研究尿激酶型血纖維蛋白溶解酶原活化因子在第二型糖尿病發生的角色，觀察尿激酶型血纖維蛋白溶解酶原活化因子基因剃除小鼠胰島素分泌與阻抗。我們亦調查尿激酶型血纖維蛋白溶解酶原活化因子在第二型糖尿病病人的濃度與胰島素分泌的相關性，藉此我們可了解尿激酶型血纖維蛋白溶解酶原活化因子在胰島素分泌與胰島細胞再生的角色，並在未來尋找出預防第二型糖尿病的策略。
材料與方法：研究一，為模擬臨床超過濾狀態，BALB/c小鼠接受高油脂飲食與streptozotocin和nicotinamide注射誘發第二型糖尿病。糖尿病小鼠血液和尿液於糖尿病發生第零、一、四週收集。腎臟組織亦收集為TCP-1?狶K疫組織化學染色與西方墨點法。同時，第二型糖尿病病人和健康對照組尿液亦收集並測量尿液中TCP-1?猁瑪@度。
研究二，各種不同腎臟疾病病人，包括微小病變腎病、糖尿病腎臟病變、膜性腎病變、慢性間質性腎炎、甲型球蛋白腎病變、紅斑性狼瘡腎炎、局部性腎絲球硬化症的病人腎臟中尿激酶型血纖維蛋白溶解酶原活化因子接受器的免疫組織化學染色表現與血液中溶解態尿激酶型血纖維蛋白溶解酶原活化因子接受器的濃度。並利用ROC曲線分析並評估不同時期糖尿病腎臟病變溶解態尿激酶型血纖維蛋白溶解酶原活化因子接受器濃度的適當切點。
研究三，我們比較BALB/c小鼠與尿激酶型血纖維蛋白溶解酶原活化因子基因剃除小鼠誘發第二型糖尿病的機率，並測量第二型糖尿病發生第0、3、30天的胰島素分泌與胰尿素阻抗，並比較胰島組織的免疫組織化學染色。另外，將尿激酶型血纖維蛋白溶解酶原活化因子質體種回尿激酶型血纖維蛋白溶解酶原活化因子基因剃除小鼠，並觀察第二型糖尿病誘導成功率。另外，使用siRNA將胰島細胞的尿激酶型血纖維蛋白溶解酶原活化因子表現沉默後，觀察胰島素分泌與胰島細胞生長狀況。最後，用口服葡萄糖耐量試驗測量第二型糖尿病病人胰島素分泌，並比較尿激酶型血纖維蛋白溶解酶原活化因子與胰島素分泌之關聯性。
結果：研究一，BALB/c小鼠在誘發糖尿病後隨時間腎絲球過濾率有顯著增加，腎臟TCP-1?狶K疫組織化學染色表現明顯增加，且TCP-1?狾銴颲氐I法在超過濾率時期於腎臟中的表現有意義增加。另外，糖尿病病人中尿液的TCP-1?瓵@度與腎絲球過濾率呈線性正相關，而超過濾率的糖尿病病人尿液中TCP-1?瓵@度有明顯增加。
研究二，比較各種腎臟疾病腎臟切片，尿激酶型血纖維蛋白溶解酶原活化因子接受器表現，和血液中溶解態尿激酶型血纖維蛋白溶解酶原活化因子接受器都明顯增加。其中糖尿病腎臟病變血中溶解態尿激酶型血纖維蛋白溶解酶原活化因子接受器濃度最高。另外，溶解態尿激酶型血纖維蛋白溶解酶原活化因子接受器濃度隨者糖尿病腎臟病變分期增加，我們並以ROC曲線算出各期糖尿病腎臟病變的適當切點。
研究三，尿激酶型血纖維蛋白溶解酶原活化因子基因剃除BALB/c小鼠在誘導糖尿病的成功率明顯增加。在誘導成功後30天，原型小鼠的胰島素分泌與胰島細胞有增加，但尿激酶型血纖維蛋白溶解酶原活化因子基因剃除小鼠卻沒增加。將尿激酶型血纖維蛋白溶解酶原活化因子質體種回尿激酶型血纖維蛋白溶解酶原活化因子基因剃除小鼠，沒有小鼠誘導成糖尿病。胰島細胞在使用siRNA將尿激酶型血纖維蛋白溶解酶原活化因子表現沉默後，胰島素分泌能力和細胞生長皆變差。第二型糖尿病病人血中尿激酶型血纖維蛋白溶解酶原活化因子與胰島素分泌能力呈正相關。
結論：糖尿病腎病變超過濾率狀態時，腎臟TCP-1?猁穛{和尿液中TCP-1?瓵@度增加。尿液中TCP-1?狴i成為糖尿病腎病變超過濾率狀態時的生物指標。而各種不同腎臟疾病中腎臟的尿激酶型血纖維蛋白溶解酶原活化因子接受器表現增加，且溶解態尿激酶型血纖維蛋白溶解酶原活化因子接受器在糖尿病腎臟病變隨著不同時期增加，溶解態尿激酶型血纖維蛋白溶解酶原活化因子接受器可成為糖尿病腎臟病變的生物指標。缺乏尿激酶型血纖維蛋白溶解酶原活化因子可能與第二型糖尿病發生有關，補充尿激酶型血纖維蛋白溶解酶原活化因子可能成為未來預防或治療第二型糖尿病的新的方法。
Background
Type 2 diabetic mellitus (DM) leads to many vascular complications, such as cardiovascular disease, cerebrovascular disease, neuropathy, retinopathy and nephropathy. Meanwhile, diabetic nephropathy (DN) is a leading cause of end-stage renal disease. When nephropathy in patients with type 2 DM progresses to end-stage renal disease, it results in both stress to the individual and a heavy socioeconomic burden for the country. However, not all patients with type 2 DM develop DN. Around one-thirds of type 2 DM patients develop DN and progress to end-stage renal disease. Therefore, early diagnosis of potential DN development is a very important issue.
Several pathophysiological mechanisms of DN have been indicated. Meanwhile, endoplasmic reticulum stress and inflammation response. When cells face hyperglycemia-related oxidative stress, misfolding proteins in endoplasmic reticulum increase. A series of adaptive responses in endoplasmic reticulum rise for removing misfolding proteins. However, when the amount of misfolding proteins overload, the cells progress to apoptosis. When glomerular mesangial cells are treated in a high glucose environment, hypocontractility of mesangial cells developed, which impairs the ability of filtration of glomerula. Glomerular hyperfiltration is the first stage of DN. Our previous study found endoplasmic reticulum stress-related chaperonin, chaperonin-containing t-complex polypeptide 1 subunit ?? (TCP-1??), may play an important role in mesangial cell hypocontractility in the development of DN. Increasing levels of TCP-1?? were noted in the cellular medium with high glucose. However, the role of TCP-1?? in DN is still unclear in the in vivo model.
Moreover, type 2 DM is associated with atherosclerosis. Normally, the systems of thrombosis and thrombolysis counterbalance in the blood. If the thrombolysis factors are defecting, the formation of thrombosis may occur. Fibrinolytic proteins play an important role in the process of atherosclerosis. Fibrinolytic proteins consist of tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA) and its receptor (uPAR). In addition, uPAR could be shed from the cellular membrane and form soluble uPAR (suPAR). Many studies show suPAR is related to inflammation and sepsis and contributes to metastasis of cancer cells. Recently, some research has shown suPAR contributes to the pathophysiology of focal segmental glomerulosclerosis. Accordingly, suPAR is associated with inflammatory response and causation of some kinds of kidney diseases. Type 2 DN is also associated with inflammation. So far, the role of suPAR on type 2 DN is still unknown.
Finally, not only increasing insulin resistance, but progressive impaired insulin secretion in patients with type 2 DM leads to hyperglycemia and eventual insulin therapy. Previous research showed regeneration of ?? cells persisted in the islet. In patients with type 2 DM, the rate of apoptosis in ?? cells is faster than that of regeneration, which contributes to impaired insulin secretion. Some studies showed uPA does not only participate in the process of fibrinolysis, but also in immune regulation and tissue repair. There are few studies exploring the effect of uPA on insulin secretion and regeneration of ?? cells, which may contribute to the development of type 2 DM
Objectives
For the specific aims, the following studies were designed. First, we established the diabetic mouse model to observe the expression of TCP-1?? in the kidney in type 2 DM. The concentration of TCP-1?? in the urine of the diabetic mouse was measured. In addition, urine in patients with type 2 DM was collected. The levels of TCP-1?? in urine in glomerular hyperfiltration in type 2 DM were compared. Second, we collected the renal biopsies from different common kidney diseases, including DN, and expression of uPAR in the kidney was measured. We also measured suPAR levels in different common kidney diseases and compared the differences among different stages of DN. In our third study, we investigated the role of uPA in development of type 2 DM. We explored the insulin secretion and resistance in the uPA knockout mouse. We also explored the association between uPA and insulin secretion in patients with type 2 DM.
Material and Methods
Study I: To mimic the clinical hyperfiltration state, a type 2 DM BLAB/c mice model was established by feeding a high-fat diet in combination with treatment of streptozotocin and nicotinamide. Blood and urine were collected at weeks 0, 1, 4 and kidney tissues were harvested for evaluation of TCP-1?? expression by immunohistochemistry (IHC) and Western Blot (WB). Meanwhile, clinical subjects including healthy controls and type 2 DM patients were recruited and their TCP-1?? levels in urine were measured.
Study II: Serum samples for suPAR and renal tissues for uPAR staining were investigated in various common kidney diseases, including minimal change disease, DN, membranous nephropathy, chronic interstitial nephritis, IgA nephropathy, lupus nephritis and focal segmental glomerulosclerosis. The levels of serum suPAR were measured and adequate cut-off values of different stage of DN were calculated by receiver operating characteristic (ROC) curve.
Study III: We explored the successful induction rate of type 2 DM between wild type and uPA knockout BALB/c mice. Insulin secretion and resistance were measured at days 0, 3, 30 after induction. Immunohistochemical stain of islet was investigated. In addition, uPA knockout mice were implanted with uPA plasmids. We also observed the successful induction rate of type 2 DM in these mice. Moreover, we explored the insulin secretion and replication of islet cell after silencing uPA by siRNA. Finally, we measured the insulin secretion in patients with type 2 DM by oral glucose tolerance test, and compared the association between uPA and insulin secretion.
Results
Study I: After induction of type 2 DM, the glomerular filtration rate in the BALB/c mice increased with time. The expression of TCP-1?? in renal tissue by immunohistochemical stain increased obviously. In addition, TCP-1?? in renal tissue by WB in hyperfiltration also increased significantly. In addition, levels of TCP-1?? in urine showed a linearly positive relation with glomerular filtration in patients with type 2 DM. Urine TCP-1?? levels significantly increased in patients with type 2 DM in the hyperfiltration phase.
Study II: In the renal biopsies in different common kidney diseases, uPAR expression increased obviously. The serum suPAR in different kidney diseases also increased, especially in DN patients. The level of suPAR increased with stages of DN. Optimal cut-off points of different stages of DN were assessed by ROC curve.
Study III: The successful induction rate in uPA knockout mice increased obviously. After induction for 30 days, the insulin secretion in wild type mice increased, but did not increase in the uPA knockout mice. After implanting uPA plasmids in uPA knockout mice, no mouse developed type 2 DM. However, in the in vitro study, the ability of insulin secretion and replication of islet cells was impaired after silencing uPA by siRNA. In clinical study, serum uPA was positively related to insulin secretion in patients with type 2 DM.
Conclusions
(1) Expression of TCP-1?? in renal tissue and urine TCP-1?? increased in the hyperfiltration phase in the type 2 DM mice. Urine TCP-1?? may be a biomarker for hyperfiltration in type 2 DM. (2) The uPAR expression in renal tissue and serum suPAR increased in different common kidney diseases. In addition, suPAR levels increased with stages of DN. Conclusively, suPAR may be a biomarker for DN. (3) Deficiency of uPA may be linked to development of type 2 DM. Supplementation of uPA might be a novel approach for prevention and treatment of type 2 DM in the future. |
