P-glycoprotein在第一型及第二型糖尿病小鼠腎臟表現量及功能之探討

Abstract

糖尿病是一種多樣性的代謝症候群，會造成全身性的代謝疾病，進而引起視網膜、腎臟、神經和心血管方面的病變。研究指出P-glycoprotein豐富地存在於腎臟中，且與藥物等異質體的代謝有密切關係。本篇研究主要是探討在糖尿病的情形下，P-gp蛋白質的表現量與功能是否會受到影響，而先前的研究指出，在腦部caveolin-1、2蛋白質的表現會影響P-gp的表現量與功能性，因此我們也探討caveolin-1、2蛋白質是否會對腎臟中的P-gp造成影響。 本研究所使用的自發性第一型和第二型糖尿病動物模式分別為NOD (non-obese diabetic) 和NZO (New Zealand obese) 小鼠，而對照組則是使用NON (non-obese, non-diabetic) 小鼠。從西方墨點法與組織免疫染色的結果顯示不論在第一型糖尿病NOD母鼠或是第二型糖尿病NZO公鼠的腎臟，其P-gp表現量皆明顯低於對照組NON小鼠。 針對caveolin-1、2蛋白質而言，其西方墨點法的結果顯示，不論在NOD母鼠或是NZO公鼠，其表現量也呈現減少的趨勢，就其組織免疫染色結果，我們可以觀察到caveolin-1、2主要分布在微血管和腎遠端小管基底側，其表現位置與P-gp並無相關，因此推論在腎臟中caveolin-1、2對於P-gp的表現量與功能可能沒有太大的影響性。我們進行小鼠腎臟刷狀絨毛膜微粒 (BBMV)的萃取，加以研究P-gp受質digoxin與腎臟BBMV攝取量的多寡，分析糖尿病對腎臟P-gp功能的影響性。實驗結果顯示糖尿病小鼠腎臟的BBMV對於P-gp受質digoxin攝取量有顯著的上升，顯示出糖尿病小鼠腎臟中P-gp的功能是下降的。 在後續的實驗中，我們使用經過基因轉殖MDR1之犬腎小管上皮細胞MDCK-MDR1，作為研究糖尿病與腎臟細胞P-gp表現量之體外試驗，研究在高血糖和高胰島素環境下，是否會造成腎臟細胞株P-gp表現量的改變。將萃取的細胞膜蛋白作西方墨點法試驗，其結果顯示給予高濃度葡萄糖和胰島素皆會造成MDCK-MDR1細胞株P-gp表現量下降。 本篇研究的結果顯示，不論在自發性第一型或是第二型糖尿病小鼠的腎臟中，P-gp的表現量與功能是顯著下降的，而就體外細胞試驗的結果顯示，高血糖的環境下會降低P-gp蛋白質的表現量，但詳細機轉與調控因子則需更進一步的研究。

Diabetes mellitus is a systematic metabolic disease and is related to a number of complications including retinopathy, nephropathy, neuropathy, and vascular diseases. Recent studies showed that diabetic nephropathy is a leading cause of end-stage kidney disease. Some reports indicated that P-gp is abundant in the kidney, and is closely related to the excretion and metabolism of xenobiotics and drugs. Moreover, previous studies showed that the levels of caveolin affect the expression and function of P-gp in the brain. Therefore, the current study is to investigate the relationship between caveolin and P-gp in the kidney of diabetes mellitus. Inbred non-obese diabetic (NOD) and New Zealand obese (NZO) mice were used as the models of type 1 and type 2 diabetes, respectively, and non-obese non-diabetic (NON) mice were used as control. Western blots and immunohistochemical staining showed that protein expressions of P-gp and caveolin were both significantly lower in the kidney of male NZO and female NOD mice than that of male and female NON mice. In addition, immunohistochemical staining indicated that P-gp is localized to the apical membrane of proximal tubule, while caveolin is localized to the endothelial cells and the basolateral membrane of the distal tubular. These results suggested that P-gp expression in the kidny may not be directly affected by caveolin. Then, we assessed the function of P-gp by using mice renal brush-border membrane vesicles (BBMVs) and digoxin was used as a substrate of P-gp. The result indicated that the uptake of digoxin in diabetic mice were higher than that in NON mice. Thus, we concluded that the activity of P-gp is decreased in the kidney of diabetic mice. To investigate the correlation between P-gp and hyperglycimia, MDCK-MDR1 cells were used as an in vitro renal model and treated with high concentration of glucose and insulin. The results showed that high concentration of glucose and insulin can reduce the expression of P-gp. In conclusion, the expression and activity of P-gp are decreased in the diabetic mice. Further studies are required to understand the regulatory pathway and mechanism of renal P-gp.