周祖述Jou, Tzuu-Shuh臺灣大學:分子醫學研究所邱麗敏Chio, Li-MinLi-MinChio2010-05-042018-07-092010-05-042018-07-092009U0001-2907200918120600http://ntur.lib.ntu.edu.tw//handle/246246/178742腎病症候群常伴隨腎絲球上皮細胞也就是足樣細胞(podocyte)的損傷，足樣細胞損傷則會造成尿蛋白的產生。尿蛋白是臨床上基本用來評估腎功能的一個指標。病理情況下，尿液中出現足樣細胞也已經被研究證實和某些腎病症候群有關聯。Podocalyxin (PC)是足樣細胞表現在其頂部細胞膜的一類蛋白，而此蛋白也被認為是足樣細胞必要的一項生化標記，判斷足樣細胞出現在尿液中的數目多寡即是透過染PC存在與否的細胞來決定的。在此我們想研究PC本身出現在尿液中量的多寡是否和腎絲球病變或和腎病病人預後的表現有所關聯，而之前的研究曾顯示PC可以低密度膜囊泡(low-density membrane vesicles)的形式出現在尿液中，在我們研發適合偵測尿中PC的capture ELISA kit過程也發現要將尿中的PC萃取出來需要界面活性劑的參與，這和前者的發現相當一致。本研究中，我們致力於研發針對偵測尿中PC的capture ELISA kit，而後將其應用在臨床上診斷為腎病症候群病人的尿液測試，包括膜性腎病(membranous nephropathy, MN), 微小病變腎病(minimal change disease, MCD)及局部性腎絲球硬化症(focal segmental glomerulosclerosis, FSGS)，結果發現在MCD/FSGS組中未治療前的尿中PC明顯低於MN組(mean ± SEM: 2.0 ± 0.6, 5.7 ± 1.6, P< 0.05)，而在治療後6個月MCD/FSGS組的尿中PC有顯著的上升(0月:2.0 ± 0.6 , 6月: 4.3 ± 1.8, P< 0.1)，我們對此現象的解釋為MCD和FSGS在臨床上同屬足樣細胞病變的腎病症候群，在診斷時足樣細胞的足突(foot processes)消失常是顯見的特徵，PC是已知在維持足突結構上為必要的蛋白，在確認病症前PC很可能早已流失於尿液中。隨著療程時間愈久，足樣細胞大量表現PC來重建足突構造，而因此也使得尿液中的PC量有一個明顯上升的結果。Nephrotic syndrome, one of the most common manifestations of renal disorders encountered in daily nephrological clinics, is characterized with profound proteinuria. Proteinuria usually herald significant glomerular epithelial cell or podocyte injury. Podocyte presence in urine has been linked to some specific glomerular disease under several pathologic conditions. Podocalyxin (PC) is expressed at apical plasma membrane of podocyte and has been considered to contribute to the organization of foot processes as well as glomerular charge barrier through its extensive negative charge. We observed that through ELISA analysis urine sample from patients with diabetic nephropathy showed a relative higher PC level than that from healthy controls. We thus investigated if presence of the essential podocyte-associated membrane protein or PC itself in urine could represent the correlation with glomerular diseases or with patients’ prognosis. In this study we established a delicate capture ELISA kit specific to detect urinary PC. During the establishment we found to extract urinary PC required detergent contribution in ELISA assay. This might respond to Pisitkun et al. study that PC could reach urine through low-density membrane vesicles. After applying the custom-made capture ELISA kit to test urine samples from patients with clinically characterized nephrotic syndromes, including membranous nephropathy (MN), minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS), PC level was observed to be significantly lesser in MCD/FSGS group than in MN group at basal time (mean ± SEM: 2.0 ± 0.6, 5.7 ± 1.6, P< 0.05) before medical treatment. In addition PC level showed an increase at sixth month in MCD/FSGS group (2.0 ± 0.6 at 0 month, 4.3 ± 1.8 at 6 month, P< 0.1) during treatment course. In MN group this undulation of PC level was not observed. We explained the alteration of urinary PC level in MCD/FSGS group should account for the histological podocyte injury. In podocyte disease group, PC loss in the urine might be a prior step than the occurrence of foot processes impairment and its observation through biopsy. After patients received therapy, vigorous PC expression by podocyte might direct its sudden increase in urine due to the fact that plausible unstable and incomplete recovery of podocyte status yet.Table of contentable of content 1文摘要 4bstract 5ntroduction 7aterials and Methods 11rine samples collection 11usion protein construct 12estern blotting 12apture Enzyme-linked immunosorbent assay (ELISA) 13tatistical Analysis 14esults 15odocalyxin Structure and Antibodies used for detection of urinary PC and recombinant protein 15equirement of Detergent to detect Urinary Podocalyxin 16he Coating Buffer Ingredients 17he Incubation Buffer Ingredients 18he Blocking Buffer Ingredients 19ensitivity and Specificity of the established capture ELISA 20orrelation with clinical characteristic of nephrotic syndrome and urinary PC 21iscussion 23eferences 30igures and tables 33ig. 1. Wild type and recombinant podocalyxin diagram 33ig. 2. Both mAb 3D3 and pAb a-PCC’recognized dN-PC and urinary PC 34ig. 3. Detergent is required for extracting podocalyxin from urine specimen 35 ig. 4. Homemade coating buffer containing carbonate-bicarbonate buffer is comparable to that of commercial kit 37ig. 5. Triton X-100 is more efficient than Tween-20 in extracting urinary podocalyxin 38ig. 6. Blocking buffer comprised of 1% gelatin is most effective in drawing the difference between controls and patients 39ig. 7. Quantitative measurement of purified standard protein dN-PC 41ig. 8. The linear range of the sensitivity of the established capture ELISA 42ig. 9. Flowchart of the established capture ELISA 44ig. 10. Higher podocalyxin level in steroid resistant group in MN and MCD/FSGS groups after treatment for a year 45ig. 11. Process of exosome formation and release into the urine 46able I. Clinical information of patients with nephrotic syndrome 47able II. The urinary PC level changes (shown as PC/Creatinine ug/mg) in response to treatment 48application/pdf901006 bytesapplication/pdfen-US酵素連結免疫吸附分析法足樣細胞podocalyxin腎病症候群膜性腎病微小病變腎病ELISApodocyteexosomenephrotic syndromemembranous nephropathyminimal change disease[SDGs]SDG3http://ntur.lib.ntu.edu.tw/bitstream/246246/178742/1/ntu-98-R96448001-1.pdf