臺灣博碩士論文加值系統

Hemojuvelin在人類醫學當中是為新興、具發展性的腎病指標，人醫的研究發現能排入尿中之可溶性(soluble) hemojuvelin可作為缺血性或橫紋肌溶解性的急性腎損傷的早期生物標記，並且與neutrophil gelatinase-associated lipocalin (NGAL)具有相似的預測準確性，其上升也與腎臟內的病理性鐵沉積變化有關。但目前在小動物（如貓）腎病臨床還未有相關研究，而老年貓常見慢性腎臟病或繼發的急性腎損傷，因此探討hemojuvelin是否能穩定於犬貓尿中偵測、而又與貓隻腎病關聯為何成為本研究之目的。
首先以reducing Western blot確認犬貓尿中hemojuvelin的存在。接著，在2015年5月至2019年5月間，本實驗納入總計94隻貓，並且分成：（1）健康控制組（18隻）、（2）單純急性腎損病組 (acute kidney injury, AKI)（10隻）、（3）慢性腎病組 (chronic kidney disease, CKD)（45隻）、及（4）慢性繼發急性腎損傷組 (acute-on-chronic kidney disease, A-on-CKD)（21隻）等4組貓咪，再以實驗室設計之sandwich ELISA進行尿中濃度之測試，並以尿中肌酸酐(creatinine)做比值(urine hemojuvelin-creatinine ratio, UHCR)。
根據Western blot結果，犬貓尿中皆有2種分子量大小的hemojuvelin，分別在15-25kDa與25-35kDa之間，這與人類研究中Type II transmembrane serine protease (TMPRSS6)所切割出來之大小相同。氮血症貓咪的出現頻率較高，後期(意即CKD stage 3-4)的CKD貓咪尿中也會有較高的出現頻率。
實驗室自製的sandwich ELISA能穩定測量貓尿中hemojuvelin的濃度。健康貓咪hemojuvelin濃度及UHCR的中位數顯著比CKD、AKI與A-on-CKD貓咪低（濃度及中位數分別為33.374 [31.107, 36.668]、41.711 [37.596, 54.902]、39.735 [38.070, 76.812]、及50.084 [39.430, 62.946]，pg/mL；UHCR分別為0.096 [0.065, 0.127]、0.664 [0.256, 1.102]、0.764 [0.385, 1.246]、及0.895 [0.543, 1.571]，x10-7），但在各組氮血症之間則無顯著差異。後期CKD貓咪hemojuvelin濃度及UHCR的中位數均較早期(意即CKD stage 1-2)貓咪要顯著的高（濃度分別為55.334 [42.598, 106.304]及38.441 [35.653, 44.939]，pg/mL；UHCR中位數分別為1.102 [0.698, 2.703]及0.355 [0.207, 0.668]，x10-7）。
以receiver operating characteristic (ROC)分析顯示hemojuvelin濃度與UHCR能將健康貓咪與CKD、AKI及A-on-CKD貓之間做區分診斷（濃度臨界值分別為34.835、36.399及44.821 pg/mL；UHCR臨界值分別為0.224、0.286及0.272 x10-7）。早期與後期的CKD貓之間亦有具顯著意義的hemojuvelin濃度與UHCR適當臨界值作為診斷上的區分（分別為38.959 pg/mL及0.440 x10-7）。同時，CKD貓中有無惡化之間的比較，亦有具顯著意義的適當臨界值（分別為43.804 pg/mL及0.661 x10-7），且UHCR能用於預測貓CKD的惡化。
尿中hemojuvelin與UHCR為貓咪腎臟疾病之生物指標，能夠作為診斷CKD、AKI與A-on-CKD的標記，UHCR亦能預測慢性腎病（CKD）惡化之可能。

Hemojuvelin is a novel biomarker for renal diseases in human medicine. Soluble-type hemojuvelin in urine had been improved to be an early biomarker for ischemic or rhabdomyolysis acute kidney injury (AKI) in human. Its increase in urine concentration was proved to be related to pathological iron deposition in kidney. But it is lack of related researches in veterinary clinical medicine. The aim of this study focused on the presence of urine hemojuvelin in cats, and the relation between urine hemojuvelin and feline renal diseases.
Ninety-four cats were enrolled into the study from May 2015 to May 2019. They were grouped into: 1) healthy control group (n=18); 2) simple acute kidney injury (AKI) group (n=10); 3) chronic kidney disease (CKD) group (n=45); and 4) acute-on-chronic kidney disease (A-on-CKD) group (n=21).
According to the results of Western blot, there were 2 subtypes of hemojuvelin in feline and canine urine in size of 15-25 and 25-35kDa. They met the sizes produced by Type II transmembrane serine protease (TMPRSS6). The presence frequency was significantly higher in 3 azotemia groups than in control group. Cats with late stage of CKD (stage 3-4) also had significantly higher presence frequency than early stage of CKD (stage 1-2).
The homemade sandwich ELISA was developed and successful to measure the concentration of feline urinary hemojuvelin. The control cats significantly had lower median urinary hemojuvelin concentration and UHCR than CKD, AKI and A-on-CKD groups respectively (medians [IQR] urinary hemojuvelin concentration were 33.374 [31.107, 36.668], 41.711 [37.596, 54.902], 39.735 [38.070, 76.812], and 50.084 [39.430, 62.946] pg/mL respectively; medians [IQR] UHCR were 0.096 [0.065, 0.127], 0.664 [0.256, 1.102], 0.764 [0.385, 1.246], and 0.895 [0.543, 1.571] x10-7 respectively). But there were no significant differences between 3 azotemia groups. The cats with late stage of CKD had significantly higher urinary hemojuvelin concentration and UHCR than the cats with early stage of CKD (medians [IQR] urinary hemojuvelin concentration were 55.334 [42.598, 106.304] and 38.441 [35.653, 44.939] pg/mL; medians [IQR] UHCR were 1.102 [0.698, 2.703] and 0.355 [0.207, 0.668] x10-7).
Receiver operating characteristic (ROC) analyses showed that urinary hemojuvelin concentration and UHCR had ability for differentiation between healthy cats and CKD, AKI or A-on-CKD cats respectively (best cut-off values of urinary hemojuvelin concentration were 34.835, 36.399 and 44.821 pg/mL respectively; best cut-off values of UHCR were 0.224, 0.286 and 0.272 x10-7 respectively). Urinary hemojuvelin concentration and UHCR also could differentiate early and late stage of CKD in cats (38.959 pg/mL and 0.440 x10-7). UHCR also had ability for prediction of progression in feline CKD.
In conclusion, feline urinary hemojuvelin and UHCR as good biomarkers for diagnosis AKI, which is similar to the results of human patients, was confirmed in this study. Additionally, it was first found that they also performed well for the diagnosing CKD in cats. And UHCR was able to differentiate the progression of feline CKD as well.

口試委員會審定書 i
致謝 …………………………………………………………………………ii
摘要 ………………………………………………………………………...iii
ABSTRACT …………………………………………………………………………v
CONTENTS ………………………………………………………………………viii
LIST OF TABLES xiv
Chapter 1. Introduction 1
Chapter 2. Literature review 3
2.1. Introduction of hemojuvelin 3
2.1.1. Cellular and physiobiological sources of hemojuvelin 3
2.1.2. The structure of hemojuvelin 3
2.2. Function of hemojuvelin 4
2.2.1. Hemojuvelin-hepcidin-ferroportin axis for iron homeostasis 4
2.2.2. Infection, inflammation and hemojuvelin 6
2.2.3. Hypoxia and hemojuvelin 7
2.3. Renal diseases review 8
2.3.1. Infection and renal diseases 8
2.3.2. Inflammation and renal diseases 9
2.3.3. Hypoxia and renal diseases 10
2.3.4. Iron metabolism and renal diseases 11
2.4. Clinical application of hemojuvelin in human medicine 11
2.4.1. Juvenile hereditary hemochromatosis 11
2.4.2. Renal diseases and hemojuvelin 12
Chapter 3. Materials and methods 15
3.1. Patients and sample collection 15
3.1.1. Sample collection and storage 15
3.1.2. Control group 15
3.1.3. Cases enrollment and exclusion criteria 16
3.1.4. Acute kidney injury (AKI) group 16
3.1.5. Chronic kidney disease (CKD) group 17
3.1.6. Acute-on-chronic kidney disease (A-on-CKD) group 18
3.1.7. Exclusion of acute kidney disease (AKD) 18
3.2. Preparation of recombinant hemojuvelin protein and its antibody for both canine and feline 20
3.2.1. Expression of recombinant hemojuvelin protein 20
3.2.2. Preparation of crude protein extract 23
3.2.3. Purification of recombinant hemojuvelin protein 24
3.2.4. Dialysis of crude extracted recombinant hemojuvelin protein 25
3.2.5. Concentrated the recombinant hemojuvelin protein 26
3.2.6. Detection of the hemojuvelin recombinant protein by SDS-PAGE 26
3.2.7. Preparation of anti-hemojuvelin antibodies 27
3.2.8. Quantitation of hemojuvelin recombinant protein concentration 27
3.3. Detection of hemojuvelin subtypes(s) in urine by Western blot analysis 29
3.3.1. Sample preparation 29
3.3.2. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 29
3.3.3. Western blotting analysis 30
3.4. Establishment of sandwich ELISA for detecting concentration of hemojuvelin in urine 31
3.4.1. Detection of hemojuvelin in feline urines by sandwich ELISA 31
3.4.2. Evaluation of the precision of in-house ELISA 32
3.5. Statistical analyses 34
Chapter 4. Results 35
4.1. Patients and samples collection 35
4.2. Production of antibodies against hemojuvelin 36
4.2.1. Quantitative analysis of the purified recombinant hemojuvelin protein 36
4.2.2. Production of antibodies 37
4.3. Detection of hemojuvelin in urine samples by Western blotting 38
4.3.1. Confirmations of subtypes of hemojuvelin in canine and feline urine samples 38
4.4. Establishment of sandwich ELISA for detecting hemojuvelin in urine samples ……………………………………………………………………………..39
4.4.1. Establishment of standard curves for hemojuvelin in urine samples 39
4.4.2. The precision of homemade sandwich ELISA 39
4.5. Statistical analysis 41
4.5.1. Comparisons of different urinary hemojuvelin subtypes between control group and azotemia group 41
4.5.2. Comparisons of different urinary hemojuvelin subtypes between groups 41
4.5.3. Comparisons of different urinary hemojuvelin subtypes between early stage of CKD group and late stage of CKD group 41
4.5.4. Comparisons between control group and azotemia group 43
4.5.5. Comparisons among all groups 46
4.5.6. Comparisons among different stages of CKD 51
4.5.7. Comparisons between different levels of urine hemojuvelin concentration and UHCR 56
4.5.8. Correlations between urine hemojuvelin, UHCR and other biochemical parameters 59
4.5.9. Receiver operating curve (ROC) analyses 61
4.5.10. Receiver operating curve (ROC) analyses of early CKD stage group and late CKD group 64
4.5.11. Receiver operating curve (ROC) analyses between CKD group and AKI group and A-on-CKD group respectively 66
4.5.12. Comparisons between progression and non-progression cases in CKD group …………………………………………………………………………..68
4.5.12.1. Receiver operating curve (ROC) analyses of progression group and non-progression group 71
4.5.12.2. Logistic regression analyses for different parameters associated with CKD progression in cats 73
4.5.12.3. Kaplan-Meire analyses and progression of CKD 76
4.5.12.4. Cox analyses and progression of CKD 78
Compared to univariate model, the multivariate Cox model analysis indicated that u-Hemojuvelin and UHCR were significantly associated with an increase in the hazard ratio for the progression. (Table 23) 78
Table 23 . Cox proportional hazard regression model for CKD progression. 78
Chapter 5. Discussion 79
5.1. Presence frequency of urinary hemojuvelin and feline renal diseases 79
5.2. Urinary hemojuvelin concentration between cats with different renal diseases ……………………………………………………………………………….80
5.3. Urinary hemojuvelin and cats with different stage of CKD 82
5.4. Correlations of urinary hemojuvelin and clinicopathology parameters 83
5.5. Urinary hemojuvelin and CKD progression in cats 86
5.6. Limitations 86
Chapter 6. Conclusions 88
References ………………………………………………………………………..89

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