- 學位論文
紅血球生成素與硫酸吲哚及纖維母細胞生長因子－23在貓慢性腎病與病程進展之相關性
The associations of plasma erythropoietin with indoxyl sulfate, fibroblast growth factor 23, and the progression in cats with chronic kidney disease
摘要
研究背景 隨著慢性腎臟病的病程進展，腎臟功能逐漸惡化會導致相關併發症如貧血、礦物質代謝與骨質異常，以及心血管疾病等。目前關於尿毒毒素、紅血球生成作用以及對於鈣磷平衡的重要因子（纖維母細胞生長因子－23）三者間的交互關係，已於細胞培養實驗、實驗鼠模型以及人類醫學中被闡述。 研究目的 由於在獸醫學領域仍缺乏相關實驗，故本實驗希望能對慢性腎病貓的血漿尿毒毒素（硫酸吲哚）、紅血球生成素以及纖維母細胞生長因子－23之交互關係進行探討，以及了解這些因子是否為貓腎臟疾病預後的指標。 研究對象 由於單次血漿檢體難以取得足量可同時檢測三者，故本實驗主要分成兩大組，分別進行硫酸吲哚－紅血球生成素（IS-EPO）組與紅血球生成素－纖維母細胞生長因子－23（EPO-FGF-23）組的檢測以了解二者的個別關連性。IS-EPO組中包含8隻健康貓與26隻慢性腎病貓，而慢性腎病貓中有3隻貧血，20隻為非貧血；另外亦區分出腎病惡化組（8隻）與非腎病惡化組（7隻）。EPO-FGF-23組中則包含7隻健康貓與15隻慢性腎病貓，而慢性腎病貓中有2隻貧血，9隻為非貧血；另外亦區分出低磷組（7隻）與高磷組（6隻）；腎病惡化組（3隻）與非腎病惡化組（6隻）。 實驗方法 以高效能液相層析儀（High Performance Liquid Chromatography）之螢光檢測法檢測貓血漿中硫酸吲哚之濃度，並以貓之商業化免疫分析套組分別檢測血漿中紅血球生成素與纖維母細胞生長因子－23之濃度。 實驗結果 與控制組相比，晚期慢性腎病貓有顯著較高的血漿硫酸吲哚的濃度（p=0.026），在腎病惡化組別也有較高濃度的趨勢（p=0.054），在經接收者操作特徵曲線（ROC）分析後顯示其具有預測腎病惡化之能力（AUC 0.804, p=0.049）。貓紅血球生成素的濃度在貓腎臟疾病組相對於控制組有上升的趨勢，但整體而言於控制組、早期腎病組與晚期腎病組間與高、低磷組間均無顯著差異，且在腎病惡化組與非腎病惡化組別間亦無達到顯著差異。而關於貓血漿中纖維母細胞生長因子－23的濃度，早期腎病組相較控制組有上升的趨勢，但整體而言於控制組、早期腎病組與晚期腎病組間與高、低磷組間均無顯著差異，且在腎病惡化組與非腎病惡化組別間亦無達到顯著差異，而以回歸分析血漿紅血球生成素與纖維母細胞生長因子－23後，於本實驗並未達到具有預測腎病惡化指標的能力。 另一方面，關於這三者與貓慢性腎病貧血的交互關係，血漿硫酸吲哚濃度於貧血與非貧血組別間並無顯著差異，然其與紅血球數之間呈現顯著負相關（p=0.040），亦與血紅素濃度（p=0.056）與血容比（p=0.083）之間具有負相關之趨勢。而在貓血漿紅血球生成素濃度的比較，雖然貧血組相較於非貧血組低，然未達顯著差異，其與紅血球相關參數之間的關係中，僅發現與平均紅血球容積具有顯著負相關（p=0.017）。最後，貓血漿纖維母細胞生長因子－23之濃度於貧血與非貧血組間未具有顯著差異，其與紅血球數成顯著正相關（Spearman: p=0.016, Linear: p=0.022），與平均紅血球容積具有顯著負相關（Spearman: p=0.011, Linear: p=0.004）。至於以這三者分別進行回歸分析預測貧血的發生之後，三者均未達到具有預測貓慢性腎病貧血的能力。 最後，關於慢性腎病貓此三者之間的相關性，本實驗結果顯示貓血漿硫酸吲哚與紅血球生成素濃度間並沒有顯著關聯性，然而在血漿紅血球生成素濃度與纖維母細胞生長因子－23之間則互相具有顯著正相關性（p=0.014）。 實驗結果與主要發現 本實驗為第一個使用專門偵測貓血漿紅血球生成素的免疫分析套組進行濃度檢測之研究。整體而言，於慢性腎病貓中，雖然未見血漿硫酸吲哚與紅血球生成素濃度的直接相關性，但顯示其與紅血球數及其他貧血相關指標（血紅素濃度、血容比）具有負相關性。此外，本實驗發現慢性腎病貓其血漿紅血球生成素與纖維母細胞生長因子－23間具有顯著正相關性。 關鍵字： 貓, 慢性腎病, 硫酸吲哚, 紅血球生成素, 纖維母細胞生長因子－23
並列摘要
Background As renal function deteriorates, several complications of chronic kidney disease will develop, such as anemia, cardiovascular diseases and disorders of minerals and bone. Associations between protein binding uremic toxins and erythropoiesis as well as fibroblast growth factor -23 have been reported through studies using cell culture, murine models, and in human medicine. Objectives Due to the lack of the related studies in the veterinary medicine, the aims of the current study are to elucidate the associations (1) between IS and erythropoietin, (2) between erythropoietin and FGF23, (3) among the three factors mentioned above, and (4) to determine whether these factors can serve as markers of progression in cats with chronic kidney disease. Samples All cases enrolled were separated into two groups for assessing possible correlations between IS, EPO (IS-EPO) and EPO, FGF23 (EPO-FGF23) in cats with CKD, respectively. In the IS-EPO group, 8 healthy cats and 26 CKD cats were enrolled. Cats with CKD were further grouped into the anemia group (n=3) and non-anemia group (n=20), and also the progression group (n=8) as well as the non-progression group (n=7). While in the EPO-FGF23 group, 7 healthy cats and 15 CKD cats were enrolled. Cats with CKD were further grouped into the anemia group (n=2) and non-anemia group (n=9), and also the progression group (n=3) as well as the non-progression group (n=6). Besides, cats with CKD in the EPO-FGF23 group were further classified into the higher phosphate group (n=6) and the lower phosphate group (n=6). Methods HPLC with fluorescence detector was used to measure feline plasma IS concentration. And commercial ELISA kits which were specific for cats were used to determine the plasma EPO and FGF23 concentration, respectively. Results Plasma IS concentration of cats with late stage CKD were significantly higher than the control group (p=0.026). Receiver operating characteristic curve (ROC) showed that plasma IS level was able to predict the progression in cats with CKD (AUC 0.804, p=0.049). Plasma EPO concentrations of cats with CKD were higher than the control group but without statistical significance. As for the feline plasma FGF23 concentration, cats with early stage CKD were higher than the control group, yet it didn’t reach statistical significance, either. The logistic regression analyses showed that plasma EPO and FGF23 weren’t able to predict the progression in cats with CKD. The associations of these three parameters and cats with anemia of CKD were analyzed. Plasma IS level correlated with RBC significantly and negatively (p=0.040), and it also showed negative correlation with hemoglobin (p=0.056) and HCT (p=0.083). The plasma EPO values were lower in the anemia group, yet it didn’t reach statistical significance. Plasma EPO concentration only correlated with MCV significantly and negatively (p=0.017) among the erythrocyte indices. Plasma FGF23 correlated with RBC significantly and positively (Spearman: p=0.016, Linear: p=0.022), and correlated with MCV significantly and negatively (Spearman: p=0.011, Linear: p=0.004). The logistic regression analyses showed that plasma IS, EPO, and FGF23 could not be able to predict the renal progression in cats. Lastly, for the association of each other in these three parameters, significantly positive association between plasma EPO and FGF23 levels was found (p=0.014). Conclusions and Clinical Importance The current study was the first study to determine the plasma EPO concentration by using ELISA kit specific for cats in cats with CKD. In conclusion, plasma IS correlated with erythrocyte indices negatively. And positive correlations between plasma EPO and FGF23 in cats with CKD were revealed. Moreover, our data suggested that there might be compensatory responses in the EPO production in the early stage of CKD in cats. Further research for elucidating the mechanisms is needed. Key word: Cat, chronic kidney disease, indoxyl sulfate, erythropoietin, fibroblast growth factor 23