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The expression pattern and concentration of urinary hemojuvelin in feline kidney diseases
|Publication Year :||2019|
|Abstract:||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。
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.
|Appears in Collections:||臨床動物醫學研究所|
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