Title
ラット腎障害モデルにおけるシスタチンCのバイオマーカ
ーとしての有用性および腎臓内局在に関する研究( 要約版
(Digest) )
Author(s)
冨樫, 裕子
Report No.(Doctoral
Degree)
博士(獣医学) 乙第140号
Issue Date
2016-03-14
Type
博士論文
Version
none
URL
http://hdl.handle.net/20.500.12099/54518
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Ặ ྡ ᶔ ⿱ Ꮚ 㢟 ┠ ࣛࢵࢺ⭈㞀ᐖࣔࢹࣝ࠾ࡅࡿࢩࢫࢱࢳࣥC ࡢࣂ࣐࣮࣮࢜࢝ࡋ࡚ࡢ᭷⏝ᛶ ࠾ࡼࡧ⭈⮚ෆᒁᅾ㛵ࡍࡿ◊✲ ࢩࢫࢱࢳࣥC ࡣศᏊ㔞 13 kDa ࡢ࣌ࣉࢳࢻ࡛㸪ᜏᖖⓗ㌟ࡢ᭷᰾⣽⬊ࡽ⾑୰ศἪࡉࢀ㸪ࡑࡢ ࢇࡀ⭈⣒⌫య࡛ࢁ㐣ࡉࢀ㸪㏆ᒀ⣽⟶ࡢ྾࠾ࡼࡧ␗ࢆཷࡅࡿࠋࡇࡢࡓࡵ㸪㏆ᒀ⣽⟶ࡀ㞀ᐖ ࡉࢀࡿࢩࢫࢱࢳࣥC ࡢ྾ࡀ┤᥋ⓗᢚไࡉࢀ㸪⣒⌫యࡀ㞀ᐖࡉࢀࡿཎᒀ₃ࢀฟࡓ⏕యෆ㧗ศ Ꮚࡼࡾ྾ࡀ➇ྜⓗ㜼ᐖࡉࢀ㸪ᒀ୰ࢩࢫࢱࢳࣥC ࡣቑຍࡍࡿࠋ㏆ᖺ㸪ᒀ୰ࢩࢫࢱࢳࣥ C ࡣ㸪㏆ ᒀ⣽⟶㞀ᐖ࠾ࡼࡧ⣒⌫య㞀ᐖࡢ୧᪉ࢆ᳨ฟࡍࡿ᪂つ⭈㞀ᐖࣂ࣐࣮࣮࢜࢝ࡋ࡚ὀ┠ࡉࢀ࡚࠸ࡿࡀ㸪᭷ ⏝ᛶ࠾ࡼࡧ≉ᚩࡘ࠸࡚༑ศゎ᫂ࡉࢀ࡚࠸࡞࠸ࠋᮏ◊✲࡛ࡣ㸪ᒀ⣽⟶㞀ᐖࢆኚࡢయࡍࡿᛴᛶ⭈ 㞀ᐖࣔࢹࣝ㸪⣒⌫య㞀ᐖࢆኚࡢయࡍࡿᛴᛶ⭈㞀ᐖࣔࢹࣝ࠾ࡼࡧ៏ᛶ⭈⮚ࣔࢹࣝࡢ୍ࡘ࡛࠶ࡿ⢾ ᒀᛶ⭈ࣔࢹࣝࢆ⏝࠸࡚㸪ᒀ୰ࢩࢫࢱࢳࣥC ࡢኚືࢆ᳨ウࡋ㸪ᚑ᮶ᆺࣂ࣐࣮࣮࢜࢝㸦⾑୰ࢡࣞࢳ ࢽࣥ㸪ᒀ⣲❅⣲㸧࠾ࡼࡧࡑࡢࡢ᪂つ⭈㞀ᐖࣂ࣐࣮࣮࢜࢝㸦ᒀ୰β2 ࣑ࢡࣟࢢࣟࣈࣜࣥ, ࢝ࣝࣅࣥࢹ ࣥ㸪ࢡࣛࢫࢸࣜࣥ㸪EGF㸪GST-α㸪GST-μ㸪KIM-1㸪NGAL㸪࢜ࢫࢸ࣏࢜ࣥࢳࣥ㸪TIMP-1㸧ẚ㍑ࡋ㸪ᒀ ୰ࢩࢫࢱࢳࣥC ࡢ⭈㞀ᐖࣂ࣐࣮࣮࢜࢝ࡋ࡚ࡢ᭷⏝ᛶ࠾ࡼࡧ≉ᚩࢆ᫂ࡋࡓࠋࡉࡽ㸪ࢩࢫࢱࢳࣥ C ࡢ⭈⮚ෆᒁᅾ⭈㞀ᐖࡢ㛵㐃ᛶࡘ࠸࡚㸪ච⤌⧊Ꮫⓗ᫂ࡋࡓࠋ ➨1 ❶࠾࠸࡚㸪ᒀ⣽⟶㞀ᐖࢆኚࡢయࡍࡿᛴᛶ⭈㞀ᐖࣔࢹࣝࡋ࡚⏝࠸ࡓ CDDP ㄏⓎᛴᛶ⭈㞀 ᐖࣛࢵࢺ࡛ࡣ㸪ᒀ୰ࢩࢫࢱࢳࣥC ࡣ㸪Day 1 ࡽቑຍࡋࡓࠋ⾑୰ࢡࣞࢳࢽࣥ࠾ࡼࡧᒀ⣲❅⣲ࡣ Day 3 ࡽቑຍࡋ㸪⌮⤌⧊Ꮫⓗࡣ㸪㏆ᒀ⣽⟶ࡢቯṚ࡞ࡢ㞀ᐖᛶኚࡀDay 3 ࡽࡳࡽࢀ㸪Day 5㹼Day7 ࡛㢧ⴭ࡞ࡗࡓࠋᚑࡗ࡚㸪ᒀ୰ࢩࢫࢱࢳࣥC ࡣ CDDP ㄏⓎᛴᛶ⭈㞀ᐖࡢ᪩ᮇ᳨ฟ᭷⏝࡛࠶ࡿࡇࡀ ♧ࡉࢀࡓࠋࡲࡓ㸪ᒀ୰KIM-1㸪GST-α ࠾ࡼࡧ EGF ࡶ Day 1 ࡽኚືࡋ㸪⭈㞀ᐖࡢ᪩ᮇ᳨ฟ᭷⏝࡛㸪≉ ᒀ୰KIM-1 ࠾ࡼࡧ GST-α ࡣቑຍ⋡ࡢⅬ࡛ᒀ୰ࢩࢫࢱࢳࣥC ࡼࡾඃࢀࡿࡇࡀ♧ࡉࢀࡓࠋ ➨2 ❶࠾࠸࡚㸪⣒⌫యኚࢆయࡍࡿᛴᛶ⭈㞀ᐖࣔࢹࣝࡋ࡚⏝࠸ࡓᢠ GBM ⣒⌫య⭈⅖ࣛࢵࢺ ࡛ࡣ㸪ᒀ୰ࢩࢫࢱࢳࣥC ࡣ Day 7 ࡛ቑຍࡋࡓࠋ⾑୰ࢡࣞࢳࢽࣥ࠾ࡼࡧᒀ⣲❅⣲ࡣDay 7 ࡲ࡛ࢇ ኚືࡏࡎ㸪ᢠGBM ⣒⌫య⭈⅖ࡢデ᩿࣐࣮࣮࡛࢝࠶ࡿᒀ୰ࣝࣈ࣑ࣥ࠾ࡼࡧ⥲ࢱࣥࣃࢡࡣ Day 7 ࡛ቑຍ ࡋࡓࠋ⌮⤌⧊Ꮫⓗࡣ㸪Day 3 ࡽ⣒⌫యࡢ⣽⬊ቑከ࠾ࡼࡧ༙᭶ᙧᡂࡀ㍍ᗘࡳࡽࢀ㸪Day 7 ࡛㢧ⴭ ࡞ࡗࡓࠋᚑࡗ࡚㸪ᒀ୰ࢩࢫࢱࢳࣥC ࡣ㸪ᚑ᮶ᆺࣂ࣐࣮࣮࢜࢝ẚ㍑ࡋ࡚㸪ᢠ GBM ⣒⌫య⭈⅖ࡢ᪩ᮇ ᳨ฟ᭷⏝࡛࠶ࡿࡇࡀ♧ࡉࢀࡓࡀ㸪ᒀ୰ࣝࣈ࣑ࣥ࠾ࡼࡧ⥲ࢱࣥࣃࢡᑐࡍࡿඃᛶࡣࡳࡽࢀ࡞ࡗ ࡓࠋᒀ୰β2 ࣑ࢡࣟࢢࣟࣈࣜࣥ㸪ࢡࣛࢫࢸࣜࣥ㸪GST-α ࠾ࡼࡧ GST-μࡣ㸪ᒀ୰ࢩࢫࢱࢳࣥC ྠࡌࡃ Day 7 ࡛ቑຍࡋࡓࠋᒀ୰ KIM-1 ࠾ࡼࡧ NGAL ࡣDay 3 ࡽቑຍࡋ㸪ᢠ GBM ⣒⌫య⭈⅖ࡢ᪩ᮇ᳨ฟᒀ୰ࢩ ࢫࢱࢳࣥC ࡼࡾඃࢀࡿࡇࡀ♧ࡉࢀࡓࠋ
➨3 ❶࠾࠸࡚㸪៏ᛶ⭈⮚ࣔࢹࣝࡋ࡚⏝࠸ࡓ⢾ᒀᛶ⭈ࢆⓎࡍࡿ ZDF ࣛࢵࢺ࡛ࡣ㸪ᒀ୰ࢩࢫ ࢱࢳࣥC ࡣᑐ↷࡛࠶ࡿ lean ࣛࢵࢺẚ㍑ࡋ࡚ 12 㐌㱋ࡽ㧗್࡛࠶ࡾ㸪㐌㱋౫Ꮡࡋ࡚ቑຍࡋࡓࠋ⾑୰ ࢡࣞࢳࢽࣥ࠾ࡼࡧᒀ⣲❅⣲ࡣ12㹼25 㐌㱋࡛ࢇኚࡋ࡞ࡗࡓࠋ⌮⤌⧊Ꮫⓗࡣ㸪12 㐌㱋࡛ ࡣ㍍ᗘࡢ⣒⌫యኚࡀࡳࡽࢀࡿࡢࡳ࡛࠶ࡗࡓࡀ㸪20 㐌㱋࡛ࡣ⣒⌫య㞀ᐖ࠾ࡼࡧᒀ⣽⟶㞀ᐖࡀ㢧ⴭ࡞ࡾ㸪
25 㐌㱋࡛ቑᝏࡋࡓࠋᚑࡗ࡚㸪ᒀ୰ࢩࢫࢱࢳࣥ C ࡣ㸪ᚑ᮶ᆺࣂ࣐࣮࣮࢜࢝ẚ㍑ࡋ࡚⢾ᒀᛶ⭈ࡢ ᪩ᮇ᳨ฟ࠾ࡼࡧែᢕᥱ᭷⏝࡛࠶ࡿࡇࡀ♧ࡉࢀࡓࠋࡲࡓ㸪⢾ᒀᛶ⭈ࡢ᭱ࡶឤᗘࡢࡼ࠸デ᩿࣐࣮ ࣮࢝ࡋ࡚⏝࠸ࡽࢀࡿᒀ୰ࣝࣈ࣑ࣥࡣ㸪lean ࣛࢵࢺẚ㍑ࡋ࡚ 12 㐌㱋ࡽ㧗್࡛࠶ࡾ㸪㐌㱋౫Ꮡ ࡋ࡚ቑຍࡋࡓࡇࡽ㸪ᒀ୰ࢩࢫࢱࢳࣥC ࠾ࡼࡧࣝࣈ࣑ࣥࡢ᭷⏝ᛶࡣྠ⛬ᗘ⪃࠼ࡿࠋᒀ୰ β2 ࣑ࢡ ࣟࢢࣟࣈࣜࣥ㸪ࢡࣛࢫࢸࣜࣥ㸪GST-μ㸪KIM-1 ࠾ࡼࡧ࢜ࢫࢸ࣏࢜ࣥࢳࣥࡶࡲࡓ㸪12 㐌㱋࡛ lean ࣛࢵࢺ ẚ㍑ࡋ࡚㧗್࡛࠶ࡾ㸪㐌㱋౫Ꮡࡋ࡚ቑຍࡋࡓࠋࡇࢀࡽࡣ㸪ᒀ୰ࢩࢫࢱࢳࣥC ྠᵝ㸪⢾ᒀᛶ⭈ ࡢ᪩ᮇ᳨ฟ࠾ࡼࡧែᢕᥱ᭷⏝࡛࠶ࡿࡇࡀ♧ࡉࢀࡓࠋ ࡉࡽ㸪➨1㸪2 ࠾ࡼࡧ 3 ❶࠾࠸࡚㸪CDDP ㄏⓎᛴᛶ⭈㞀ᐖࣛࢵࢺ㸪ᢠ GBM ⣒⌫య⭈⅖ࣛࢵࢺ࠾ࡼ ࡧZDF ࣛࢵࢺ࡛ࡣ㸪ࢩࢫࢱࢳࣥ C ࡣ㏆ᒀ⣽⟶ᒁᅾࡋ㸪⭈㞀ᐖࡢ㐍⾜㛵ࢃࡽࡎ㸪ࢩࢫࢱࢳࣥ C ච ᰁⰍᛂᛶ࠾ࡼࡧ㝧ᛶ㡿ᇦࡣࢇኚࡋ࡞࠸ࡇࡀ♧ࡉࢀࡓࠋ ௨ୖ㸪ᒀ୰ࢩࢫࢱࢳࣥC ࡣ㸪ᒀ⣽⟶㞀ᐖ࠾ࡼࡧ⣒⌫య㞀ᐖࡢ࠸ࡎࢀࢆኚࡢయࡍࡿᛴᛶ⭈㞀ᐖ㸪 ࡞ࡽࡧ៏ᛶ⭈⮚ࡢ୍ࡘ࡛࠶ࡿ⢾ᒀᛶ⭈ࡢ᪩ᮇ᳨ฟ᭷⏝࡛࠶ࡾ㸪⢾ᒀᛶ⭈ࡢែᢕᥱࡀྍ ⬟⪃࠼ࡿࠋᒀ୰ࣝࣈ࣑ࣥ࠾ࡼࡧ⥲ࢱࣥࣃࢡẚ㍑ࡍࡿ㸪⣒⌫య㞀ᐖࢆኚࡢయࡍࡿᛴᛶ⭈㞀 ᐖ࠾ࡼࡧ⢾ᒀᛶ⭈࠾࠸࡚㸪ᒀ୰ࢩࢫࢱࢳࣥC ࡢ᭷⏝ᛶࡣࡑࢀࡽྠ⛬ᗘ⪃࠼ࡿࠋ᪂つ⭈㞀ᐖࣂ ࣐࣮࣮࡛࢜࢝࠶ࡿᒀ୰KIM-1 ࡣ㸪ᒀ୰ࢩࢫࢱࢳࣥ C ྠᵝ㸪3 ࡘࡢࢱࣉࡢ⭈㞀ᐖࡢ᪩ᮇ᳨ฟ࠾ࡼ ࡧ⢾ᒀᛶ⭈ࡢែᢕᥱ᭷⏝࡛࠶ࡿࡇࡀ♧ࡉࢀࡓࠋᒀ୰ β2 ࣑ࢡࣟࢢࣟࣈࣜࣥࡣ㸪ᒀ୰ࢩࢫࢱࢳ ࣥC 㢮ఝࡢኚືࢆ♧ࡋࡓࠋࡑࡢ㸪ᒀ⣽⟶㞀ᐖኚࡢయࡍࡿᛴᛶ⭈㞀ᐖ࡛ࡣGST-α ࠾ࡼࡧ EGF㸪 ⣒⌫య㞀ᐖࢆኚࡢయࡍࡿᛴᛶ⭈㞀ᐖ࡛ࡣࢡࣛࢫࢸࣜࣥ㸪GST-α㸪GST-μ ࠾ࡼࡧ NGAL ࡀ᪩ᮇ᳨ฟ ᭷⏝࡛࠶ࡾ㸪⢾ᒀᛶ⭈࡛ࡣࢡࣛࢫࢸࣜࣥ㸪GST-μ ࠾ࡼࡧ࢜ࢫࢸ࣏࢜ࣥࢳࣥࡀ᪩ᮇ᳨ฟ࠾ࡼࡧែ ᢕᥱ᭷⏝࡛࠶ࡿࡇࡀ♧ࡉࢀࡓࠋࡉࡽ㸪ࢩࢫࢱࢳࣥC ࡣ㸪⭈⮚࠾࠸࡚㸪ච⤌⧊Ꮫⓗ㏆ᒀ ⣽⟶ᒁᅾࡋ㸪ࢩࢫࢱࢳࣥC චᰁⰍᛂᛶ࠾ࡼࡧ㝧ᛶ㡿ᇦࡣ㸪ᒀ⣽⟶㞀ᐖ࠾ࡼࡧ⣒⌫య㞀ᐖࡢ࠸ࡎࢀ ࢆኚࡢయࡍࡿᛴᛶ⭈㞀ᐖ࡞ࡽࡧ⢾ᒀᛶ⭈ࡢ㐍⾜㛵㐃ࡋ࡞࠸ࡇࡀ♧ࡉࢀࡓࠋ
Ꮫ ㄽ ᩥ せ ⣙
Ặ ྡ TOGASHI, Yuko
㢟 ┠ Studies on the Usefulness of the Cystatin C as a Renal Biomarker and its Localization in Kidney
㸦ࣛࢵࢺ⭈㞀ᐖࣔࢹࣝ࠾ࡅࡿࢩࢫࢱࢳࣥ C ࡢࣂ࣐࣮࣮࢜࢝ࡋ࡚ࡢ᭷⏝ ᛶ࠾ࡼࡧ⭈⮚ෆᒁᅾ㛵ࡍࡿ◊✲㸧
Cystatin C, a peptide of molecular weight 13 kDa, is produced and secreted to blood at a constant rate by all karyocytes, freely filtrated from blood into glomeruli, and completely reabsorbed and catabolized in proximal renal tubules. Due to these features, tubular damage directly reduces reabsorption of cystatin C to the proximal renal tubules, and glomerular alterations competitively inhibits tubular uptake of cystatin C by the leakage of high molecular weight protein, which causes the increase in urinary level. Recently, urinary cystatin C has attracted attention as a novel biomarker for detection of both proximal renal tubular and glomerular damage, however, the usefulness and characteristics have not been investigated enough. In this study, using acute kidney injury (AKI) model whose main lesion was renal tubular injury, AKI model whose main lesion was glomerular injury, and diabetic nephropathy model, one of chronic kidney disease (CKD) models, we measured urinary cystatin C level and compared it to the conventional biomarkers (blood creatinine, urea nitrogen (UN)) and the other novel biomarkers (β2-microglobulin, calbindin, clusterin, EGF, GST-α, GST-μ, KIM-1, NGAL, osteopontin, and TIMP-1) in order to clarify the usefulness and characteristics of urinary cystatin C as a renal biomarker for damage. In addition, the relationship between the immunohistochemical localization of cystatin C in kidney and progression of renal damage was evaluated.
In Chapter 1, CDDP-induced AKI rat was used as a model of AKI whose main lesion was renal tubular injury. Urinary cystatin C level increased from Day 1. Blood creatinine and UN did from Day 3, and histopathological alternations of proximal renal tubule such as necrosis were observed from Day 3, and became more severe and spread on Days 5 and 7. These results indicated that urinary cystatin C was useful for the early detection of renal damage. Urinary KIM-1, GST-α, and EGF also changed from Day 1, indicating the usefulness for the early detection of renal damage, and KIM-1 and GST-α were superior to cystatin C in terms of high-fold increase. In Chapter 2, anti-GBM glomerulonephritis rat was used as a model of AKI whose main lesion was glomerular injury. Urinary cystatin C level increased on Day 7. Blood creatinine and UN hardly changed until Day 7. Urinary albumin and total protein, diagnostic markers of anti-GBM glomerulonephritis, increased on Day 7. Histopathological alternations of glomeruli such as hypercellularity and crescent formation were observed from Day 3, and became apparent on Day 7. These results indicated that urinary cystatin C was useful for the early detection of renal damage compared to the conventional biomarkers, but it did not have the superiority to urinary albumin and total protein. Urinary β2-microglobulin, clusterin, GST-α, and GST-μ also increased on Day 7. Urinary KIM-1 and NGAL increased from Day 3, indicating their superiority to cystatin C in terms of the early
detection of anti-GBM glomerulonephritis.
In Chapter 3, ZDF rat which developed diabetic nephropathy was used as a model of CKD. Urinary cystatin C level in ZDF rats were higher than that in lean rats (control rats) on Week 12, and it further increased age-dependently. Blood creatinine and UN hardly changed from Week 12 to Week 25. Histopathological slight change of glomeruli was observed on Week 12, and both glomeruli and renal tubule showed apparent alternations on Week 20, and it worsened on Week 25. These results indicated that urinary cystatin C was useful for the early detection of diabetic nephropathy compared to conventional biomarkers, and could monitor the progression of nephropathy. Urinary albumin in ZDF rats, the most sensitive marker of diabetic nephropathy in clinical settings, was also higher than lean rats on Week 12, and further increased age-dependently, indicating the usefulness of urinary cystatin C and albumin was similar. Urinary β2-microglobulin, clusterin, GST-μ, KIM-1, and osteopontin in ZDF rats were also higher than those in lean rats on Week 12, and it further increased, indicating the usefulness for the early detection and monitor the progression of diabetic nephropathy.
Additionally, in Chapters 1, 2, and 3, cystatin C was immunohistochemically localized in the proximal renal tubule in the kidney, and the immunoreactivity and positive area for cystatin C were hardly changed regardless of the progression of renal damage in CDDP-induced AKI rats, anti-GBM glomerulonephritis rats, and ZDF rats. In conclusion, urinary cystatin C was proved to be useful for the early detection of renal damage in AKI whose main lesion was renal tubular injury, AKI whose main lesion was glomerular injury, and diabetic nephropathy, one of CKD, and monitor the progression of diabetic nephropathy. Compared to urinary albumin and total protein, the usefulness of cystatin C was similar to those in AKI whose main lesion was glomerular injury and diabetic nephropathy. Regarding with novel renal biomarkers, urinary KIM-1 was also useful for the early detection for the three types of renal damages in this study, and monitor the progression of diabetic nephropathy. Urinary β2-microglobulin level was similar to urinary cystatin C. Urinary GST-α and EGF were useful for the early detection of AKI whose main lesion was renal tubular injury, urinary clusterin, GST-α, GST-μ, and NGAL were useful for that of AKI whose main lesion was glomerular injury. Urinary clusterin, GST-μ, and osteopontin were useful for the early detection and monitor the progression of diabetic nephropathy. Additionally, cystatin C was immunohistochemically localized in the proximal renal tubule in the kidney, and the immunoreactivity and positive area for cystatin C were not related to the progressions of AKI whose main lesion was renal tubular injury, AKI whose main lesion was glomerular injury, and diabetic nephropathy.
