[보고서]말기신부전(ESRD)에서 자식작용(Autophagy) 조절 인자 발굴 및 기능 규명

정주영

[국가R&D연구보고서] 말기신부전(ESRD)에서 자식작용(Autophagy) 조절 인자 발굴 및 기능 규명
Identification of autophagy regulatory factor and function in End Stage Renal Disease 원문보기

보고서 정보
주관연구기관	충남대학교 Chungnam National University
연구책임자	정주영
보고서유형	최종보고서
발행국가	대한민국
언어	한국어
발행년월	2019-06
과제시작연도	2018
주관부처	과학기술정보통신부 Ministry of Science and ICT
연구관리전문기관	한국연구재단 National Research Foundation of Korea
등록번호	TRKO201900021372
과제고유번호	1711072118
사업명	개인기초연구(과기정통부)(R&D)
DB 구축일자	2020-05-16
키워드	만성신부전.자식작용.혈관사이세포.당뇨병성신증.만성신부전 동물모델.

초록 ▼

□ 연구개요
말기신부전(ESRD)은 급성신부전을 포함한 다양한 원인에 의해 발병하며 발병 후 적절하고 지속적인 처치를 받지 못하면 회복이 어려우며 환자의 고통과 사회적 비용증가로 다방면의 치료법 개발이 활발히 진행되고 있음
- 자식작용(Autophagy)는 세포기관과 단백질의 합성과 분해사이의 균형을 조절하는 역할을 하며 활성산소와의 상호작용을 통해 cell survival 기전을 조절하는 양면성을 가지고 있으며 신경변성질환 및 암, 심혈관질환등 다양한 병리상태에 관여하는 것으로 보고되고 있음
- 본 과제는 당뇨병성 신증의 말기신부전에서 자식작용의 역할을 확인하고 이들의 조절 메카니즘을 규명하여 만성신부전의 보호기전 및 치유기전을 규명하는데 있음

□ 연구 목표대비 연구결과
본 연구의 내용은 말기신부전의 발병 기전을 중심으로
○ 1차년도: 말기신부전에서 상피세포 및 내피세포의 변이 기전 연구 및 Autophagy 조절 기전 연구
- 만성 신부전에서의 신장상피세포-내피세포간의 변이 기전에서 Autophagy 작용 기전 연구 (in vitro)
- 산화스트레스에 의한 신장상피세포-내피세포간의 변이 기전에서 만성신부전 발생시 EMT 발생기전에 있어 Autophagy 조절 기전 확립 (in vitro)
○ 2차년도: 만성신부전에서 혈관사이세포(mesangial cell)의 증식으로 인한 사구체 비대, EMT 발생 기전 연구 및 Autophagy 조절 기전 연구
- 혈관간세포의 증식 및 신장상피세포-내피페관의 변이 기전에서의 Autophagy 작용 기전 연구 (in vitro)
- 만성 신부전 동물모델 (당뇨병성 신병증 동물모델)에서 혈관간세포의 증식, 사구체의 변화, 관상간질섬유화의 증가에서 Autophagy 조절 기전 연구 (in vivo)
- 산화스트레스에 의한 혈관간세포의 증식성 기전 변이 기전을 토대로 만성신부전 동물모델(NQO1Knock out mice in Diabetic nephropathy) 을 이용한 산화스트레스 → 혈관간세포의 증식 → EMT 발생기전 확립 및 Autophagy 조절 기전 확립 (in vivo)
○ 3차년도: 말기신부전에서 사구체 구조적 변화를 통한 임상증상 발현기전에 있어 사구체족돌기(podocytic process) 기능 부전에 관한 연구 및 Autophagy 조절 기전 연구
- 만성신부전 동물모델 (IL-17 Knock out mice in Diabetic nephropathy) 을 이용한 산화스트레스 → 족세포 탈착 및 사구체의 구조적 변화 → 만성신부전 발병 및 발병기전에 있어 Autophagy 조절 기전 동물조직 연구 (in vivo)
○ 연구목표의 흐름에 따라 만성신부전 발생기전 및 진행에서 만성신부전 발생 시 증상 및 형태학적 변화에서 Autophagy 조절기전 연구를 체계적으로 수행하였고, 연구수행기간 중 본 연구과제를 통하여 석박사과정 학생 각 1명을 배출하였고, 연구결과를 국제 저널 및 신장관련 국제학술대회에 참가하여 발표하였음.

□ 연구개발결과의 중요성
1. 자식작용의 조절기전을 규명하여 당뇨병성 신증의 발병기전에 대한 지식을 확보
2. 말기신부전 치료에서 자식작용의 역할을 세포 및 동물 모델을 통하여 확인하였고 이를 근거로 새로운 치료제 개발에 도움이 되는 기술을 개발하여 국가적 경쟁력을 확보함
3. 본 과제의 성공적 수행결과에 따라 신부전 발병기전에 새로운 Mechanism을 이용한 발병의 원인에 입각한 예방적 치료가 가능한 새로운 개념의 신부전 치료제의 기전을 제공함으로서 국내외의 제약시장에 최고의 고부가 가치 상품 개발을 통해 자리매김할 것으로 기대함

(출처 : 연구결과 요약문 2p)

목차 Contents

표지 ... 1
연구결과 요약문 ... 2
목차 ... 3
1. 연구개발과제의 개요 ... 4
가. 연구개발의 필요성 ... 4
나. 연구의 최종목표 ... 6
다. 연구의 추진 전략 및 범위 ... 7
2. 연구수행내용 및 연구결과 ... 7
가. 1년차(2016)연구수행결과 ... 7
나. 2년차(2017) 연구수행결과 ... 11
다. 3년차(2018)연구수행결과 ... 14
3. 연구개발결과의 중요성 ... 17
4. 참고문헌 ... 18
5. 연구성과 ... 19
대표적 연구실적 ... 19
끝페이지 ... 22

표/그림 (22)

표 TGF-β1 mechanism에서 EMT로의 이행과정 시 상피세포의 손상 기전
표 산화적 스트레스와 신손상과의 관계
표 항산화효소의 발현과 tubulo-interstitial nephropathy와 의 관계
표 Cytotoxicity of glucose treated by time and concentration in proximal tubular epithelial cells(HK2). HK2 cells were harvested 24h, 48h and 72h after glucose treatment(0,10,30,50,100mM). Cytotoxicity in HK2 cells was observed using MTT assay. (A) 24h treatment by glucose concentration. (B) 48h treatment by glucose concentration. (C) 72h treatment by glucose concentration. There was no cytotoxicity by glucose in HK2 cells
표 Expression of TGF-β and α-SMA by treatment of glucose with concentration and time in proximal tubular epithelial cells(HK2). HK2 cells were harvested 24h, 48h and 72h after glucose treatment(0,10,20,30mM). Expression of TGF-β and α-SMA was observed using western blot analysis. Protein levels were determined by western blot analysis (A) Band intensties was quantified by densitometry and adjusted according to the respective β-actin band intensity (B)
표 Expression of Autophagy-related factors in high glucose treated proximal tubular epithelial cells(HK2). HK2 cells were harvested 3h,6h,12h and 24h after high glucose treatment(20mM). Expression of Autophagy-related factors was observed using westein blot analysis. Protein levels were determined by western blot analysis (A). Band intensities was quantified by densitometry and adjusted accoring to the respective β-actin band intensity (B)
표 Expression of LC3 in high glucose treated proximal tubular epithelial cells(HK2). HK2 cells were harvested 1h, 3h and 6h after high glucose teratment(20mM). Expression of LC3 was observed using immunofluorescence. (A) 1h teratment by glucose concentration. (B) 3h treatment by glucose concentration. (C) 6h treatment by glucose concentration
표 NADPH: quinone oxidoreductase (NQO1) knockdown in proximal tubular epithelial cells(HK2). HK2 cells were harvested 24h after si NQO1 or si scramble. Expression of NQO1 was observed using western blot analysis. Protein levels were determined by western blot analysis(A). Band intensities was quantified by densitometry and abjusted according to the respective β-actin band intensity (B)
표 Expression of Autophagy-related factors in si NQO1 or si scramble and high glucose treated proximal tubular eqithelial cells(HK2). HK2 cells were harvested 3h,12h and 24h after high glucose teratment(20mM). Expression of Autophagy-related factors was observed using western blot analysis. Protein levels were determined by western blot anallysis (A). Band intensities was quantified by densitometry and adjusted according to the respective β-actin band intensity (B)
표 Profibrotic signal expression after high concentration glucose treatment in HK2 cells and human renal mesangial cells (HRMCs). HK2 cells and HRMCs were treated with various concentrations of glucose for 24 h, and the total cell lysates were subjected to western blotting. The expression intensities of profibrotic signal proteins (A, C) were determined by densitometry, and were normalized to those of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (B, D). Values are expressed as the means ± SD of triplicate experiments
표 Autophagy-related protein expression following treatment of HK2 cells and human renal mesangial cells (HRMCs) with high concentrations of glucose. HK2 cells and HRMCs were treated with various concentrations of glucose for 24 h, and the total cell lysates were subjected to western blotting. The expression intensities of autophagy-related proteins (A, C) were determined by densitometry, and were normalized to those of β-actin (B, D). Values are expressed as the means ± SD of triplicate experiments
표 Treatment with high glucose concentrations enhanced mechanistic target of rapamycin (mTOR) suppression in HK2 cells. HK2 cells were pretreated with rapamycin (100 nM) for 12 h, and treated with glucose (25 mM) for 24 h. Total cell lysates were subjected to western blotting. The expression intensities of proteins (A) were determined by densitometry and were normalized to those of β-actin (B). Values are expressed as means ± SD of triplicate experiments
표 High glucose concentrations altered autophagosome expression in HK2 cells pretreated with 3-methyladenine (3MA) or ethanolamine (EA). HK2 cells were pretreated with 3MA (5 mM) or EA (10 mM) for 3 or 12 h, respectively. After pretreatment, the HK2 cells were treated with glucose (25 mM) for 24 h. Total cell lysates were subjected to western blotting. The expression intensities of proteins (A) were determined by densitometry, and were normalized to those of β-actin (B). Values are expressed as the means ± SD of triplicate experiments
표 Autophagy-related protein expression in the kidney tissue of streptozotocin (STZ)-treated mice. C57BL/6N mice were injected with STZ (50 mg/kg) for 5 consecutive days. After 1 month, the C57BL/6N mice were sacrificed and their kidney tissues were subjected to western blot analysis. The tissue homogenates were analyzed for autophagy-related protein expression by western blot analysis (A). Band intensities were determined by densitometry and were normalized to those of β-actin (B). Values are expressed as the means ± SD of triplicate experiments
표 Autophagy-related protein expression in the kidney tissue of streptozotocin (STZ)-treated mice. C57BL/6N mice were injected with STZ (50 mg/kg) for 5 consecutive days. After 3 month, the C57BL/6N mice were sacrificed and their kidney tissues were subjected to western blot analysis. The tissue homogenates were analyzed for autophagy-related protein expression by western blot analysis (A). Band intensities were determined by densitometry and were normalized to those of β-actin (B)
표 Effect of NQO1 knockout on kidney histopathology determined via Hematoxylin and Eosin (H&E) satining of tissue. C57BL/6N and NQO1 knockout mice were injected with STZ (50 mg/kg) for 5 consecutive days. After 3 month, the C57BL/6N and NQO1 knockout mice were sacrificed. Kidney tissue from wild type control (A) and NQO1 knockout control (C) groups showed normal histological patterns. After streptozotocin treatment, wild type STZ (B) and NQO1 knockout STZ (D) group displayed significant glomerular basement membrane thickening
표 Effect of IL-17 knockout on renal physiology. C57BL/6N and IL-17 knockout mice were injected with STZ (50 mg/kg) for 5 consecutive days. After 3 month, the C57BL/6N and IL-17 knockout mice were sacrificed. After STZ treatment food intake, water intake and urination was increased. Diabetic IL-17-/- mice are relatively reduced from food intake, water intake and urination compared to WT diabetic mice
표 Effect of IL-17 knockout on renal physiology. C57BL/6N and IL-17 knockout mice were injected with STZ (50 mg/kg) for 5 consecutive days. After 3 month, the C57BL/6N and IL-17 knockout mice were sacrificed
표 Effect of IL-17 knockout on serum HbA1c. C57BL/6N and IL-17 knockout mice were injected with STZ (50 mg/kg) for 5 consecutive days. After 3 month, the C57BL/6N and IL-17 knockout mice were sacrificed. Diabetic IL-17-/- mice are relatively reduced serum HbA1c compared to WT diabetic mice
표 Effect of IL-17 knockout on kidney histopathology determined via Hematoxylin and Eosin (H&E) staining of tissue. C57BL/6N and IL-17 knockout mice were injected with STZ (50 mg/kg) for 5 consecutive days. After 3 month, the C57BL/6N and IL-17 knockout mice were sacrificed. Kidney tissue from wild type control (A) and NQO1 knockout control (C) groups showed normal histological patterns. After streptozotocin treatment, wild type STZ (B) and NQO1 knockout STZ (D) group displayed significant glomerular basement membrane thickening
표 Effect of 17 knockout on kidney histopathology determined via Sirius red staining of tissue. C57BL/6N and IL-17 knockout mice were injected with STZ (50 mg/kg) for 5 consecutive days. After 3 month, the C57BL/6N and IL-17 knockout mice were sacrificed. Kidney tissue from wild type control (A) and NQO1 knockout control (C) groups showed normal histological patterns. After streptozotocin treatment, wild type STZ (B) and NQO1 knockout STZ (D) group displayed significant accumulation of collagen in glomerular basement membrane
표 Effect of IL-17 knockout on kidney histopathology determined via LC3 immunohistochemistry staining of tissue. C57BL/6N and IL-17 knockout mice were injected with STZ (50 mg/kg) for 5 consecutive days. After 3 month, the C57BL/6N and IL-17 knockout mice were sacrificed. Kidney tissue from wild type control (A) and NQO1 knockout control (C) groups showed normal histological patterns. After streptozotocin treatment, LC3 positive were increased wild type STZ (B) and NQO1 knockout STZ (D) group. Diabetic IL-17-/- mice are relatively reduced LC3 positive cells compared WT diabetic mice

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[국가R&D연구보고서] 말기신부전(ESRD)에서 자식작용(Autophagy) 조절 인자 발굴 및 기능 규명
Identification of autophagy regulatory factor and function in End Stage Renal Disease 원문보기