신장의 허혈 재관류 손상에서 원격 허혈 전처리의 효과: Microarray analysis를 이용한 허 혈 전처리의 메커니즘 이해

Abstract

Remote ischemic preconditioning (IP) is a potential renoprotective strategy. However, the results of this strategy have not been demonstrated in large animals, and the mechanism of remote IP remains to be defined. Using a single-kidney porcine model, we evaluated the organ protective action of IP, and attempted to demonstrate the mechanism underlying remote IP in renal ischemia reperfusion injury (IRI). Fifteen 20-week-old Yorkshire pigs weighing 35–38 kg were used. One week after left nephrectomy, we performed remote IP (clamping of the right external iliac artery for two cycles of 10 minutes) and right renal artery clamping (warm ischemia; 90 minutes). We divided the animals into three experimental groups, each containing five animals: control group = 90 minutes of warm ischemia without IP (n = 5); group 1 (remote IP with early window [IP-E group], n = 5) = 20 minutes IP followed by 90 minutes warm ischemia after a 10-minute time window; and group 2 (remote IP with late window [IP-L group], n = 5) = 20 minutes IP followed by 90 minutes warm ischemia after a 24-hour time window. Serum creatinine and urinary neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), and microalbumin levels were measured before renal ischemia and 6, 24, and 72 hours post-ischemia. The Porcine (V2) Gene Expression Microarray 4 x 44 (Agilent Technologies, Palo Alto, CA) was used for differential gene expression analysis. The list of altered gene expression levels was uploaded to DAVID (Database for Annotation, Visualization and Integrated Discovery v6.7), and Functional Annotation was used to analyse and categorise the differentially expressed genes and to find genes that participate in common biological pathways with a significant probability of being involved. There were no differences in serum creatinine changes between the different study groups. The IP-L group had lower urinary NGAL than the control and IP-E groups at 72 hours post-ischemia. At 72 hours post-ischemia, the urinary KIM-1 level was lower in the IP-L group than in the control and the IP-E group, and in the IP-L group, KIM-1 was near pre-ischemic levels, whereas in the control and IP-E group, KIM-1 levels increased. Microalbumin levels also were lower in the IP-L group. Among 43,603 probes, a total of 1,053 probes were selected as they represented significant data. Compared to the control group, the IP-E and IP-L groups had 179 genes with altered expression levels in common, most of which were related to apoptosis, inflammation, and oxidation. Among these genes, 848 gene expression levels were altered only in the IP-L group, and most of these were associated with immune response. When we uploaded the list of genes, DAVID suggested several pathways, including the complement system, coagulation cascade, and cytokines and cytokine receptors. Remote IP can mitigate renal damage from IRI. To effectively provide renoprotection, remote IP requires a considerable rather than a short time window before ischemia. Furthermore, IP elicits its renoprotective effect on renal IRI through various mechanisms rather than one specific pathway. |원격 허혈 전처리 (ischemic preconditioning, IP)는 조직이 허혈 재관류 손상 (ischemia reperfusion injury, IRI)에 잘 견딜 수 있도록 하는 방법으로 이미 많은 연구결과가 발표된바 있다. 하지만, 이전 연구는 대부분 설치류와 같은 소형동물에 그치며, 그 메커니즘은 아직 명확하게 밝혀지지 않았다. 이에 저자는 대형 포유동물인 단일신 돼지 모델을 이용해 신장의 IRI에서 IP의 효과와 메커니즘을 밝히고자 한다. 실험 1주일 전 복강경하 좌측 신장 제거술을 한 뒤, 총 15마리의 암퇘지 (35-40Kg)를 3군으로 분류하였다. 대조군은 IP 없이 우측 신장의 동맥을 클램프하여 90분간의 IRI를 주었다. 실험1군 (remote IP with early window [IP-E])은 우측 장골 동맥을 클램프하여 IP 처리를 한 뒤 바로 90분의 IRI를 주었고, 실험2군 (remote IP with late window [IP-L])은 IPC 후 24시간의 공백기를 두고 IRI를 가하였다. IP는 우측 장골 동맥을 10분간 2번 클램프하는 것으로 하였다. 신장기능 평가를 위하여, 혈중 크레아티닌, 요중 neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), microalbumin 등을 측정하였다. Porcine (V2) Gene Expression Microarray 4 x 44 (Agilent Technologies, Palo Alto, CA)를 이용하여 변형된 유전자 발현을 분석하였으며, 대조군과 2배 이상의 차이가 날 때를 유의한 차이로 정의하였다. 변형된 유전자 발현을 DAVID (Database for Annotation, Visualization and Integrated Discovery v6.7, National Institute of Allergy and Infectious Diseases; http://david.abcc.ncifcrf.gov) 프로그램을 이용하여 기능 주석(Functional Annotation) 및 관여하는 특정 pathway를 분석하였다. 세 실험군 간 혈중 크레아티닌의 차이는 관찰되지 않았다. 하지만, IRI 72시간 뒤 IP-L 군에서 요중 NGAL과 KIM-1이 대조군과 IP-E 군에 비하여 낮게 측정 되었다. Microalbumin도 IP-L 군에서 더 낮은 경향을 보였다. Microarray에서 총 43,603개의 raw data가 검출 되었으며, 그 중 1,053개의 유전자가 각 군 간에 차이가 있는 것으로 나타났다. IP-E 군과 IP-L 군에서 공통적인 변화를 보이는 유전자는 179개 였으며, 848개의 유전자는 2군에서만 변형된 유전자 발현을 보였다. 기능 주석 (Functional Annotation) 분석 결과 IP-E 군과 IP-L 군에서 공통으로 관찰된 유전자는 대부분 세포자멸사, 항산화, 면역반응, 염증반응 등에 분류 되었으며, IP-L 군에서만 변형된 유전자는 면역 반응, 단백질 분해 등에 분류되었다. 기능적 분류에 따른 pathway는 도움체 계통(complement system), 응고 폭포 (coagulation cascade), 시토카인과 시토카인 수용체 등이 선별되었다. 신장의 IRI에서 IP는 신장손상을 줄인다. 또, 이러한 작용은 면역이나 염증 반응이 충분히 일어날 수 있는 공백기가 있을 때 더욱 효과적이다. IP는 어떠한 한가지의 pathway라기 보다는 다양한 메커니즘을 통하여 발현되는 것으로 사료된다.; and group 2 (remote IP with late window [IP-L group], n = 5) = 20 minutes IP followed by 90 minutes warm ischemia after a 24-hour time window. Serum creatinine and urinary neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), and microalbumin levels were measured before renal ischemia and 6, 24, and 72 hours post-ischemia. The Porcine (V2) Gene Expression Microarray 4 x 44 (Agilent Technologies, Palo Alto, CA) was used for differential gene expression analysis. The list of altered gene expression levels was uploaded to DAVID (Database for Annotation, Visualization and Integrated Discovery v6.7), and Functional Annotation was used to analyse and categorise the differentially expressed genes and to find genes that participate in common biological pathways with a significant probability of being involved. There were no differences in serum creatinine changes between the different study groups. The IP-L group had lower urinary NGAL than the control and IP-E groups at 72 hours post-ischemia. At 72 hours post-ischemia, the urinary KIM-1 level was lower in the IP-L group than in the control and the IP-E group, and in the IP-L group, KIM-1 was near pre-ischemic levels, whereas in the control and IP-E group, KIM-1 levels increased. Microalbumin levels also were lower in the IP-L group. Among 43,603 probes, a total of 1,053 probes were selected as they represented significant data. Compared to the control group, the IP-E and IP-L groups had 179 genes with altered expression levels in common, most of which were related to apoptosis, inflammation, and oxidation. Among these genes, 848 gene expression levels were altered only in the IP-L group, and most of these were associated with immune response. When we uploaded the list of genes, DAVID suggested several pathways, including the complement system, coagulation cascade, and cytokines and cytokine receptors. Remote IP can mitigate renal damage from IRI. To effectively provide renoprotection, remote IP requires a considerable rather than a short time window before ischemia. Furthermore, IP elicits its renoprotective effect on renal IRI through various mechanisms rather than one specific pathway.