Early diagnosis of postoperative acute renal failure –
from bench to bedside
早期诊断术后急性肾脏衰竭研究—
从基础到临床
20100402
Progress Report
吴明达
Introduction
Acute Renal Failure (ARF) = Acute Kidney Injury (AKI)
Relationship between the clinical phases and the cellular phases of ischemic acute renal failure (ARF), and the temporal impact on organ function as represented by the glomerular filtration rate (GFR).
Modified from Timothy A_Sutton et al., Kidney International (2002)
Devarajan P (2005) Curr Opin Pediatr
Metabolic consequences of acute IRI to kidney tubule cells
The animal model of ischemia-reperfusion injury for AKI
40 min
3
6
12
24
48
(hr)
The animal model of ischemia-reperfusion injury for AKI
Pre 0
After IR 24 hr
ACT
Netrin-1
Neogenin
DCC
UNC5B
Pre 0 hr
I/R 0 hr
I/R 3 hr
Cortex
Effect of IRI on the mRNA expression of Netrin-1 and its receptors in rat kidney
Netrin-1 (LRSP) mRNA expression were determined in control kidneys and at 0 and 3 h after reperfusion following 40 min of renal ischaemia (Pre 0, I/R 0 hr and I/R 3 hr kidneys, respectively). RT-PCR products were obtained with use of specific primer pairs for rat ACT, Netrin-1 and its receptors.
Ligand
Receptor
DCC family, 64% consensus sequence
50% identity
Hemojuvelin
Effect of ischaemia/reperfusion on netrin-1 and DCC protein level in rat kidney, blood and urine
Immunoblot of netrin-1 and its receptor DCC in acute kidney injury induced by 40 min I/R after 6, and 24 h. A decrease was observed in kidney and blood and 0 hr refers to kidney extracts from sham-operated rats. Interesting, the decrease of netrin-1 was observed in postoperative urine.
Whole kidney
Blood
Urine
(hr) 0-3 8-24
(No.) 1 2 3 4
(hr) 0 0 0 6 6 6 24 24 24
(No.) 1 2 3 4 5 6 7 8 9
(hr) 0 0 3 3 24 24 24
(No.) 1 2 3 4 5 6 7
Netrin-1
DCC
Lotus tetragonolobus lectin (LTL), a marker for proximal tubules
DAPI, a marker for nuclei
Immunohistochemical colocalization of proximal tubules marker, Netrin-1 and its receptor DCC
Netrin-1
DAPI
DCC
Merge
DAPI
DCC
Merge
DAPI
DCC
Merge
Sham
I/R after 6 hr
I/R after 24 hr
Netrin-1 and its receptor were decrease after I/R
Netrin-1
Netrin-1
Time-response of HK-2 cells under ischemia injury
0 6 24 (hr)
Normal
ATP deprivation
Normal
ATP deprivation
Normal
ATP deprivation
WB: Anti-Netrin-1
WB: Anti-Actin
Michael Bennett et al., (2008) CJASN
Urine NGAL predicts severity of AKI After Cardiac Surgery
Serum creatinine measurements obtained at various time points post-cardiac surgery from NSARF databank
Urine netrin-1 from 9 AKI patients in NSARF databank
The setup of SNP databank from AKI patients

备注
82
累计收案人数
±3.18
标准差
5.25 ug
样本平均总量
26.23 ng/ul
样本平均浓度
Genomic DNA
Study
2009.10~2010.3
Date
急性肾衰竭,现在又称作急性肾损伤,他的特色是急性肾功能丧失,在ICU的病房中,发生率高达35-50%,并且与死亡率有密切的关系.其中,ICU手术过程的缺血再灌流伤害是主要造成AKI的致病机转之ㄧ.
我们以因为IRI导致AKI的病人,根据他们的肾丝球过滤率,对照术后时间,可以发现在术后2天内,他们的GFR会降到最低,随后肾脏启动自我修复机制,假设修复及时,则肾脏可以回复正常功能,否则,则有可能导致慢性肾病甚至是需要依赖肾脏替代治疗.
由此,我们可以知道在术后的两天内为AKI是否发生的黄金观察期,如何评估肾脏受损的程度,是否可以凭藉自我的修复,或是需要外在的医疗及早介入,其中,合适的生物标记将有助於判断肾脏的状态.
*
以细胞的层次来分析,Ischemia 以及reperfusion整个过程都会对於细胞造成伤害,例如缺血导致氧气供应中断,降低呼吸链的ATP制造,引发caspase活化造成Apotosis/Necrosis,细胞膜极性的改变,细胞骨架的受损,氧化的伤害甚至是NO所造成的protein nitrosylation
*
我们首先分析Netrin-1以及相对应的接受器在IRI前后在大鼠肾脏的mRNA表现,从RT-PCR的结果,我们可以看到Netrin-1在IRI3小时之后表现量显著降低,但是对照他相对应的接受器,Neo1以及UNC5B却没有显著变化,这似乎暗示著肾脏细胞因为IRI的压力导致缺乏Netrin-1这种ligand,可能会促使相对应的接受器自行走向Aptosis的方向.
在蛋白质层次的分析中,我们同时观察kidney, blood以及urine在相同蛋白质量的情况下,以专一性对抗netrin-1的抗体分析蛋白质量变化.蛋白质量呈现随著I/R之后的时间而减少,然而,当我们继续检测尿液中的蛋白质,却发现上述随著时间而减少的netrin-1,在尿液中随著I/R术后的时间而增加,这和我们蛋白质体分析尿蛋白的结果是相符的.
免疫组织定位的染色结果显示,肾脏中的netrin-1以及其中之一的receptor DCC都是出现在肾脏的近端肾小管中.
原本出现在近端肾小管的netrin-1以及其接受器DCC,蛋白质量会随著I/R术后而逐渐减少,而在尿液中发现他们的存在,这与我们先前以western检测kidney tissue 的结果相符.
*
因此,我们以人类近端肾小管细胞株HK2做为材料,利用antimycinA 抑制呼吸链ATP的制造模拟缺氧环境,同样发现细胞株会走向apotosis,并且netrin-1会从HK-2细胞上消失.
既然Netrin-1能够在早期的尿液中被发现,我们尝试与现有的biomarker NGAL进行比较,根据Michael Bennett在2008年於CJASN发表的研究,NGAL对於开心手术的病人有良好的预测能力,大约在术后4-6小时可以准确预测AKI的发生.
我们根据NSARF现有收集的病患在术后不同时间点的尿液,以RIFLE将他们区分为发生AKI与否的族群,其中再挑出根据血清CRE上升所判别的族群作为与NGAL比较的样本.
将上述AKI患者术后不同时间收集的尿液样本,分别以NGAL以及netrin-1两套ELISA kit进行测定,NGAL的结果显示AKI病人主要的高峰点同样出现在术后6-9小时之间,有趣的是,Netrin-1在术后3小时已经到达高峰,随著术后的时间下降回baseline,这样的结果告诉我们Netrin-1可以在早期术后即时反应出肾脏受损的状态.
*