【作者】 张懿；

【导师】 梅长林； 叶朝阳； 赵学智； 王雪琦；

【作者基本信息】 第二军医大学 ， 内科学， 2010， 博士

【摘要】 背景及目的近期一项报告指出,血清肌酐的轻度升高(0.5mg/dl)使成人住院率增加14%,死亡率增加6.5倍。尽管近年来对于AKI有了进一步地理解,但在重症病人,其死亡率仍然高达50%以上。AKI是一个复杂的综合征,病因复杂,包括缺血再灌注、毒物及脓毒血症等。迄今,尚缺乏有效治疗AKI的方法。在AKI中,肾小管上皮细胞死亡多少对于AKI的严重程度及预后是非常重要的。研究证实,不管是体外细胞凋亡还是体内肾小管细胞坏死都依赖于细胞周期中的某些酶的激活。在细胞周期中,周期素(cyclin)和周期素依赖性激酶(CDK)形成复合物促进细胞周期的进程,周期素依赖性激酶抑制剂(CDKI)则作用于各种CDK负向调节细胞周期的进程。一般认为,细胞周期的阻滞利于损伤细胞的修复,减轻损伤的致死性和致畸性,避免损伤的DNA进入复制期并传给子代细胞。所以,在多种肾脏疾病的研究中,包括AKI, CDKI的研究颇多并被证明在AKI中是具有细胞保护作用的。一些抑制CDK酶活性的抑制剂在肾毒性药物诱导的肾小管上皮细胞损伤中也显示出较好的保护作用。因此,越来越多的研究认为,对于细胞周期活动和细胞周期调控蛋白生物学功能的进一步理解利于探求AKI早期治疗中的潜在靶点。目前已知的七种CDKI中,根据其共享序列和对于CDK的抑制分为二大家族。CIP/KIP家族,包括P21,P27,P57等,可以和多种CDK作用,对于细胞周期的抑制相对较为广泛；INK4家族,包括P16,P15,P18,P19等,只与CDK4／6作用,特异性地阻滞细胞周期于G1期。在AKI研究领域,CDKI的研究主要集中于CIP/KIP家族,很少涉及INK4家族成员。由于在多种肿瘤中缺失或突变,INK4家族既往被认为是一类肿瘤抑制因子。但近年来研究显示,INK4家族成员还具有许多其它新的生物学功能,所以我们设想INK4家族成员在AKI中可能起着一定的保护作用。在INK4家族中,各成员间大约有40%的同源氨基酸相似度。与P15,P16,P19相比,P18相对较为保守。人和鼠的P18多肽都含有168个氨基酸,其中至少有153个残基是相同的,在最不保守的C末端区存在7个残基的不同。所以,我们选取INK4家族成员P18做为主要研究对象,观察INK4家族成员P18在顺铂诱导的AKI中的保护作用。方法首先,观察P18是否参与AKI的发病过程。顺铂单次腹腔注射(12.5mg/kg)制备AKI小鼠模型,顺铂(60μM)诱导小鼠肾脏上皮细胞(TCMK-1)的损伤,利用Realtime-PCR和Western Blot明确P18在AKI小鼠和TCMK-1细胞损伤中的变化；其次,明确P18在急性肾损伤中的保护作用。同样方法诱导P18基因敲除小鼠(p18-／-)和其野生型同窝小鼠(p18+／+)的AKI模型,通过对p18-／-和p18+/+AKI小鼠肾脏组织学形态,肾功能和生存率的比较,明确P18基因对于AKI小鼠的肾脏保护作用；小鼠P18质粒和空载体分别转染猪的肾小管上皮细胞(LLC-PK1),顺铂(1001μM)诱导两种细胞损伤,通过对细胞LDH释放,caspase3酶激活,凋亡细胞比例和凋亡蛋白表达等结果的分析,明确P18基因对于顺铂诱导的肾小管上皮细胞损伤的保护作用；第三,探讨P18肾脏保护作用的机制。利用Realtime-PCR和Western Blot证明AKI小鼠和LLC-PK1细胞损伤中内质网应激的存在,观察P18基因缺失和P18蛋白过表达对于顺铂诱导的内质网应激程度的影响。结果(1)在顺铂诱导的AKI小鼠肾组织中以及在顺铂诱导的肾脏细胞(TCMK-1)损伤中,p18mRNA和p18蛋白的表达均呈上调趋势；(2)与p18+／+小鼠相比,顺铂诱导的p18-／-小鼠的肾损伤明显加重,肾功能恶化加剧,生存率明显降低,两组相比具有明显的统计学差异；(3)与空载体转染相比,小鼠P18质粒转染使顺铂诱导的LLC-PK1凋亡细胞明显减少,尽管两组细胞相比,LDH释放实验结果无明显差别；与空载体转染相比,小鼠P18质粒转染使顺铂诱导的LLC-PK1细胞中caspase 3酶的激活明显减少,活性caspase 3蛋白和其底物PARP的表达也明显减少；(4)在顺铂诱导的AKI小鼠(p18+／+和p18-/-)肾组织中,grp78 mRNA和grp78蛋白的表达均明显上调,提示在顺铂诱导的肾损伤中伴有内质网应激的参与,而生理盐水和顺铂注射的p18-／-小鼠肾皮质区grp78 mRNA和grp78蛋白的表达均强于其p18+／+对照鼠,说明P18基因缺失加重AKI小鼠肾组织内的内质网应激。除了grp78, p18-/-小鼠肾皮质区grp94 mRNA和CHOP mRNA水平也明显高于其p18+／+小鼠；小鼠P18质粒转染使顺铂诱导的LLC-PK1细胞中grp78和caspase 12的表达明显减少；内质网应激通过三条途径诱导细胞凋亡,western blot结果证实,P18-／-小鼠肾组织中PERK和eIF2α蛋白的磷酸化水平强于P18+／+小鼠,小鼠P18质粒转染的LLC-PK1细胞中PERK和eIF2α蛋白的磷酸化水平弱于空载体转染的LLC-PK1细胞。这一结果提示,P18基因通过影响PERK和eIF2a途径调节顺铂诱导的内质网应激,从而在AKI中发挥肾脏保护作用。结论INK4家族成员P18参与顺铂诱导的AKI,P18在顺铂诱导的AKI中具有肾脏保护作用,P18通过细胞周期调控和对内质网应激水平的调节在顺铂诱导的AKI中发挥保护作用。更多还原

【Abstract】 BackgroundAccording to a recent study, increased serum creatinine (0.5mg/dl) was reported in more than 14% of adult admission, and was associated with a 6.5-fold increased mortality. Despite an increased understanding about the incidence and consequences of acute kidney injury (AKI) in recent years, morbidity and mortality associated with this syndrome in critically ill patients remain as high as 50% or more. AKI is a heterogeneous syndrome, and possible contributing causes are similarly heterogeneous, ranging from ischemia/perfusion, poisons, pyemia and so on. Up to now, there is no effective treatment for AKI.Cell death in company with AKI contributes significantly to the severity of the syndrome. Studies have demonstrated that both apoptotic cell death in vitro and necrotic tubular cell death in vivo are dependent on the activation of certain enzymes of the cell cycle, during which cyclins bind to their partner cyclin-dependent kinases (CDK) and promote progression of the cell cycle, and on the other hand cyclin-dependent kinase inhibitors (CDKI) negatively control the cell cycle via inhibiting the activity of CDK. It is common sense that cell cycle arrest is beneficial for injured cells to repair themselves and attenuate the lethality and teratogenicity by preventing injured DNA from repeating and transferring to its daughter cells. Many studies on kidney diseases including AKI confirmed that CDKI had cytoprotective effects in AKI. Other studies also found that some drugs that inhibited the activities of CDK also had renoprotective effects in toxin-induced tubular cell injury. Therefore, more investigators believe that a further understanding about cell cycle activity and the characters of cell cycle proteins may help find potential targets for early treatment of AKI.Seven CDKI have been identified and are divided into two families according to the same sequence that they share and the target CDKs that they control. The CIP/KIP family, including p21, p27 and p57, is believed to respond to many CDKs and inhibit the cell cycle. The INK4 family, including p16, p15, p18 and p19, only responds to CDK4/6, and specifically blocks the cell cycle in early Gl phase. Studies of CDKI have mainly focused on the CIP/KIP family, and few studies are concerned with the INK4 family.As INK4 members are often mutant and deleted in many tumors, they are thought of as tumor-inhibiting factors, however, recent studies found that INK4 members possess some novel functions. We therefore supposed that INK4 members may have a potential cytoprotective effect in AKI.In the members of the INK4 family, there is approximately 40% identical amino acid homology. Compared with p16, p15 and p19, p18 is relatively conserved. Human and mouse p18 polypeptides, each having 168 amino acids, are identical over 153 residues, with seven of the substitutions occurring in the least conserved C-terminal domain. So, we selected p18 as our major target to investigate the potential cytoprotective effect of the INK4 family in AKI.Our study was attempted to identify the role of INK4 family member p18 in cisplatin-induced AKI.MethodsFirstly, we explored the involvement of p18 in AKI. An AKI mouse model was constructed by a single-dose intraperitoneal injection of cisplatin (12.5mg/kg). Mouse kidney epithelial cell (TCMK-1) injury was induced by 24-h incubation of cisplatin (60μM) with the cells. The expression of p18 was identified by real-time polymerase chain reaction (real-time PCR) and Western blot at mRNA and protein levels.Secondly, we identified the protective effect of p18 in renal injury. The same method was used to induce p18 gene knock-out (p18-/-) and their wild type brood (WT) AKI mice. Analysis of kidney histology, renal function and survival between p18-/-and pl8+/+mice 3 days after cisplatin injection was made to identify the role of p18 in cisplatin-induced AKI. In cell experiments, mouse p18 plasmid and its vehicle were transfected in pig proximal tubular cell line LLC-PK1, and cisplatin (100μM) was added into the medium and incubated with the cells for 24 h. LDH release, caspase 3 enzymatic activity and apoptosis between cells transfected with mouse p18 plasmid and cells transfected with the vehicle were analyzed to identify the protective role of p18 in renal cell injury.Thirdly, we primarily investigated the protective mechanisms of p18 in cisplatin-induced AKI. Endoplasmic reticulum stress (ERS) was demonstrated the contribution in cisplatin-induced AKI by real-time PCR and western blot. Mechanism analysis was performed by comparing ERS severity under the conditions of p18 gene knockout and p18 protein overexpression.ResultsThe results of the present study included:1) P18 mRNA and protein expressions were upregulated in cisplatin-induced kidney injury and renal cell injury.2) Compared with p18+/+mice, kidney injury and renal function of p18-/-mice were worse, and the survival rate of p18-/-mice was also significantly shorter. Deletion of p18 exacerbated cisplatin-induced AKI.3) Compared with vehicle transfection, mouse p18 plasmid transfection significantly decreased cisplatin-induced LLC-PK1 apoptosis, though there was no significant difference in LDH release between them. The same results were obtained in caspase 3 enzyme activity, active caspase 3 protein and the release fragment of its substrate PARP.4) Grp78 mRNA and protein expressions were both increased in cisplatin-induced p18-/-and p18+/+AKI mice, implying that ERS existed in cisplatin-induced kidney injury. Compared with p18+/+mice, grp78 mRNA and protein expressions were both significantly higher in p18-/-mice regardless of saline or cisplatin injection, suggesting that p18 deletion aggravated ERS level in normal or cisplatin-induced AKI mice. Besides grp78, grp94 mRNA and CHOP mRNA were both higher in p18-/-mice than those in p18+/+mice. Western blot showed that mouse p18 plasmid transfection decreased cisplatin-induced grp78 protein and active caspase 12 protein expression in LLC-PK1 cells as compared with vehicle transfection. The proteins in ERS induced apoptosis signal pathways, p-PERK, p-eIF2a, higher in p18-/-kidney than p18+/+kidney and lower in mouse p18 plasmid transfection cells than vehicle transfection, suggesting that p18 regulated cisplatin-induced kidney ERS via PERK-eIF2a pathway and exerted renoprotection in AKI.ConclusionsINK4 family member p18 was also involved in cisplatin-induced AKI and played a renoprotective role in AKI. The protective action of p18 was exerted via regulating the cell cycle and severity of ERS in cisplatin-induced AKI.更多还原