Protection Of Cyclin-dependent Kinase Inhibitor P18^INK4C In Cisplatin-induced Acute Kidney Injury

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.