The Mechanism Study Of Different Drug Combination Preventing Urografin Induced Acute Kidney Injury

Background&Objective: Contrast-induced acute kidney injury(CIAKI), also called contrast induced nephropathy(CIN), is an important complication by the application of contrast. It has become the third most important complications following stent thrombosis, stent restenosis after percutaneous coronary intervention. Contrast medium in clinical application includes urografin, ioversol, iohexol. The pathogenesis of CIAKI is complex. Studies have shown that the cell apoptosis induced by endoplasmic reticulum stress(ERS) play an important role in the renal tubular injury of CIAKI. It was suggested that atorvastatin, probucol and alprostadil can inhibit renal tubular cell apoptosis by antioxidant and anti-inflammatory effect or improving vascular perfusion to prevent CIAKI. Previous study have shown atorvastatin can successfully suppress apoptosis induced by ERS and then prevent CIAKI. However, there is no specific research about the above effect of the drug combination. In recent decades of research found many new biomarkers to early detect CIAKI. Previous research has intiatively proved clusterin can predict the occurrence of CIAKI, but lack of further argument. Therefore, this study intends to establish the rat CIAKI model by urografin and add drug intervention, to compare the influence of combined drugs with the single atorvastatin for CIN. The purpose is to discuss whether various drug combinations can play a protective role of CIAKI by inhibiting ERS specific molecular chaperone, and from the perspective of drug combination, to define diagnostic significance of clusterin for CIAKI and effect of drug on it.Methods: 50 Wistar rats were randomly divided into 5 groups: A group(atorvastatin group); P group(atorvastatin + probucol group); Q group(atorvastatin + alprostadil group); NC group(contrast media control group) and N group(control group). After adaptive feed for 7 days, 2ml blood from subclavian vein was get to measure renal function. P group was given by gavage probucol for 14 days, and then atorvastatin was added with probucol for 10 days. Q and A groups were given the same volume 0.5% sodium carboxymethyl cellulose for 14 days, and then atorvastatin for 10 days. N and NC were given same volume solution. On the 22 nd day, urografin was injected to rats in the four groups and the same dose normal saline to N group. Q group was injected alprostadil on 24 hours and 30 minutes prior to surgery, 24 hours after surgery respectively. Other groups were injected same dose normal saline. 48 hours after surgery, 2ml blood was obtained to measure renal function. 72 hours after surgery, following 2ml blood drawn, rats of every groups were killed and both kidneys were obtained, of which the left was fixed in formalin for HE, TUNEL staining and immunohistochemitry of GRP78, Clusterin, GADD153/CHOP, Caspase-12 and the right cryopresrved in-80℃ refrigerator for real-time RT-PCR and western blot of the targets above to detect their expression on protein and nucleic levels. SPSS19.0 was applied to statistic analysis. Continuous variables are expressed as mean value ± SD. Paired t-test were used to compare variables within group. Comparison between groups was determined by One-way ANOVA test, MannWhitney U test and Kruksal-Wallis test. P<0.05 was considered statistically different.Result: 1. Urografin can significantly increase serum creatinine, leading to the highest creatinine value in NC group. There is no statistical difference between the combined groups and N group. Compared to A group, the protective effect of Q group is stronger. 2. Nucleic expression levels of GRP78, GADD153/CHOP and Caspase-12 in PCR were all highest in NC group, with statistical difference in GADD153/CHOP and Caspase-12. Expression in the combined group was lower than A group, without different significance. 3. Protein expression levels of GRP78, GADD153/CHOP and Caspase-12 in Western Blot were all remarkably highest in NC group. The level in A group was significantly higher than P for GRP78,and significantly higher than Q for Caspase-12, and higher than the both combined groups for GADD153. The expression of the protein above in the combined groups were similar as even lower than N group. For GADD153 and Caspase-12, expression in Q group was remarkably lower than P group.4. For HE staining and electronic microscope, cavity change in tubular epithelial cells appeared, necrosed cells, thickened basement membrane, narrow lumen, decreased and swelling endoplasmic reticulum and mitochondrion, rambling and shedding microvilli can be seen in NC group. Degree of renal injury in three drug groups is reduced and the combined group is lower than A group. 5. The apoptosis index is NC group>A group > Q group >P group >N group, with statistical significance. Index in A group is significantly higher than P and Q group. There is no different significance between P and Q group. 6. In immunohistochemitry, the three targets also expressed highest in NCgroup, and expression of the three targets in A group was higher than that in the combined groups, with significant difference of GADD 153 compared to Q group and of Caspase-12 compared to the both combined groups. 7. There is no difference among the five groups on the nucleic expression of clusterin. Protein expression of clusterin were highest in NC group in western blot and immunohistochemitry compared to other four group. Expression in A group was significantly higher than that in drug combination groups and in P group was higher than in Q group.Conclusion: 1. The cell apoptosis induced by ERS pathway may play an important role in pathogenesis of CIAKI induced by urografin. Atorvastatin, probucol and alprostadil can prevent the occurrence of CIAKI, may through the inhibition of cell apoptosis induced by ERS pathway. The protective effect for CIAKI of probucol or alprostadil combined with atorvastatin may be stronger than the single atorvastatin through the mechanism of ERS, with the more effect of alprostadil combined with atorvastatin. 2. It can be further illustrated that clusterin can be an early predictive biomarker of CIAKI. The effect for kidney protection of probucol or alprostadil combined with atorvastatin may be stronger than the single atorvastatin, leading to down-regulation of clusterin, with the more effect of alprostadil combined with atorvastatin.