| Background(1) The risk of contrast induced acute kidney injury was increasing year by year.Along with the increase use of Computed Tomography and cardiac catherization with contrast, the risk of contrast induced acute kidney injury (CI-AKI) was increasing year by year. The reported incidence of CI-AKI was 1%-5%, and the incidence was as high as 30-50% in high risk patients. The development of CI-AKI is strongly associated with prolonged hospitalization, additional healthcare expenses, late renal and cardiovascular adverse events, and death. The percutaneous coronary intervention (PCI) report of 2013 in our country reported that the patients with PCI had increased to 200 thousand. Therefore, the risk of CI-AKI will still increase year by year.(2) There was no effective therapy for the preventions of CI-AKINow, very few therapeutic options of CI-AKI have consistently shown benefit, except for periprocedural hydration and use of a small amount of low-osmolality contrast media. Accordingly, an essential part of reducing CI-AKI is the ability to identify patients who are at great risk of CI-AKI, so that closer monitoring and swift intervention might be implemented.(3) Discovering new risk factor would be helpful to identifying patients at high risk of CI-AKI.Although some risk factors such as chronic kidney disease, and diabetic mellitus, are strongly associated with CI-AKI, the search for new risk factors that can be used to improve the identification of CI-AKI remains an active area of great interest. Our previous studies demonstrated that the inflammation related biomarker, such as c-reactive protein, N-terminal pro-brain natriuretic peptide and uric acid, were strongly associated with the development of CI-AKI. This might demonstrate that inflammation and endothelial dysfunction contributed to the CI-AKI development. Low density lipoprotein cholesterol (LDL-C) is the most common and strong cardiovascular risk factor associated with incident myocardial infarction and ischemic coronary heart disease. LDL-C induces dysfunction of the endothelium, causes vascular wall infl ammation and induces up-regulation of vaso-constrictive endothelin type B receptor expression with increased vasoconstriction, which play ma-jor role in the development of CI-AKI. However, few studies have been conducted to evaluate the rela-tionship between LDL-C and CI-AKI. Therefore, we hypothesized that LDL-C may be associated with CI-AKI through above mechanisms involved in CI-AKI development. Then, we conducted the present study to evaluate whether LDL-C is an independent risk factor of CI-AKI in patients undergoing PCI.(4) The mechanism of contrast induced acute kidney injury remained unclear, and new mechanism would be helpful to find new CI-AKI therapyIn addition, the mechanism of CI-AKI remained unclear, although many researches reported that the renal toxicity of contrast and renal medulla ischemia hypoxia were important contributors to the development of CI-AKI. Unfortunately, most previous CI-AKI models are not appropriate since they were induced in rats by the intravenous injection of a high osmolality CM, in addition to reagents that inhibit prostaglandin (indomethacin) and nitric oxide synthesis, which are associated with the greatest risk of developing CI-AKI and are not used in clinical practice. In contrast, a low or iso-osmolality CM is widely used in clinical settings. Therefore, it is important to develop an experimental model for LOCM-induced AKI with different definitions for AKI based on SCr and cys-C in order to develop new mechanisms or strategies for preventing CI-AKI in the future.(5)The relationship between miRNAs and acute kidney injuryTo date, there were not biomarkers of early-predicting CI-AKI. Therefore, more and more researches amied to identify new biomarker of CI-AKI, such as Neutrophil gelatinase-associated lipocalin, KIM-1, IL-8, miRNA. miRNA has some following advantages:tissue specificity, easy amplification pathway, able to withstand repeated freeze-thaw and very stable form in the blood and other body fluids. More and more study demonstrated that miRNA might be the new biomarker for CI-AKIGodlwin J et al performed a research to examine expression profiles of miRNAs following renal renal ischemia reperfusion injury. Global miRNA expression profiling on samples prepared from the kidneys of C57BL/6 mice that underwent unilateral warm ischemia revealed nine miRNAs (miR-21, miR-20a, miR-146a, miR-199a-3p, miR-214, miR-192, miR-187, miR-805, and miR-194) that are differentially expressed fol-lowing IRI when compared with sham controls. Among them miR-21 and miR-20a increased quickly after renal renal ischemia reperfusion injury,and sustained for 7 days.Either the levels of tissues or serum of miRNAs could predict the progressing of AKI and might be used to early identify AKI. However, the mechasim of AKI with different causes would be not all the same. Therefore, the expression of miRNAs would also be different. Now, few studies were performed to evaluate the expression of miRNAs in CI-AKI, especially for CI-AKI based on the new model. The findings of expression of miRNAs in CI-AKI would be important to research the mechasim of CI-AKI.(6)The relationship between autophagy and acute kidney injuryAutophagy is a self-eating response to stress and plays important roles in the pathogenesis of a variety of diseases. Many previous researches focused on cancer, and demonstrated the important role of autophagy in development of cancer. Few researches focus on CI-AKI. However, recent years, more and more researches began to focus on the role of autophagy in AKI.In the model of cisplatin induced AKI, the authors found autophagosome formation and LC3-II accumulation. In addition, using the autophagy inhibitor chloroquine, can significantly increased the number of renal tubule cell apoptosis and improve the level of blood urea nitrogen and creatinine. However, using the rapamycin with upregulation of autophagy can lead to opposite result. The result was consistent with Periyasamy-Thandava et al, and demonstrated the protective role of autophagy in AKI. However, some previous studies reported the opposite results. Inone K et al demonstrated that the expression of autophagy and apoptosis in the renal proximal tubule increased after the use of cisplatin. The levels of apoptosis increased after inhibiting the autophagy or silent the Beclin-1. These result indicated that autophagy could induce the apoptosis of renal proximal tubule. Therefore, the role of autophagy would be influenced by the different models of AKI. However, more and more researchers or researches supported the protective role of autophagy on AKI. To date, few studies were performed to evaluate the impact of autophagy on CI-AKI.(7)The relationship among miRNAs, autophagy and acute kidney injuryPickering MT et al inhibited miRNA-20a to lead to a Gl checkpoint due to an accumulation of DNA double strand breaks, resulting from premature temporal accumulation of the E2F1 transcription factor. These data provide a mechanistic view of miRNA-20a regulation of E2F1 in the context of the emerging model that miRNAs coordinate the timing of cell cycle progression. These results demonstrated that E2F1 was the key gene of autophagy. In addition, miRNA20a might regulate autophagy through E2F1. Therefore, we hypothesized that miRNA20a would influence the development of CIAKI through the same methods.Atg7, as the E1-like enzyme, was the important autophagy related gene. The first pathway involves the covalent conjugation of Atg12 to Atg5 and the E2-like enzyme Atg10. This conjugate is organized into a complex by associating with Atg16 in a noncovalent fashion to form the multimeric Atg12-Atg5-Atg16 complex, which functions as the E3 ligase of LC3. Therefore, Atg7 was the important authophagy related gene. To evlaute the relationship of Atg7 and miRNAs on the development of AKI, we hypothesized that miRNA-20a might regulate the Atg7 through the bioinformatic analysis.Therefore, we also hypothesized that miRNA-20a could regulate autophagy by targeting Atg 7 in the CI-AKI rat model.Based on the above background, the present study aimed to evaluate the mechanism of CI-AKI and the preventinon of CI-AKI in clinical practice. First, we amied to establish the novel CI-AKI rat model based on low osmolar nonionic contrast medium and was accordance with clinical practice. Second, we aimed to identify the expression of miRNAs of CI-AKI through the gene chip technology, and identify the different expression of miRNA20a between groups with CI-AKI and control. Third, we aimed to evaluate the expression of autophagy and miRNA20a in CI-AKI model, and then demonstrate the Targeted relationship of miRNA20a and Atg 7, and demonstrate that miRNA20a might mediate autophagy by targeting Atg 7 in the CI-AKI rat model. Last, we investigated the relationship of LDL-C and CI-AKI in our prospective observational research, and to identify whether LDL-C was the novel risk factor of CI-AKI.Part1. A novel simple experimental model for low-osmolar contrast-induced acute kidney injury using different definitions based on the levels of serum creatinine and cystatin CObjectivesTo develop a novel simple experimental model in rat for low-osmolar contrast-induced acute kidney injury (CI-AKI) with different definitions for AKI that would be comparable to CI-AKI in humans.MethodsWe used adult male Sprague-Dawley rats aged 8-10 weeks old (190-230g). The rats were kept in standard housing facilities in a 12 h light/12 h dark cycle at 24 ± 1 ℃ and 45 ± 5% humidity. The rats were acclimatized for 7 days before the start of the study. The experimental procedures were performed in accordance with our institutional and national guidelines for animal research.The rats were randomly allocated into the following five groups (n=6 for each group and each experiment):(1) sham (normal saline [NS]+NS), (2) NS plus contrast medium (CMi5) (NS+CM15), (3) furosemide (FM) plus NS (FM+NS), (4) FM+CM10, and (5) FM+CMi5. After they were acclimatized for 7 days, and 6 h before CM administration, groups 3,4 and 5 received furosemide (Harvest Pharmaceutical Co., GuangZhou, China), which was injected intramuscularly (10 mL/kg). The other groups received the same volume of saline. Then, all the groups had restricted access to water for 6 h. After 6 h, groups 2 and 5 received LOCM (350 mg I/mL, Bayer, Guangzhou, China) in the tail vein at a dose of 15 mL/kg, while group 4 received 10 mL/kg of LOCM for under ether anesthesia. Groups 1 and 3 received the same dose of normal saline. Subsequently, all the rats were given unlimited access to standard rat chow and water. The rats were weighed before the furosemide and LOCM injections, and at 24h after CM administration. After 24h following CM administration, all the rats’ blood was sampled to determine the SCr levels, and they were killed by decapitation. Their kidneys were removed for histological analyses. The SCr and cys-C measurements were performed by using a biochemical automatic analyzer. CI-AKI was defined as an absolute increase in serum creatinine of≥0.5 mg/dL or≥25% over the baseline value within 48-72 h after contrast exposure.ResultsIn the FM+CMi5 group, SCr concentration was significantly higher after CM exposure than before (32.9 ± 4.57 vs.158.7 ± 14.48 μmol/L, p<0.001). In the other groups, there were minor changes in the SCr levels between the pre- and post-CM or NS exposure. In addition, the cys-C levels were also higher after CM exposure than that before in the FM+CM15 group (0.08 ± 0.03 vs.0.18 ± 0.05 mg/L, p< 0.001). There were minor changes in the FM+NS group before and after NS administration. Furthermore, only rats in the FM+CMi5 group developed CI-AKI based on the definitions for SCr or cys-C. The histopathological scores were significantly higher in the FM+CMi5 group than in the FM+NS group. The result of HE analysis in CI-AKI group demonstrated that renal tubular was disorder, and vacuole degeneration was found in epithelium cells; The lumen of renal tubular was changed into small, even occlusion. And it exists pyknosis and nuclei splitting. However, the control group was reported that enal tubular, and the lumen and structure of renal tubular were relatively normal. In addition, the result of SD rats with transmission electron microscopy reported that basilar membrane, mitochondria and microvilli structure were relatively normal. But the result CI-AKI group reported that the basal membrane of renal tubular was thickened; mitochondrial was swelling and disorder; and the microvilli showed partly falling and vacuolating.ConclusionsWe developed a simple and reliable animal model for LOCM-induced AKI that is similar to clinical CI-AKI based on different definitions for AKI.Part 2.1dentify the expression of miRNA between CI-AKI and control groups, and the expression of miRNA20a in groups with CI-AKI or without CI-AKI.ObjectivesTo identify the expression of miRNA between groups with Contrast induced acute kidney injury (CI-AKI) and without CI-AKI(control), and to identify the expression of miRNA20a in groups with CI-AKI or without CI-AKI.MethodsBased on the previous method, the rats were randomly allocated into the following 2 groups (n=3 for each group and each experiment):(1) furosemide (FM) plus NS (FM+NS, contol gourp), and (2) FM+CM15 (CI-AKI groups). After 24 h following CM or NS administration, all the rats’blood was sampled to determine the SCr levels, and they were killed by decapitation. Their kidneys were removed for analyses. The expression of miRNAs in the kidney tissue of rats was analysised by miRNA microarray assays. Real-time PCR was performed to validate results of the autophagy related miRNA.ResultsBased on analysis of miRNA microarray assays, we found that there were 230 miRNAs were differently expressed in the kidney tissues between CI-AKI and control groups. Among these, there were 108 up-expressed miRNAs (such asmiRNA148a-3p, miRNA25a-3p, and miRNA20a-5p) and 108 down-expressed miRNAs (miRNA3596d, miRNA3584-3p, and miRNA340-3p) between two groups with more than two-fold changes. Through the analysis of the network of target-gene, and based on the previous researches, we choosed the miRNA-20a as the target of the future researches. In addition, Real-time PCR demonstrated that the expression of miRNA-20a was higher in CI-AKI group than control group, either in the renal tissues or serum sample.ConclusionsmiRNA microarray assays demonstrated that there were many different expressions of miRNAs between CI-AKI and control groups. It might be the first to demonstate this result based on the new simple CI-AKI aminal model. To evaluate the mechanism of miRNA in the development of CI-AKI, we choosed miRNA20a based on the previous researches and bioinformatic analysis.Part3. miRNA-20a mediates contrast-induced autophagy bytargeting Atg7 in contast induced acute kidney injury rat model.ObjectivesTo demonstrate MiR-20a mediates contrast-induced autophagy by targeting ATG7 in contast induced acute kidney injury rat model.MethodsBased on our previous established CI-AKI rat model and the resut of of miRNA microarray assays, we performed the bioinformatics analysis to find the possible autophagy related target gene of miRNA-20a. And the gene must be proven in preivous researches. We used the Dual-luciferase reporter gene assay to demonstrate the relationship of miRNA20a and target gene. Then we use real-time PCR, wester-blot and transmission electron microscopy to find the expression of autophagy in CI-AKI rat model.And then we used the antagomir miRNA20a to down-regulated the expression of miRNA-20a to demonstrate that miRNA20a might mediate the autophagy through Atg7. And we also measured the levels of serum creatinine and HE analysis to find the impact of autophagy on renal tissues.ResultsAccording to the result of database of TargetScan and MicroRNA.ORG, we found the target gene of the miRNA20a was Atg7, which has been proven to be an important autophagy related gene. And the Dual-luciferase reporter gene assay demonstrated that Atg 7 was the target gene of the miRNA20a. The western-blot demonstrated that the expression of autophagy related protein Belcin-1 was higher in CI-AKI group than the control group. And the real-time PCR demonstrated that the level of Atg7 was also higher in CI-AKI group than control group. In addition, the transmission electron microscopy reported that the number of autophagosomes were also more in CI-AKI group than control group. Furthermore, antagomiR-20a increased the expression of Atg7 and beclin-1 in renal tissues. The level of serum creatinine of was lower in antagomiR-20a group than control group. The HE analysis also demonstrated that the injury of renal was lighter in antagomiR-20a group than control group.ConclusionsWe might be the fisrt to demonstate that the autophagy exists in the development of CI-AKI. In addition, we used our resut of of miRNA microarray assays to find miRNA-20a and used the dual-luciferase reporter gene assay to demonstrate the target gene of miRNA-20a was Atg7, which has been proven to be an important autophagy related gene. Furthermore, considering the protection of atuophagy in the AKI based on previous researches, we also demonstrated that interventions of miRNA-20a can mediate the autophagy by targeting Atg7 in the CI-AKI development.Part4. LDL cholesterol as a novel risk factor for contrast-induced acute kidney injury in patients undergoing percutaneous coronary interventionObjective:To evaluate whether Low density lipoprotein cholesterol (LDL-C) is an independent risk factor of contrast-induced acute kidney injury (CI-AKI) I in patients undergoing percutaneous coronary intervention (PCI) is unknown.MethodsWe prospectively enrolled 3236 consecutive patients undergoing PCI between January 2010 and September 2012. Univariate and multivariate logistic regression analyses were performed to identify independent risk factors of CI-AKI. Univariate analysis included variables such as LDL-C, diabetes, he-moglobin, renal failure, CM volume, Mehran risk score and intra-aortic balloon pump. Variables with p values <0.15 in the univari-ate analysis were entered into multivariate logistic regression analysis by forward stepwise selection. In addition, the risk factors that were proven to be associated with the CI-AKI in previous researches were also included. The primary end point was the development of CI-AKI, defined as an absolute increase in serum creatinine of≥0.5 mg/dL or≥25% over the baseline value within 48-72 h after contrast exposure.ResultsA total of 3236 consecutive patients undergoing PCI were analyzed (mean age 63.3±11.0 years; mean LDL-C level,2.7 ± 0.9 mmol/L; mean eGFR 81.4 ± 25.3 mL/min/1.73 m2 and mean Mehran score 4.5 ± 3.8). CI-AKI was observed in 338 patients (10.4%). Patients with CI-AKI had a significantly higher rate of in hospital mortality (0.5% vs.4.4%, p<0.001), and significantly higher rates of other in hospital complications compared with those without CI-AKI. The LDL-C quartiles were as follows:Ql (<2.04 mmol/L), Q2 (2.04-2.61 mmol/L), Q3 (2.61-3.21 mmol/L) and Q4 (>3.21 mmol/L). Patients with high baseline LDL-C levels were more likely to develop CI-AKI and composite end points (8.9%,9.9%,10.5%,12.6%, p=0.001, and 5.0%,5.2%,6.1%,8.1%, respectively; p=0.007). Univariate logistic analysis showed that LDL-C levels (increment 1 mmol/L) were significantly associated with CI-AKI (odds ratio=1.25,95% confidence interval (CI),1.11-1.39, p<0.001). Furthermore, LDL-C was still remained a strong significant risk factor of CI-AKI (odds ratio=1.23, 95% CI,1.04-1.45, p=0.014), even after adjusting for potential confounding risk factors. In addition, chronic heart failure, hypo-albuminemia and primary PCI were other independent risk factors of CI-AKI in our population.ConclusionsThe present study demonstrated that the incidence of CI-AKI was as high to 10.2% in unselected patients undergoing PCI. And patients with CI-AKI presented poor in-hospital outcomes. The present study might be the first to report that LDL-C was an new risk factor for CI-AKI, and measurement of plasma LDL-C concentrations in patients undergoing PCI may be helpful to identify those who are at risk of CI-AKI and poor in hospital outcomes.In addition, we also fond that chronic heart failure, hypo-albuminemia and primary PCI were other independent risk factors of CI-AKI in our population... |
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