| Chronic kidney disease(CKD) is a main cause of end stage renal failure which affects human’s health seriously. Long term of rational therapy might help to block multiple pathways involved in renal damage so that the progression of renal failure could be prevented. As a result, large amount of researches were carried out to clarify the detailed mechanism of progressive renal disease.The renin-angiotensin-aldosterone system(RAAS) is a humoral regulation cascade that functions in the homeostatic control of blood pressure, extracellular volume and tissue perfusion. In addition to the circulation system, members of RAAS are also detected in tissues such as heart, vessel and kidney which participate in the development of hypertensive target organ damage. Dysregulation of Angiotensin Ⅱ(Ang Ⅱ), the main effector of RAAS, could lead to the elevation of blood pressure as well as high perfusion pressure of kidney which will then result in renal pathophysiological changes. In addition to hemodynamic effects, Ang Ⅱ is proven to play an important role in processes of inflammation and fibrosis. It is validated that Ang Ⅱ-induced renal inflammation and fibrosis are mediated via multiple mechanisms. Transforming growth factorβ1(TGFβ1)/Smad, Rho/Rho kinase and NF-κB are main signaling pathways involved in the production of extracellular matrix and recruitment of inflammatory cells. Renal inflammation and fibrosis are markedly suppressed by blocking these pathways. Importantly, angiotensin-converting enzyme inhibitors(ACEI) or AT1 receptor blockers(ARB) have been suggested to ameliorate the progression of hypertensive renal injury and bring clinical benefits. However, the progression of renal damage could not be completely controlled by ACEI or ARB. As a result, it is important to elucidate the precise molecular mechanism of acute renal injury in Ang Ⅱ-induced hypertension so that effective therapy strategy could be established.Recently, microarray analysis has been wildly used to investigate the global alterations of gene expression and identify genes that are important to cardiovascular and other diseases. Therefore, microarray analysis may help us understand crucial factors for kidney damage and elucidate the precise molecular mechanisms of acute renal injury. In this study, microarray assay and biological analysis are used in an Ang Ⅱ-induced hypertension mouse model for the investigation of important genes participate in the development of early renal injury.Objective:To establish a mouse model of hypertensive early renal injury with Ang Ⅱ infusion. Base on this model, to investigate the significant genes, gene functions and pathways associated with renal injury by microarray assay and biological analysis.Methods:1. Eight to ten-week-old C57BL/6 male mice were implanted with osmotic pumps and infused with Ang Ⅱ at 1500ng/kg/min for 1, 3, and 7 days. Systolic blood pressure was measured by the tail-cuff method every day before and after Ang Ⅱ infusion.2. After infusion of Ang Ⅱ for 1, 3, 7 days, H&E, immunohistochemical staining of Mac-2, Masson’s trichrome, and periodic acid-Schiff staining(PAS) were carried out to evaluate the degree of renal inflammation, fibrosis and glycogen deposition.3. Gene expression profiling was performed using Affymetrix Gene Chip Mouse Genome 430 2.0 Array according to the manufacturer’s instructions.4. The random-variance model(RVM) F-test was applied to filter the differentially expressed genes for the control and Ang Ⅱ-treated group(at days 1, 3, and 7). q PCR and correlation analysis were performed to confirm the reliability of microarray data.5. GO analysis was used to organize genes into hierarchical categories and uncover the gene regulatory network on the basis of biological process and molecular function. Specifically, two-side Fisher’s exact test and χ2 test were used to classify the GO category, and FDR was calculated to correct the P-value, the smaller the FDR, the small the error in judging the P-value.6. Pathway analysis was used to find out the significant pathway of the differential genes according to KEGG. Fisher’s exact test and χ2 test were used to select the significant pathway, and the threshold of significance was defined by P-value(P<0.05) and FDR.7. The series test of cluster(STC) algorithm of gene expression dynamics was used to profile the gene expression time series and to identify the most probable set of clusters generating the observed time series. The expression model profiles are related to the actual or the expected number of genes assigned to each model profile.8. The normalized expression value of significant differential genes from the five most significant profiles, namely profiles 19, 21, 8, 22 and 6, was used to build a co-expression network in order to find out the most important genes that participate in the process of Ang Ⅱ-induced renal injury.9. Quantitative real-time PCR(q PCR) was used to verify the differential expression of 17 selected genes that were detected by microarray. The gene expression levels were quantified relative to the expression of GAPDH.Results:1. Hypertensive mouse model was successfully established by Ang Ⅱ infusion. The blood pressure was markedly increased in a time-dependent manner(at days 1, 3 and 7).2. The number of polymorphonuclear cells increased in renal tissue after Ang Ⅱ infusion as shown by H&E staining.3. The number of Mac-2-positive macrophages increased in renal tissue after Ang Ⅱ infusion as shown by immunohistochemical staining.4. The area of collagen deposition enlarged after continuous infusion of Ang Ⅱ as shown by Masson’s trichrome staining.5.No significant difference was detected between Ang Ⅱ infused group and control with PAS staining.6.As shown by microarray analysis, a total of 1,511 genes significantly expressed in Ang Ⅱ-infused mice at least at one time point when compared to control. 640 probes significantly increased and 434 probes decreased in the 1 d group; 380 probes markedly increased and 277 probes decreased in the 3 d group; 224 probes increased and 190 probes decreased in the 7 d group. A significant correlation between microarray and q PCR results was detected by correlation analysis.7. High enrichment GO terms yielded from GO analysis included response to stimuli(including drug, nutrient, glucocorticoid, lipopolysaccharide, hypoxia, peptide hormone, c AMP, mechanical, ethanol, cytokine, estradiol, hydrogen peroxide, cold, organic nitrogen and insulin), aging, organ regeneration, immune response, cell adhesion, metabolic processes(including lipid, fatty acid, cholesterol and lipoprotein), kidney development, regulation of blood pressure, cellular processes(cell proliferation and differentiation), and ion transport.8. 93 pathways were significantly modulated as shown by biological pathway analysis. Several key pathways associated with metabolic pathways(including PPAR, cytochrome P450, retinol, amino acids, fatty acid, nitrogen and lipid), immune/inflammatory processes(including cytokine-cytokine receptor interaction, antigen processing and presentation, natural killer cell mediated cytotoxicity, chemokine signaling pathways, adipocytokine, leukocyte transendothelial migration and TLR), ECM-receptor interaction, rennin-angiotensin system, MAPK, m TOR, Jak-STAT and Wnt signaling pathways as well as apoptosis were significantly overrepresented.9. After STC analysis, 1,511 genes were classified into 26 profiles based on expression patterns. Among these profiles, 5 significant profiles were identified including No. 19, 21, 8, 22 and 6. Gene expressions in profiles No. 19, 21 that are comprised of 275 and 176 genes respectively, were increased at early time points(1 or 3 d) and then gradually decreased at later times(3 or 7 d); gene expressions in profile 22 were constantly increased at all times(1, 3, and 7 d); while the genes in profiles No. 8 and 6 had opposite effects compared with genes in profiles No. 19, 21. Among these patterns, one most significant pattern was profile 19 according to ascending P-values.10. Fatty acid binding protein 1(Fabp1) appeared at the center of the large-scale gene co-expression network and the 46 k-core subnetwork. It directly regulated 63 neighboring genes that interact according to their degrees. These interactions depended in large part on Fabp1 because the clustering coefficient of this gene was 0.85, which was lower than for other genes.11. The expression levels of genes(including Fabp1、Fgl1、Rgn、Ccdc69、BB144871、Slc36a2、OTTMUSG00000008561, Tmem30a、Tmem25、Fads3、Plagl1、Ace、Acy1、Hk2、Hba-a1、Fkbp5、Bcl2l11) detected by q PCR were consistent with microarray analysis.Conclusions:1. The systolic blood pressure can increase to 150-170 mm Hg after Ang Ⅱ infusion.2. Renal inflammation is aggravated by Ang Ⅱ infusion featured as increased polymorphonuclear cells and macrophages infiltration.3. Renal fibrosis is aggravated by Ang Ⅱ infusion.4. Ang Ⅱ infusion has no effect on renal glycogen deposition.5. Total 1,511 genes are differentially expressed in kidney during Ang Ⅱ infusion at days 1, 3 and 7, while the number of differentially expressed genes is decreasing.6. The most significant GO terms include response to stimuli, aging, organ regeneration, immune response, cell adhesion, metabolic process, kidney development, regulation of blood pressure, cellular processes, and ion transport, suggesting that these GO terms may play critical roles in Ang Ⅱ-induced early renal injury.7. Several key pathways such as metabolic pathways, immune/inflammatory process, ECM-receptor interaction, rennin-angiotensin system, MAPK, m TOR, Jak-STAT and Wnt signaling pathways are significantly overrepresented after Ang Ⅱ infusion.8. Profile No. 19 that exhibits an expression pattern of increased expression at 1 d and decreased expression at days 3 and 7 of Ang Ⅱ infusion is considered to be the main potential expression pattern in our experiment.9. Fabp1 appears at the center of co-expression network indicating an important role in the development of acute renal injury in Ang Ⅱ-induced hypertension. |
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