The Effect Of RAS Activation On Macrophage Polarization In Adipose Tissue In Rat With Chronic Renal Failure

The relationship between inflammation and activation of renin-angiotensin system activation in adipose tissue in CRF and its mechanismBackgroundChronic kidney disease (CKD) is increasing in epidemic form throughout the world, primarily due to the overall increase in the prevalence of CKD as a major cause of hypertension (HTN), diabetes mellitus (DM) and obesity.1-3According to data from the National Health and Nutrition Examination Surveys (NHANES), the prevalence of CKD (excluding terminal CKD) in the United States was13.1%in the period1999-2004.1.3The prevalence of CKD stages3-5showed up around5%, based on data from NHANES1999-2004in the United States and in studies conducted in Australia, China and India.Cytokine expression and immune cell infiltration causing renal inflammation accompanies almost all kidney diseases. Patients with chronic kidney disease (CKD) are considered to be at significantly increased risk of cardiovascular disease (CVD), especially those with end-stage renal disease (ERSD) treated with dialysis or renal transplantation even if their graft function within normal range. Angiotensin (Ang) II, the main effector of the renin-angiotensin system (RAS), is one of the major mediators of vascular remodeling in hypertension. Besides being a potent vasoactive peptide, Ang II exerts proinflammatory effects on the vasculature by inducing integrins, adhesion molecules, cytokines and growth and profibrotic mediators through activation of redox-sensitive pathways and transcription factors. The renin-angiotensin system (RAS) plays a key role in the development and pathophysiology of hypertension and cardiovascular disease (CVD).Properly functioning adipose tissue is essential to human health, serving as a source of energy during fasting and providing insulation for temperature regulation, among other roles. In the past, adipocytes were recognized as an inert energy source; however, adipocytes are now known to synthesize and secrete proinflammatory factors such as cytokines, acute-phase response proteins, chemotactic/chemoattractants, eicosanoids, prostaglandins, and potentially anti-inflammatory effectors (eg, adiponectin, interleukin [IL]-6, and IL-10)[1]. Collectively, these adpocyte-derived factors have been termed adipokines They have increased steadily ovier the years; more than50different adipokines are now known to be secreted from adipocytes. Adipose tissue comprises adipocytes (30-50%), preadipocytes and fibroblasts, collagen fibre matrix, blood vessels and immune cells (monocytes/macrophages and lymphocytes). Adipokines are peptides secreted by adipocytes (adiponectin, leptin, visfatin, etc.) whereas cytokines refer to the peptides secreted from the stromavascular fraction of cells (MCP-1, macrophage infiammatoryprotein, TNFa and ILs1(3,6,8,10, etc.). There is an overlap, however, because an adipokine may be secreted from both (e.g. apelin and resistin). It is well known that mortality rates among adults with end-stage renal disease (ESRD), particularly those receiving dialysis, are significantly greater than among agematched controls in the general population. Enhanced chronic systemic inflammation and reduced insulin sensitivity are often associated in patients with chronic renal failure, contributing to cardiovascular morbidity and mortality in these patients.Macrophage infiltration is a common feature of obesity and many kidney diseases. However, macrophages have significant heterogeneity in their functions. Macrophages have recently been classified into two polarization states, Ml and M2. M1or "classically activated" macrophages are induced by classic immune pathways (such as LPS and interferon-y), and enhance proinflammatory cytokine production, such as TNF-α, IL-1β, and IL-6. M2or "alternatively activated" macrophages are generated by exposure to IL-4and IL-13. M2macrophages are important in resolution of inflammation and tissue repair through synthesis of anti-inflammatory cytokines IL-10and IL-1decoy receptor and endocytic clearance capacities. Inhibition of proinflammatory macrophages has beneficial effects in experimental chronic inflammatory renal injury. M1macrophages with M2macrophages in the body in a state of dynamic equilibrium.For a long time, people think that the RAS regulates only the systolic blood pressure and renal electrolyte balance, however, nearly a decade of research found that a variety of components of the renin-angiotensin system expression in multiple tissues,such as adrenal gland, kidney, brain, heart and blood vessels, and plays an important role in various organs of local. With the increase in the incidence of obesity and metabolic syndrome, the expression of RAS and its function have been taken seriously. A large number of studies have demonstrated that human and mouse adipocytes express angiotensinogen (AGT), angiotensin-converting enzyme (ACE) and angiotensin receptor1(AT1) and2(AT2). Briefly, renin (protease) secreted from the granular cells of the juxtaglomerular apparatus reacts with angiotensinogen (AGT) produced by the liver to release Ang I (1-10), which is further cleaved by a dipeptidyl carboxypeptidase, angiotensin-converting enzyme (ACE), released from capillary endothelial cells of the lung, to convert Ang I to Ang II (1-8). Ang II is considered the major physiologically active component of RAS. The biological actions of Ang II are transmitted by two seven-transmembrane G-protein-coupled receptors-AT1R and AT2R. Most of the physiological effects of Ang II are conveyed by AT1R. AT1R activation induces an increase in blood volume and BP by stimulating vasoconstriction, along with adrenal aldosterone secretion, renal sodium reabsorption and sympathetic neurotransmission. Adipose tissue renin-angiotensin system has had a tremendous impact on the body, such as caused by abnormal lipid metabolism, hypertension, insulin resistance, and other parts of inflammatory diseases, including atherosclerosis, inflammatory lung disease, kidney failureand tumors. In addition, the local RAS also play an important role in adipose tissue metabolism and physiological functions. Adipose tissue angiotensin Ⅱ (AngⅡ) inhibits lipolysis and stimulates lipid synthesis through binding to its receptor, thus contributing to the accumulation of adipose tissue lipid and causing obesity. Local AngⅡ inhibits the differentiation of preadipocytes to reduce the number of adipocytes, promote the hypertrophy of adipocyte; angiotensin II also promotes the release of inflammatory cytokines, causing inflammation reaction of the adipose tissue. Ang Ⅱ promotes the release of these proinflammatory cytokines from both mature adipocytes and preadipocytes through activation of the NF-kB pathway, Ang Ⅱ may also function on stromal vascular immune cells, such as monocytes, macrophages, and T cells, that are known to express local RAS. Therefore, activation of adipose tissue RAS caused the adipose tissue local and the systemic inflammatory response.We have demonstrated that inflammation and activation of RASexists in white adipose tissue in chronic renal failure rats,but the relationship between inflammation and activation of RAS in unknown.In this study,weare should to reveal the relationship between inflammation and activation of RAS and tis mechanism. MethodsStudies in vivo1. Animal model preparationsSD male rats were maintained on a12/12h light/dark cycle at25℃and fed adlibitum a standard laboratory chow for one week. Subsequently, the animals were randomly assigned to two groups:sham (n=8).chronic renal failure (n=24). Surgical five-sixth nephrectomy (5/6Nx) and sham operations in rats were performed. Briefly, under pentobarbital anesthesia, two-thirds of the left kidney was removed in the first stage of the procedure, and a week later the right kidney was totally excised (totally5/6Nx). Control animals were sham-operated with only decapsulation of the kidney.2. Angiotensin receptor blocked (Telmisartan) stimulation12weeks after the operation, the CRF rats were randomized into3groups (n=8in each group) matched for body weight and received the following treatment: intragastric administration of Telmisartan, or hydralazine alone, or without anything. The treatments lasted4weeks.3. The collection of blood, urine and adipose tissueAt the16weeks after opertation, the consecutive24-hour urine samples were collected in metabolic cages before the animals were sacrificed. The animals (n=6in each group) were anesthetized with sodium pentobarbital. The blood was collected, and the adipose tissue was removed and the outer connective tissues were stripped. Then cut the tissue into small pieces and store in liquid nitrogen.4. Determination of the phosphorylation of NF-κB P65, STAT1/3andexpression of SOCS1/3Adipose tissue was homogenized in ice-cold fractionation buffer. The lysate was incubated on ice for15min and then centrifuged at13,000×g for40min at4℃. The cytosolic fraction was localized below the layer of the fat cake. The cytosolic fraction was aspired from below the solidified fat cake, and an aliquot was used to measure protein by the Bradford method. Samples were diluted4:1(v/v) with5×Laemmli buffer, heated to95℃and then the phosphorylation of NF-κB P65,STAT1/3and SOCS1/3protein was detected by western blotting.5. Determination of IL-6、MCP-1、TNF-α、MCP-1、iNOS、IL-1β、Arg1、 CD206mRNA dectected by real-time PCRTotal RNA of adipose tissue was extracted with TRIZOL reagent. IL-6mRNA, TNF-amRNA, MCP-1、iNOS、IL-1β, Arg1、CD206mRNA were detected by real-time PCR according to the manufacturer’s protocol.6. StatisticsAll values are mean±SEM. The significance of differences among mean values was determined by One-way ANOVA. Statistical comparison of the control group with treated groups was performed using statistical soft ware SPSS13.0.The accepted level of significance was P<0.05.Studies in vitro1. Cell cultureRAW264.7macrophage were obtained from American Type Culture Collection (NO.TIB-71) and were cultured in high glucose Dulbecco’s minimum essential medium(DMEM) with10%fetal bovine serum(PPA,German), at37℃in a5%CO2incubator, After reaching confluence, RAW264.7cells were incubated in DMEM overnight and treatment with LPS or mIL-4and AngⅡ for the indicated concentration and time.2. The effect of AngⅡ on LPS or mIL-4induced polarization of macrophagesRAW264.7cells were exposed to LPS (10ng/ml) or mIL-4(100ng/ml) and AngⅡ for indicated concentration,0.01,1,10, umol/L; or for indicated time,16,20,24h. Total RNA of cell was extracted with TRIZOL reagent. IL-1β mRNA, CD11c mRNA,iNOS mRNA, Argl mRNA, CD206mRNA, dectin mRNA were detected by real-time PCR according to the manufacturer’s protocol.3. StatisticsAll values are presented as mean±SEM. The significance of differences among mean values was determined by One-way ANOVA. Statistical comparison of the control group with treated groups was performed using statistical soft ware SPSS13.0.The accepted level of significance was P<0.05.ResultsStudies in vivo1. General dataData are summarized in Table l.As expected, the CRF group exhibited a significant increase in serum creatinine and urine protein concentration (P<0.001), and a significant lower creatinine clearance and body weight (P<0.001) when compared with the sham-operated normal control group.2. The effect of CRF on inflammation in adipose tissue and macrophage polarization2.1. IL-6,TNF-a,MCP-1, IL-1β,iNOS,Arg1,CD206,dectin gene expression in SC and VC adipose tissueThe gene expressions of IL-6,TNF-α,MCP-1in SC and VC adipose tissue from CRF rats were increased relative to sham-operated animals, and telmisartan restrains inflammation and polarize macrophage in adipose tissue (SC:IL-6F=10.634,P=0.004;TNF-a:F=7.986, P=0.009; MCP-1:F=44.408,P<0.001;IL-1β: F=78.067, P<0.001;iNOS:F=26.558, P<0.001;Argl:F=21.272, P <0.001;CD206:F=24.090, P<0.001;dectin:F=77.401, P<0.001;VC:IL-6F=,8.178,P=0.008; TNF-a:F=7.830,P=0.009; MCP-1:F=7.951, P=0.009; IL-1β:F=11.529,P=0.003,iNOS:F=41.300, P<0.001;Argl:F=45.307, P<0.001, CD206:F=7.098,P=0.012;dectin:F=34.616,P<0.001),2.2.Phosphorylation of NF-κB P65and STAT1/3protein expression of SOCS1/3in SC and VC adipose tissueThe phosphorylation level of NF-κB P65, STAT1/3and expression of SOCS1/3in SC and VC adipose tissue from CRF rats were increased relative to sham-operated animals (SC:P65:F=14.088,P=0.002;STAT1:F=26.698, P<0.001;STAT3:F=48.309, P<0.001;SOCS1:F=27.093; P<0.001;SOCS3:F=22.869;VC:P65:F=8.885,P=0.032; STAT1:F=29.260, P=0.001;STAT3:F=29.853,P<0.001;SOCS1:F=62.087; P<0.001;SOCS3:F=10。421, P=0.004).Conclusion1.Inflammation exists in SC and VC fat pads in CRF.2.Telmisartan contributed to macrophage M1to M2conversion and restrained inflammation through the inhibition of SOCS1/3and then increased phosphorylation of STAT1/3in CRF rat adipose tissue.1. Studies in vitro Effect of AngⅡ on polarization of macrophagesAfter the stimulation of RAW264.7with lOng/ml LPS or100ng/ml mIL-4and lumol/L AngⅡ for16,20,24hours, the gene expressions of IL-1β,CD11c,iNOS mRNA increase gradually with the prolonging of time (IL-10:F=116.085, P<0.001, CD11c:F=61.221, P<0.001,iNOS:F=36.728,P<0.001),and the gene expression of Argl,CD206,dectin decrease gradually with the prolonging of time(Arg1:F=148.706, P<0.001;CD206:F=129.299, P<0.001;dectin:F=36.196, P<0.001). The effect was maximal at24h treatment. Moreover, the gene expressions of IL-1β,CD11c,iNOS increase gradually with the increase of10ng/ml LPS and AngⅡ doses (IL-1β: F=168.363,P<0.001, CD11c:F=23.389, P<0.001,iNOS:F=60.192,P<0.001), the gene expression of Argl,CD206,dectin decrease gradually with the AngⅡ doses and100ng/ml mIL-4(Argl:F=66.050, P<0.001;CD206:F=35.013, P<0.001;dectin:F=42.032, P<0.001). The effect was maximal at10umol/L AngⅡ treatment.ConclusionOur results revealed that AngⅡ have synergy on the polarization induced by LPS and mIL-4in RAW264.7.