Inflammation and Fibrosis in Perirenal Adipose Tissue of Patients With Aldosterone-Producing Adenoma

Inflammation and Fibrosis in Perirenal Adipose Tissue of Patients With Aldosterone-Producing Adenoma Abstract The prevalence of primary aldosteronism is much higher than previously thought. Recent studies have shown that primary aldosteronism is related to a higher risk of cardiovascular events. However, the underlying mechanism is not yet clear. Here we investigate the characteristics, including inflammation, fibrosis, and adipokine expression, of adipose tissues from different deposits in patients with aldosterone-producing adenoma (APA). Inflammation and fibrosis changes were evaluated in perirenal and subcutaneous adipose tissues obtained from patients with APA (n = 16), normotension (NT; n = 10), and essential hypertension (EH; n = 5) undergoing laparoscopic surgery. We also evaluated the effect of aldosterone in isolated human perirenal adipose tissue stromal vascular fraction (SVF) cells and investigated the effect of aldosterone in mouse 3T3-L1 and brown preadipocytes. Compared with the EH group, significantly higher levels of interleukin-6 (IL-6) and tumor necrosis factor-α messenger RNA (mRNA) and protein were observed in perirenal adipose tissue of patients with APA. Expression of genes related to fibrosis and adipogenesis in perirenal adipose tissue was notably higher in patients with APA than in patients with NT and EH. Aldosterone significantly induced IL-6 and fibrosis gene mRNA expression in differentiated SVF cells. Aldosterone treatment enhanced mRNA expression of genes associated with inflammation and fibrosis and stimulated differentiation of 3T3-L1 and brown preadipocytes. In conclusion, these data indicate that high aldosterone in patients with APA may induce perirenal adipose tissue dysfunction and lead to inflammation and fibrosis, which may be involved in the high risk of cardiovascular events observed in patients with primary aldosteronism. To date, accumulating evidence has demonstrated that the incidence of primary aldosteronism (PA) is much higher than previously believed (1). Compared with patients with essential hypertension (EH) matched for blood pressure, sex, and age, patients with PA have an increased rate of cardiovascular events and mortality (2, 3). And the prevalence of type 2 diabetes and metabolic syndrome are higher in PA than in matched patients with EH (4). Adipose tissue inflammation and fibrosis play an important role in cardiovascular disease. In patients with high-risk heart disease, epicardial adipose tissue expresses significantly higher levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) compared with subcutaneous adipose tissue (5). A previous study has reported that aldosterone worsens metabolism, whereas a mineralocorticoid receptor (MR) antagonist reduced insulin resistance in 3T3-L1 preadipocytes (6, 7). Rondinone et al. (8) reported that 1 to 10 nM aldosterone can induce 3T3-L1 preadipocyte differentiation. Penfornis et al. (9) found that aldosterone induces T37i cells into mature brown adipocytes via an MR. What’s more, research has shown that aldosterone and high-sensitivity C-reactive protein concentrations predict incident metabolic syndrome (10). Aldosterone increases expression of IL-6 and TNF-α in mature 3T3-L1 adipocytes, and MR blockade reduces expression of IL-6 and TNF-α (11). However, the mechanism underlying this phenomenon is still not clear. Adipose tissue inflammation and fibrosis play an important role in cardiovascular disease. Human epicardial adipose tissue induces atrial myocardium fibrosis (12). In addition, a study found that atrial fibrillation is associated with subepicardium adipose tissue remodeling in humans (13). Whether chronic aldosterone increase in patients with PA directly affects adipose tissue inflammation and fibrosis has not yet been clarified. In this study, we explored the fibrosis and inflammation change in perirenal and subcutaneous adipose tissue of patients with aldosterone-producing adenoma (APA). We also evaluated the effect of aldosterone in isolated human perirenal adipose tissue stromal vascular fraction (SVF) cells and investigated the effect of aldosterone in mouse 3T3-L1 and brown preadipocytes. Methods Patients From October 2014 to December 2016, 31 consecutive patients who were referred to our hospital were enrolled in the study. Among them, 16 patients with APA were studied. In addition, 10 patients with normotension (NT) and 5 with EH served as controls. The diagnosis of APA and EH was based on international diagnostic guidelines (14). The characteristics of the patients are described in Table 1. Perirenal and subcutaneous adipose tissues were obtained from patients with APA, NT, and EH undergoing laparoscopic adrenalectomy, nephrolithotomy, or ureterolithotomy. The study was approved by the Institutional Review Board of Nanfang Hospital, and all participating individuals provided written informed consent. Table 1. Patient Characteristics   NT  EH  APA  NT vs APA  EH vs APA  Sex (% female)  50.0%  20.0%  56.3%  —  —  Age (y)  47.2 ± 15.8  55.2 ± 7.2  45.7 ± 9.6  0.763  0.057  BMI (kg/m2)  22.5 ± 2.7  23.9 ± 3.1  23.2 ± 3.2  0.585  0.668  SBP (mm Hg)  123.3 ± 12.9  140.8 ± 16.1  149.4 ± 20.8  0.02  0.413  DBP (mm Hg)  74.6 ± 10.4  89.6 ± 4.7  97.5 ± 14.1  <0.001  0.243  Duration of hypertension (y)  —  4.0 (3.3–8.5)  4.0 (1.0–7.0)  —  0.935  Glycemia (mmol/L)  5.1 ± 0.5  5.2 ± 0.4  5.5 ± 2.1  0.626  0.78  K+ (mmol/L)  3.9 ± 0.7  4.2 ± 0.2  3.6 ± 0.9  0.337  0.132  WBC (109/L)  7.5 ± 2.7  8.5 ± 2.7  7.0 ± 2.1  n.s.  0.195  Creatinine (umol/L)  95.7 ± 64.3  114.8 ± 31.7  68.0 ± 19.3  0.128  0.001  ALT (U/L)  20.2 ± 8.2  22.8 ± 9.3  17.5 ± 2.9  0.215  0.312  AST (U/L)  19.5 ± 13.5  23.0 ± 4.9  19.3 ± 5.6  0.490  0.315  Aldosterone (ng/dL)  —  11.0 (10.3–11.0)  25.6 (23.3–40.9)  —  0.004  Plasma renin activity (ng/mL/h)  —  5.0 (0.6–5.0)  0.2 (0.1–0.5)  —  0.025  ARR  —  2.2 (1.9 ± 2.2)  124.3 (77.7–364.2)  —  0.004    NT  EH  APA  NT vs APA  EH vs APA  Sex (% female)  50.0%  20.0%  56.3%  —  —  Age (y)  47.2 ± 15.8  55.2 ± 7.2  45.7 ± 9.6  0.763  0.057  BMI (kg/m2)  22.5 ± 2.7  23.9 ± 3.1  23.2 ± 3.2  0.585  0.668  SBP (mm Hg)  123.3 ± 12.9  140.8 ± 16.1  149.4 ± 20.8  0.02  0.413  DBP (mm Hg)  74.6 ± 10.4  89.6 ± 4.7  97.5 ± 14.1  <0.001  0.243  Duration of hypertension (y)  —  4.0 (3.3–8.5)  4.0 (1.0–7.0)  —  0.935  Glycemia (mmol/L)  5.1 ± 0.5  5.2 ± 0.4  5.5 ± 2.1  0.626  0.78  K+ (mmol/L)  3.9 ± 0.7  4.2 ± 0.2  3.6 ± 0.9  0.337  0.132  WBC (109/L)  7.5 ± 2.7  8.5 ± 2.7  7.0 ± 2.1  n.s.  0.195  Creatinine (umol/L)  95.7 ± 64.3  114.8 ± 31.7  68.0 ± 19.3  0.128  0.001  ALT (U/L)  20.2 ± 8.2  22.8 ± 9.3  17.5 ± 2.9  0.215  0.312  AST (U/L)  19.5 ± 13.5  23.0 ± 4.9  19.3 ± 5.6  0.490  0.315  Aldosterone (ng/dL)  —  11.0 (10.3–11.0)  25.6 (23.3–40.9)  —  0.004  Plasma renin activity (ng/mL/h)  —  5.0 (0.6–5.0)  0.2 (0.1–0.5)  —  0.025  ARR  —  2.2 (1.9 ± 2.2)  124.3 (77.7–364.2)  —  0.004  Abbreviations: ALT, alanine transaminase; ARR, aldosterone-to-renin ratio; AST, glutamic oxal(o)acetic transaminase; BMI, body mass index; DBP, diastolic blood pressure; K+, serum potassium; n.s., nonsignificant; SBP, systolic blood pressure; WBC, white blood cell count. View Large Isolation of cells from the SVF of human perirenal fat Human perirenal adipose tissue was obtained from a 52-year-old normotensive female who underwent laparoscopic ureterolithotomy. The primary SVF from human perirenal fat was isolated as described previously (15). The primary SVF cells from human perirenal adipose tissue were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). For adipocyte differentiation, cells were grown to 100% confluence and then differentiated with a standard brown preadipocyte differentiation protocol (16). Twelve hours before treatment, differentiated cells were cultured in serum-free DMEM. For the aldosterone stimulation study, at the time of treatment, cells were incubated with vehicle or aldosterone (10−8 mol/L; Sigma) for 24 hours. The selected concentration of aldosterone was based on previous studies (11). Cell culture Mouse 3T3-L1 preadipocytes were obtained from the type culture collection of the Chinese Academy of Sciences. The brown preadipocytes were established as previously described (17, 18). Briefly, interscapular brown adipose tissue was isolated from new-born C57BL/6 mice and digested with collagenase, and then filtered and spun down. The pelleted SVF was plated on culture dishes, immortalized with pBabe-SV40T-puro retroviral vector (expressing large T antigen and puromycin resistant gene), and selected with puromycin. The established brown preadipocyte cell line was tested and verified for their capability of adipogenic differentiation and uncoupling protein 1 (UCP1) expression. The differentiation of 3T3-L1 preadipocytes was performed according to a standard differentiation protocol [DMEM supplemented with 10% FBS, 0.5 mM isobutylmethylxanthine (Sigma), 1 µM dexamethasone (Sigma), and 20 nM insulin (Sigma) for 2 days, and then cells were cultured in DMEM with 10% FBS and 20 nM insulin]. Brown preadipocyte differentiation was initiated with 0.5 mM isobutylmethylxanthine, 1 µM dexamethasone, 20 nM insulin, and 1 nM 3,3′,5-triiodo-l-thyronine (Sigma) for 2 days, and then cells were cultured in DMEM containing 10% FBS, 20 nM insulin, and 1 nM 3,3′,5-triiodo-l-thyronine. Cells were maintained in the presence or absence of aldosterone from day 0 to day 8. In addition, in another set of experiments, after differentiation and 12 hours before treatment, differentiated cells were cultured in serum-free DMEM. At the time of treatment, cells were treated with vehicle or aldosterone. RNA isolation/quantitative real-time polymerase chain reaction and Western blotting RNA was isolated from adipose tissue and cultured cells using Trizol (TAKARA), and complementary DNA synthesis was performed with a PrimeScript RT Reagent Kit (TAKARA). Quantitative polymerase chain reaction (PCR) was performed using a Roche LightCycler 480 Real-Time PCR System; 18S was used for normalization in human adipose tissue and SVF cells, and Arbp (38B4) was used as internal control in mouse 3T3-L1 and brown preadipocytes. Western blotting was performed as previously described (19). An anti-IL-6 antibody [Cell Signaling Technologies; catalog no. 12153; Research Resource Identifier (RRID): AB_2687897] was used for detection of IL-6. Immunohistochemistry, immunofluorescence, and Masson staining Paraffin-embedded sections of perirenal adipose tissues from patients with APA, NT, and EH were stained with Masson’s trichrome as described previously (20). TNF-α, fibronectin (FN), and collagen I (COLI) protein expression was analyzed by immunohistochemistry as described earlier (20). Perirenal adipose tissue sections were incubated with antibodies against TNF-α (Abcam; catalog no. ab6671; RRID: AB_305641), FN (Abcam; catalog no. ab23751; RRID: AB_447656), or COLI (Abcam; catalog no. ab6308; RRID: AB_305411). For immunofluorescence, paraffin-embedded sections of perirenal adipose tissues from patients with APA, NT, and EH were incubated with antibodies against CD68 (Abcam; catalog no. ab955; RRID: AB_307338) or TNF-α (Abcam; catalog no. ab6671; RRID: AB_305641), followed by incubation with fluorophore-conjugated secondary antibodies. Statistical analysis Student t test was used to analyze data with a normal distribution, and the results are presented as the mean ± standard error of the mean. Wilcoxon signed-ranks test was used for nonnormal distribution data analysis, and the results are presented as the median (25% to 75%). P < 0.05 was considered statistically significant. Results Patient characteristics Table 1 describes the clinical and laboratory characteristics of the study patients. Plasma aldosterone and the aldosterone-to-renin ratio were higher and serum potassium and plasma renin activity were lower in patients with APA than in patients with EH (P < 0.05). Blood pressure and duration of hypertension were similar in both patients with APA and patients with EH. Creatinine in all patients is within normal range. Human adipose tissue Inflammatory burden in perirenal adipose tissue of patients with APA We investigated whether perirenal adipose tissue of patients with APA differed from that of patients with NT and EH and found that IL-6 and TNF-α messenger RNA (mRNA) levels were significantly higher in perirenal adipose tissue of patients with APA than in NT and EH patient tissues (Fig. 1A). Western blotting confirmed increased expression of IL-6 protein in perirenal adipose tissue of patients with APA (Fig. 1B). Immunohistochemistry analysis of perirenal adipose tissue showed that, compared with NT and EH individuals, TNF-α expression was significantly increased in patients with APA (Fig. 1D). Figure 1. View largeDownload slide Inflammatory and fibrosis burden in perirenal adipose tissue (peri-N) of patients with APA. (A) IL-6 and TNF-α mRNA expression levels in perirenal adipose tissues from different patient groups. +, vs NT; *, vs EH. ++P < 0.01; **P < 0.01. (B) Western blot for IL-6 protein in perirenal adipose tissue. (C) Expression of early growth response factor-1 (EGR1), FN, COLI, and transforming growth factor β1 (TGFβ1) mRNA in perirenal adipose tissue between groups. +P < 0.05; *P < 0.05; #P < 0.05. (D) Representative perirenal adipose tissue histological findings demonstrated by immunohistochemical and Masson trichrome staining (blue indicates positive staining) (×400). +, vs NT; *, vs EH; #, vs NT. Scale bar = 50 μm. Figure 1. View largeDownload slide Inflammatory and fibrosis burden in perirenal adipose tissue (peri-N) of patients with APA. (A) IL-6 and TNF-α mRNA expression levels in perirenal adipose tissues from different patient groups. +, vs NT; *, vs EH. ++P < 0.01; **P < 0.01. (B) Western blot for IL-6 protein in perirenal adipose tissue. (C) Expression of early growth response factor-1 (EGR1), FN, COLI, and transforming growth factor β1 (TGFβ1) mRNA in perirenal adipose tissue between groups. +P < 0.05; *P < 0.05; #P < 0.05. (D) Representative perirenal adipose tissue histological findings demonstrated by immunohistochemical and Masson trichrome staining (blue indicates positive staining) (×400). +, vs NT; *, vs EH; #, vs NT. Scale bar = 50 μm. Fibrotic changes in perirenal adipose tissue of patients with APA Immunohistochemistry analysis of perirenal adipose tissue showed that FN and COLI expression was significantly higher in tissues from patients with APA than in tissues from patients with NT and EH. Masson’s staining showed that perirenal adipose tissues from patients with APA developed more extensive collagen deposition compared with tissues from individuals with NT and EH (Fig. 1D). Moreover, we detected the mRNA expression of fibrosis-related genes, including FN, COLI, early growth response factor-1 (EGR1), and transforming growth factor-β1 (TGFβ1). The mRNA levels of these genes were significantly increased in perirenal adipose tissues of the APA group (Fig. 1C). Adipokine mRNA is upregulated in adipose tissues of patients with APA To assess the characteristics of perirenal adipose tissue, we examined the molecular phenotype of white and brown fat. The expression of peroxisome proliferator–activated receptor γ (PPARγ), CCAAT-enhancer-binding protein (C/EBP) α, C/EBPβ, UCP1, PPARγ coactivator-1α (PGC1α), and cell death–inducing DFFA-like effector A (CIDEA) in perirenal adipose tissue of patients with APA were significantly increased compared with that of patients with NT and EH (Fig. 2A and 2B). We also found that mRNA levels of C/EBPβ, PGC1α, and CIDEA in subcutaneous adipose tissues of patients with APA were dramatically increased compared with individuals with NT and EH (Fig. 2C and 2D). Figure 2. View largeDownload slide Adipokine mRNA is upregulated in adipose tissues of patients with APA. (A, B) Expression of adipokine mRNA in white fat and brown fat in perinephric adipose tissues from different patient groups. (C, D) Expression of adipokine mRNA in white fat and brown fat in subcutaneous adipose tissue from different patient groups. +, vs NT; *, vs EH; #, vs NT. +P < 0.05; ++P < 0.01; *P < 0.05; **P < 0.01; #P < 0.05; ##P < 0.01. peri-N, perirenal adipose tissue; sub-Q, subcutaneous adipose tissue. Figure 2. View largeDownload slide Adipokine mRNA is upregulated in adipose tissues of patients with APA. (A, B) Expression of adipokine mRNA in white fat and brown fat in perinephric adipose tissues from different patient groups. (C, D) Expression of adipokine mRNA in white fat and brown fat in subcutaneous adipose tissue from different patient groups. +, vs NT; *, vs EH; #, vs NT. +P < 0.05; ++P < 0.01; *P < 0.05; **P < 0.01; #P < 0.05; ##P < 0.01. peri-N, perirenal adipose tissue; sub-Q, subcutaneous adipose tissue. Human perirenal adipose tissue SVF cell isolation and adipocyte differentiation in vitro To investigate the effect of aldosterone on SVF cells’ characteristics and function, we isolated primary preadipocytes from human perirenal adipose tissue (Fig. 3A and 3B). In addition, we treated differentiated SVF cells with aldosterone (10 nM) for 24 hours to measure mRNA expression of genes related to adipokines, inflammation, and fibrosis. Aldosterone significantly induced IL-6, FN, COLI, and TGFβ1 mRNA expression in differentiated SVF cells and induced a marked upregulation of PPARγ, C/EBPα, and CIDEA mRNA expression (Fig. 3C and 3D). Figure 3. View largeDownload slide Effects of aldosterone (ALD) on mRNA expression related to adipokines, inflammation, and fibrosis in fully differentiated human perirenal adipose tissue SVF cells. (A) Culture of isolated cells from the SVF of human perirenal fat (×100). (B) Differentiated SVF cells from human perirenal adipose tissue. (C, D) Effects of aldosterone on mRNA levels of genes associated with adipokines, lipid metabolism and inflammation, and fibrosis in differentiated human perirenal adipose tissue SVF cells. Well-differentiated cells were treated with aldosterone (10–8 M) for 24 hours. *P < 0.05 vs CON (control). Scale bar = 200 μm. Figure 3. View largeDownload slide Effects of aldosterone (ALD) on mRNA expression related to adipokines, inflammation, and fibrosis in fully differentiated human perirenal adipose tissue SVF cells. (A) Culture of isolated cells from the SVF of human perirenal fat (×100). (B) Differentiated SVF cells from human perirenal adipose tissue. (C, D) Effects of aldosterone on mRNA levels of genes associated with adipokines, lipid metabolism and inflammation, and fibrosis in differentiated human perirenal adipose tissue SVF cells. Well-differentiated cells were treated with aldosterone (10–8 M) for 24 hours. *P < 0.05 vs CON (control). Scale bar = 200 μm. Effect of aldosterone on the expression of genes related to adipokines, inflammation, and fibrosis in cultured mouse adipocytes To determine whether elevation of inflammation and fibrosis in perirenal adipose tissue of patients with APA were induced by high aldosterone levels, we treated 3T3-L1 and brown preadipocytes with aldosterone during differentiation. Aldosterone stimulated differentiation of 3T3-L1 and brown preadipocytes into mature adipocytes (Fig. 4A and 4C). Moreover, aldosterone induced an increase in adipokine gene expression in mature 3T3-L1 and brown preadipocytes, which indicated that aldosterone influences mature adipocytes (Fig. 5A and 5C). According to the previous results, aldosterone significantly increased the expression of proinflammatory cytokines such as IL-6 and TNF-α. The mRNA levels of fibrosis genes including FN, COLI, TGFβ1, and EGR1 were higher after aldosterone treatment (Fig. 4B and 4D and Fig. 5B and 5D ). Figure 4. View largeDownload slide Effect of aldosterone (ALD) on mouse 3T3-L1 preadipocytes and brown preadipocytes during differentiation. (A–D) Cells were treated with aldosterone from day 0 to day 8. Aldosterone stimulated mouse 3T3-L1 and brown preadipocyte differentiation. Effect of aldosterone treatment on gene expression on day 8. *P < 0.05 vs CON (control). Figure 4. View largeDownload slide Effect of aldosterone (ALD) on mouse 3T3-L1 preadipocytes and brown preadipocytes during differentiation. (A–D) Cells were treated with aldosterone from day 0 to day 8. Aldosterone stimulated mouse 3T3-L1 and brown preadipocyte differentiation. Effect of aldosterone treatment on gene expression on day 8. *P < 0.05 vs CON (control). Figure 5. View largeDownload slide Effect of aldosterone (ALD) on mature 3T3-L1 and brown preadipocytes. Well-differentiated 3T3-L1 and brown preadipocytes were treated with aldosterone for 24 hours. (A–D) Effect of aldosterone on gene expression in mature 3T3-L1 and brown preadipocytes. *P < 0.05 vs CON (control). Figure 5. View largeDownload slide Effect of aldosterone (ALD) on mature 3T3-L1 and brown preadipocytes. Well-differentiated 3T3-L1 and brown preadipocytes were treated with aldosterone for 24 hours. (A–D) Effect of aldosterone on gene expression in mature 3T3-L1 and brown preadipocytes. *P < 0.05 vs CON (control). Macrophages in SVF cell and perirenal adipose tissue of patients with APA We analyzed the mRNA expression of macrophage cell surface marker (CD68, CD11b) and CD4+ in SVF cells. As observed in Fig. 6A, CD68 mRNA expression was similar in the control and aldosterone groups (P > 0.05). CD11b and CD4+ mRNA could barely be detected in SVF cells (cycle threshold values were ∼35; data not shown). We further analyzed the previous genes’ expression in perirenal adipose tissue in patients with NT, EH, and APA (Fig. 6B). The mRNA levels of CD68, CD11b, and CD4+ were similar in perirenal adipose tissue in NT, EH, and APA. Coimmunofluorescence of CD68 and TNF-α in perirenal adipose tissues of patients with NT, EH, and APA showed that only a very small number of CD68 immunopositive cells were detected (Fig. 6C). TNF-α immunofluorescence results showed that TNF-α protein was significantly higher in perirenal adipose tissue of patients with APA than in that of patients with NT and EH. Using immunofluorescence, we also found that these CD68+ cells coexpressed the TNF-α (Fig. 6C). Figure 6. View largeDownload slide Macrophages in SVF cell and perirenal adipose tissue (peri-N) of patients with APA. (A) Gene expression of macrophage cell marker (CD68) in fully differentiated human perirenal adipose tissue SVF cells. Well-differentiated cells were treated with aldosterone (ALD; 10–8 M) for 24 hours. (B) Perirenal adipose tissue gene expression of macrophage cell surface markers (CD68, CD11b) and CD4+ in patients with NT, EH, and APA. (C) Triple immunofluorescence of CD68 (green), TNF-α (red), and 4′,6-diamidino-2-phenylindole (DAPI; blue) in perirenal adipose tissues between groups (×400). Scale bar = 50 μm. Figure 6. View largeDownload slide Macrophages in SVF cell and perirenal adipose tissue (peri-N) of patients with APA. (A) Gene expression of macrophage cell marker (CD68) in fully differentiated human perirenal adipose tissue SVF cells. Well-differentiated cells were treated with aldosterone (ALD; 10–8 M) for 24 hours. (B) Perirenal adipose tissue gene expression of macrophage cell surface markers (CD68, CD11b) and CD4+ in patients with NT, EH, and APA. (C) Triple immunofluorescence of CD68 (green), TNF-α (red), and 4′,6-diamidino-2-phenylindole (DAPI; blue) in perirenal adipose tissues between groups (×400). Scale bar = 50 μm. Discussion In this study, we demonstrated inflammation and fibrosis in perirenal adipose tissues of patients with APA, and these factors may be involved in the incidence and development of cardiovascular events observed in patients with APA. Recent data show that perirenal fat has unique properties and that its function may be different from adipose tissue in other deposits. Specifically, Svensson et al. (21) and Mohsen-Kanson et al. (22) reported that perirenal adipose tissue in healthy subjects possesses the molecular characteristics of brown adipose tissue. A recent study showed that the expression of UCP1 protein and mRNA was lower in perirenal adipose tissues from hypertensive patients compared with normotensive patients, but there was no statistical significance in subcutaneous adipose tissue (23), which indicated that perirenal adipose tissue may have a distinct characteristic. However, available data on perirenal adipose tissue in patients with APA are limited. We observed that mRNA expression of fat-related genes such as PPARγ, C/EBPα, C/EBPβ, UCP1, PGC1α, and CIDEA in perirenal adipose tissue was significantly higher in the APA group than in the NT and EH groups. Recent studies have found that the prevalence rate of PA is much higher than was previously; the incidence of PA is 4.6% to 16.6% among patients with hypertension (24). A previous study found that high-sensitivity C-reactive protein was markedly higher in patients with PA compared with matched patients with EH (25). Another study found that the mRNA level of IL-6 and MCP-1 was similar in omental adipose tissue of patients with APA and patients with nonfunctioning adenoma (26). A previous study demonstrated that expression of IL-6, TNF-α, and PPARγ genes in visceral adipose tissues from patients with APA and patients with nonfunctioning adenoma was similar (26). As patients with PA present a high cardiovascular disease risk compared with EH, we used patients with EH as controls to compare inflammation, and all control patients with adrenal adenoma were excluded. Moreover, we studied perirenal adipose tissue rather than visceral adipose tissue. Our results show that expression of IL-6 and TNF-α protein and mRNA in perirenal adipose tissue was significantly higher in the APA group than in the NT and EH groups, suggesting that excess aldosterone in vivo may be related to increased perirenal adipose tissue inflammation. Eplerenone (compared with placebo) reduced the risk of death and hospitalization in patients with systolic heart failure (27), and aldosterone induced cardiac endothelial cell proliferation and perivascular fibrosis (28, 29). Compared with EH, myocardial fibrosis is more common in patients with PA (30). Adipose tissue fibrosis plays an important role in adipocyte dysfunction (31). And studies have demonstrated that the extracellular matrix is associated with white adipose tissue metabolic and endocrine dysfunction (32–34). However, there are no data available regarding perirenal adipose tissue fibrosis changes in patients with APA. Our study demonstrated that expression of genes and proteins related to fibrosis such as FN and COLI was higher in perirenal adipose tissue in patients with APA than in patients with NT and EH. Aldosterone classically acts in a genomic manner through induction and modulation of gene transcription in target tissues expressing the cytoplasmic/nuclear MR (35). Previous studies have demonstrated that in animal models, aldosterone-induced inflammation occurs via a genomic mechanism in which the expression and activation of NFκB are increased (36). Moreover, multiple studies have demonstrated that administration of eplerenone reduced mRNA levels of IL-6 and TNF-α in obese mice adipose tissue (36, 37). In addition, the study also found that in diet-induced obese mice, endothelial MR activation mediates endothelial dysfunction and eplerenone alleviates obesity-induced inflammation (37). Our results reveal that excess aldosterone in patients with APA may induce perirenal adipose tissue inflammation and that inflammatory factors such as IL-6 and TNF-α may cause vascular endothelium damage. In vitro, aldosterone not only enhanced the differentiation of white and brown preadipocytes but also affected the function of mature adipocytes. In accordance with a previous study (11), our results show that aldosterone increases expression of IL-6 and TNF-α in mature 3T3-L1 adipocytes. As mentioned before, the previous study reported that aldosterone promoted T37i cells differentiation via MR (9), but Viengchareun et al. (38) reported that MR inhibits UCP1 expression in brown adipocytes (T37i cells). The authors point out that MR both allows brown preadipocytes to enter into the differentiation program and inhibits UCP1 expression (promoting intracellular triglyceride accumulation) (38). Furthermore, a recent study reported that MR antagonist treatment induced brown adipocyte-specific markers in primary murine preadipocytes, 3T3-L1, and human primary preadipocytes (39, 40). In 3T3-L1 cells, aldosterone decreased apelin and adiponectin levels and increased plasminogen activator inhibitor-1 (PCI-1) expression via glucocorticoid receptor but not MR (41, 42). Furthermore, the impact of aldosterone on human perirenal adipocytes is unknown. SVF cells isolated from human perirenal adipose tissue was used to analyze the effect of aldosterone, confirming the effect of aldosterone on human adipose cells. Macrophage accumulation in adipose tissue plays a key role in inflammation (43, 44). Hence, we examined macrophage cell surface marker in SVF cells and perirenal adipose tissue. The results show that CD68, one of the macrophage cell surface markers, can also be detected in SVF cells. Coimmunofluorescence revealed a high density of TNF-α but detected only a very small number of CD68 immunopositive cells (Fig. 6C). We did find that most of these CD68 immunopositive cells coexpressed TNF-α. Because the majority of TNF-α immunopositive cells did not colocalize with CD68, the current results suggest that in addition to macrophages, adipocytes play an important role in the inflammation of perirenal adipose tissue in patients with APA. It has been reported that adipose tissue macrophage accumulation was associated with obesity, which contributes to TNF-α expression (43). They performed CD68 real-time PCR and immunohistochemical analysis on human subcutaneous adipose tissues [body mass index (BMI) ranged from 19.4 to 60.1 kg/m2]; the results showed that BMI and adipocyte area were predictors for CD68-positive cells. They also found that obese subjects have more CD68+ cells compared with lean subjects. Another study also reported that epididymal adipose tissue has more macrophage infiltration in obese mice than lean mice (45). However, whether or not adipose tissue macrophage accumulation occurs in patients with APA is unknown. A previous study reported that elperenone suppressed macrophage infiltration in adipose tissue of obese mice (7). In our study, patients’ BMIs are lower than 27 kg/m2, which may be one of the reasons that we only found very few CD68-positive cells along with inflammation burden in perirenal adipose tissue of patients with APA. As we all know, both adipocytes and macrophages can release several cytokines, such as IL-6 and TNF-α (46). Our results in 3T3-L1, brown preadipocytes, and SVF-derived adipocytes also confirmed that adipocytes could release IL-6 and TNF-α with aldosterone stimulation. In conclusion, high aldosterone in patients with APA may induce perirenal adipose tissue dysfunction and lead to inflammation and fibrosis. Future studies will need to increase the sample size and investigate the relationship of regional adipose tissue inflammation, circulating inflammatory factors, and cardiovascular events in patients with APA. Appendix. Antibody Table Peptide/Protein Target  Name of Antibody  Manufacturer, Catalog No.   Species Raised in; Monoclonal or Polyclonal  Dilution Used  RRID  TNF-α  Anti-TNF-α antibody  Abcam, ab6671  Rabbit; polyclonal to TNF-α  1:100  AB_305641  COLI  Anti-Collagen I antibody (COL-1)  Abcam, ab6308  Mouse; monoclonal (COL-1) to Collagen I  1:40  AB_305411  FN  Anti-Fibronectin antibody  Abcam, ab23751  Rabbit; polyclonal to Fibronectin  1:500  AB_447656  IL-6  IL-6 (D3K2N) rabbit mAb  Cell Signaling Technology, 12153  Rabbit; monoclonal to IL-6 (D3K2N)  1:500  AB_2687897  CD68  Anti-CD68 antibody (KP1)  Abcam, ab955  Mouse; monoclonal (KP1) to CD68  1:25  AB_307338  TNF-α (immunofluorescence)  Anti-TNF-α antibody  Abcam, ab6671  Rabbit; polyclonal to TNF-α  1:25  AB_305641  Peptide/Protein Target  Name of Antibody  Manufacturer, Catalog No.   Species Raised in; Monoclonal or Polyclonal  Dilution Used  RRID  TNF-α  Anti-TNF-α antibody  Abcam, ab6671  Rabbit; polyclonal to TNF-α  1:100  AB_305641  COLI  Anti-Collagen I antibody (COL-1)  Abcam, ab6308  Mouse; monoclonal (COL-1) to Collagen I  1:40  AB_305411  FN  Anti-Fibronectin antibody  Abcam, ab23751  Rabbit; polyclonal to Fibronectin  1:500  AB_447656  IL-6  IL-6 (D3K2N) rabbit mAb  Cell Signaling Technology, 12153  Rabbit; monoclonal to IL-6 (D3K2N)  1:500  AB_2687897  CD68  Anti-CD68 antibody (KP1)  Abcam, ab955  Mouse; monoclonal (KP1) to CD68  1:25  AB_307338  TNF-α (immunofluorescence)  Anti-TNF-α antibody  Abcam, ab6671  Rabbit; polyclonal to TNF-α  1:25  AB_305641  Abbreviation: mAb, monoclonal antibody. View Large Abbreviations: APA aldosterone-producing adenoma BMI body mass index C/EBP CCAAT-enhancer-binding protein CIDEA cell death–inducing DFFA-like effector A COLI collagen I DMEM Dulbecco’s modified Eagle medium EGR1 early growth response factor-1 EH essential hypertension FBS fetal bovine serum FN fibronectin IL-6 interleukin-6 MR mineralocorticoid receptor mRNA messenger RNA NT normotension PA primary aldosteronism PCR polymerase chain reaction PGC1α peroxisome proliferator-activated receptor γ coactivator-1α PPARγ peroxisome proliferator–activated receptor γ RRID Research Resource Identifier SVF stromal vascular fraction TGFβ1 transforming growth factor β1 TNF-α tumor necrosis factor-α UCP1 uncoupling protein 1. Acknowledgments We thank Yu-Hua Tseng (Joslin Diabetes Center, Harvard Medical School, Boston, MA) for providing the mouse brown preadipocytes and Dang Qiang and Tong Chen (Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou, China) for their help in obtaining human adipose tissue samples. Financial Support: This work was supported by National Natural Science Foundation of China Grants 81628004, 31400992, 81470047, and 81500656, and Guangdong Science and Technology Project 2013B022000061. Disclosure Summary: The authors have nothing to disclose. References 1. Funder JW, Carey RM, Mantero F, Murad MH, Reincke M, Shibata H, Stowasser M, Young WF, Jr. The management of primary aldosteronism: case detection, diagnosis, and treatment: an endocrine society clinical practice guideline. J Clin Endocrinol Metab . 2016; 101( 5): 1889– 1916. Google Scholar CrossRef Search ADS PubMed  2. Milliez P, Girerd X, Plouin PF, Blacher J, Safar ME, Mourad JJ. 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Inflammation and Fibrosis in Perirenal Adipose Tissue of Patients With Aldosterone-Producing Adenoma

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Endocrine Society
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Copyright © 2018 Endocrine Society
ISSN
0013-7227
eISSN
1945-7170
D.O.I.
10.1210/en.2017-00651
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Abstract

Abstract The prevalence of primary aldosteronism is much higher than previously thought. Recent studies have shown that primary aldosteronism is related to a higher risk of cardiovascular events. However, the underlying mechanism is not yet clear. Here we investigate the characteristics, including inflammation, fibrosis, and adipokine expression, of adipose tissues from different deposits in patients with aldosterone-producing adenoma (APA). Inflammation and fibrosis changes were evaluated in perirenal and subcutaneous adipose tissues obtained from patients with APA (n = 16), normotension (NT; n = 10), and essential hypertension (EH; n = 5) undergoing laparoscopic surgery. We also evaluated the effect of aldosterone in isolated human perirenal adipose tissue stromal vascular fraction (SVF) cells and investigated the effect of aldosterone in mouse 3T3-L1 and brown preadipocytes. Compared with the EH group, significantly higher levels of interleukin-6 (IL-6) and tumor necrosis factor-α messenger RNA (mRNA) and protein were observed in perirenal adipose tissue of patients with APA. Expression of genes related to fibrosis and adipogenesis in perirenal adipose tissue was notably higher in patients with APA than in patients with NT and EH. Aldosterone significantly induced IL-6 and fibrosis gene mRNA expression in differentiated SVF cells. Aldosterone treatment enhanced mRNA expression of genes associated with inflammation and fibrosis and stimulated differentiation of 3T3-L1 and brown preadipocytes. In conclusion, these data indicate that high aldosterone in patients with APA may induce perirenal adipose tissue dysfunction and lead to inflammation and fibrosis, which may be involved in the high risk of cardiovascular events observed in patients with primary aldosteronism. To date, accumulating evidence has demonstrated that the incidence of primary aldosteronism (PA) is much higher than previously believed (1). Compared with patients with essential hypertension (EH) matched for blood pressure, sex, and age, patients with PA have an increased rate of cardiovascular events and mortality (2, 3). And the prevalence of type 2 diabetes and metabolic syndrome are higher in PA than in matched patients with EH (4). Adipose tissue inflammation and fibrosis play an important role in cardiovascular disease. In patients with high-risk heart disease, epicardial adipose tissue expresses significantly higher levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) compared with subcutaneous adipose tissue (5). A previous study has reported that aldosterone worsens metabolism, whereas a mineralocorticoid receptor (MR) antagonist reduced insulin resistance in 3T3-L1 preadipocytes (6, 7). Rondinone et al. (8) reported that 1 to 10 nM aldosterone can induce 3T3-L1 preadipocyte differentiation. Penfornis et al. (9) found that aldosterone induces T37i cells into mature brown adipocytes via an MR. What’s more, research has shown that aldosterone and high-sensitivity C-reactive protein concentrations predict incident metabolic syndrome (10). Aldosterone increases expression of IL-6 and TNF-α in mature 3T3-L1 adipocytes, and MR blockade reduces expression of IL-6 and TNF-α (11). However, the mechanism underlying this phenomenon is still not clear. Adipose tissue inflammation and fibrosis play an important role in cardiovascular disease. Human epicardial adipose tissue induces atrial myocardium fibrosis (12). In addition, a study found that atrial fibrillation is associated with subepicardium adipose tissue remodeling in humans (13). Whether chronic aldosterone increase in patients with PA directly affects adipose tissue inflammation and fibrosis has not yet been clarified. In this study, we explored the fibrosis and inflammation change in perirenal and subcutaneous adipose tissue of patients with aldosterone-producing adenoma (APA). We also evaluated the effect of aldosterone in isolated human perirenal adipose tissue stromal vascular fraction (SVF) cells and investigated the effect of aldosterone in mouse 3T3-L1 and brown preadipocytes. Methods Patients From October 2014 to December 2016, 31 consecutive patients who were referred to our hospital were enrolled in the study. Among them, 16 patients with APA were studied. In addition, 10 patients with normotension (NT) and 5 with EH served as controls. The diagnosis of APA and EH was based on international diagnostic guidelines (14). The characteristics of the patients are described in Table 1. Perirenal and subcutaneous adipose tissues were obtained from patients with APA, NT, and EH undergoing laparoscopic adrenalectomy, nephrolithotomy, or ureterolithotomy. The study was approved by the Institutional Review Board of Nanfang Hospital, and all participating individuals provided written informed consent. Table 1. Patient Characteristics   NT  EH  APA  NT vs APA  EH vs APA  Sex (% female)  50.0%  20.0%  56.3%  —  —  Age (y)  47.2 ± 15.8  55.2 ± 7.2  45.7 ± 9.6  0.763  0.057  BMI (kg/m2)  22.5 ± 2.7  23.9 ± 3.1  23.2 ± 3.2  0.585  0.668  SBP (mm Hg)  123.3 ± 12.9  140.8 ± 16.1  149.4 ± 20.8  0.02  0.413  DBP (mm Hg)  74.6 ± 10.4  89.6 ± 4.7  97.5 ± 14.1  <0.001  0.243  Duration of hypertension (y)  —  4.0 (3.3–8.5)  4.0 (1.0–7.0)  —  0.935  Glycemia (mmol/L)  5.1 ± 0.5  5.2 ± 0.4  5.5 ± 2.1  0.626  0.78  K+ (mmol/L)  3.9 ± 0.7  4.2 ± 0.2  3.6 ± 0.9  0.337  0.132  WBC (109/L)  7.5 ± 2.7  8.5 ± 2.7  7.0 ± 2.1  n.s.  0.195  Creatinine (umol/L)  95.7 ± 64.3  114.8 ± 31.7  68.0 ± 19.3  0.128  0.001  ALT (U/L)  20.2 ± 8.2  22.8 ± 9.3  17.5 ± 2.9  0.215  0.312  AST (U/L)  19.5 ± 13.5  23.0 ± 4.9  19.3 ± 5.6  0.490  0.315  Aldosterone (ng/dL)  —  11.0 (10.3–11.0)  25.6 (23.3–40.9)  —  0.004  Plasma renin activity (ng/mL/h)  —  5.0 (0.6–5.0)  0.2 (0.1–0.5)  —  0.025  ARR  —  2.2 (1.9 ± 2.2)  124.3 (77.7–364.2)  —  0.004    NT  EH  APA  NT vs APA  EH vs APA  Sex (% female)  50.0%  20.0%  56.3%  —  —  Age (y)  47.2 ± 15.8  55.2 ± 7.2  45.7 ± 9.6  0.763  0.057  BMI (kg/m2)  22.5 ± 2.7  23.9 ± 3.1  23.2 ± 3.2  0.585  0.668  SBP (mm Hg)  123.3 ± 12.9  140.8 ± 16.1  149.4 ± 20.8  0.02  0.413  DBP (mm Hg)  74.6 ± 10.4  89.6 ± 4.7  97.5 ± 14.1  <0.001  0.243  Duration of hypertension (y)  —  4.0 (3.3–8.5)  4.0 (1.0–7.0)  —  0.935  Glycemia (mmol/L)  5.1 ± 0.5  5.2 ± 0.4  5.5 ± 2.1  0.626  0.78  K+ (mmol/L)  3.9 ± 0.7  4.2 ± 0.2  3.6 ± 0.9  0.337  0.132  WBC (109/L)  7.5 ± 2.7  8.5 ± 2.7  7.0 ± 2.1  n.s.  0.195  Creatinine (umol/L)  95.7 ± 64.3  114.8 ± 31.7  68.0 ± 19.3  0.128  0.001  ALT (U/L)  20.2 ± 8.2  22.8 ± 9.3  17.5 ± 2.9  0.215  0.312  AST (U/L)  19.5 ± 13.5  23.0 ± 4.9  19.3 ± 5.6  0.490  0.315  Aldosterone (ng/dL)  —  11.0 (10.3–11.0)  25.6 (23.3–40.9)  —  0.004  Plasma renin activity (ng/mL/h)  —  5.0 (0.6–5.0)  0.2 (0.1–0.5)  —  0.025  ARR  —  2.2 (1.9 ± 2.2)  124.3 (77.7–364.2)  —  0.004  Abbreviations: ALT, alanine transaminase; ARR, aldosterone-to-renin ratio; AST, glutamic oxal(o)acetic transaminase; BMI, body mass index; DBP, diastolic blood pressure; K+, serum potassium; n.s., nonsignificant; SBP, systolic blood pressure; WBC, white blood cell count. View Large Isolation of cells from the SVF of human perirenal fat Human perirenal adipose tissue was obtained from a 52-year-old normotensive female who underwent laparoscopic ureterolithotomy. The primary SVF from human perirenal fat was isolated as described previously (15). The primary SVF cells from human perirenal adipose tissue were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). For adipocyte differentiation, cells were grown to 100% confluence and then differentiated with a standard brown preadipocyte differentiation protocol (16). Twelve hours before treatment, differentiated cells were cultured in serum-free DMEM. For the aldosterone stimulation study, at the time of treatment, cells were incubated with vehicle or aldosterone (10−8 mol/L; Sigma) for 24 hours. The selected concentration of aldosterone was based on previous studies (11). Cell culture Mouse 3T3-L1 preadipocytes were obtained from the type culture collection of the Chinese Academy of Sciences. The brown preadipocytes were established as previously described (17, 18). Briefly, interscapular brown adipose tissue was isolated from new-born C57BL/6 mice and digested with collagenase, and then filtered and spun down. The pelleted SVF was plated on culture dishes, immortalized with pBabe-SV40T-puro retroviral vector (expressing large T antigen and puromycin resistant gene), and selected with puromycin. The established brown preadipocyte cell line was tested and verified for their capability of adipogenic differentiation and uncoupling protein 1 (UCP1) expression. The differentiation of 3T3-L1 preadipocytes was performed according to a standard differentiation protocol [DMEM supplemented with 10% FBS, 0.5 mM isobutylmethylxanthine (Sigma), 1 µM dexamethasone (Sigma), and 20 nM insulin (Sigma) for 2 days, and then cells were cultured in DMEM with 10% FBS and 20 nM insulin]. Brown preadipocyte differentiation was initiated with 0.5 mM isobutylmethylxanthine, 1 µM dexamethasone, 20 nM insulin, and 1 nM 3,3′,5-triiodo-l-thyronine (Sigma) for 2 days, and then cells were cultured in DMEM containing 10% FBS, 20 nM insulin, and 1 nM 3,3′,5-triiodo-l-thyronine. Cells were maintained in the presence or absence of aldosterone from day 0 to day 8. In addition, in another set of experiments, after differentiation and 12 hours before treatment, differentiated cells were cultured in serum-free DMEM. At the time of treatment, cells were treated with vehicle or aldosterone. RNA isolation/quantitative real-time polymerase chain reaction and Western blotting RNA was isolated from adipose tissue and cultured cells using Trizol (TAKARA), and complementary DNA synthesis was performed with a PrimeScript RT Reagent Kit (TAKARA). Quantitative polymerase chain reaction (PCR) was performed using a Roche LightCycler 480 Real-Time PCR System; 18S was used for normalization in human adipose tissue and SVF cells, and Arbp (38B4) was used as internal control in mouse 3T3-L1 and brown preadipocytes. Western blotting was performed as previously described (19). An anti-IL-6 antibody [Cell Signaling Technologies; catalog no. 12153; Research Resource Identifier (RRID): AB_2687897] was used for detection of IL-6. Immunohistochemistry, immunofluorescence, and Masson staining Paraffin-embedded sections of perirenal adipose tissues from patients with APA, NT, and EH were stained with Masson’s trichrome as described previously (20). TNF-α, fibronectin (FN), and collagen I (COLI) protein expression was analyzed by immunohistochemistry as described earlier (20). Perirenal adipose tissue sections were incubated with antibodies against TNF-α (Abcam; catalog no. ab6671; RRID: AB_305641), FN (Abcam; catalog no. ab23751; RRID: AB_447656), or COLI (Abcam; catalog no. ab6308; RRID: AB_305411). For immunofluorescence, paraffin-embedded sections of perirenal adipose tissues from patients with APA, NT, and EH were incubated with antibodies against CD68 (Abcam; catalog no. ab955; RRID: AB_307338) or TNF-α (Abcam; catalog no. ab6671; RRID: AB_305641), followed by incubation with fluorophore-conjugated secondary antibodies. Statistical analysis Student t test was used to analyze data with a normal distribution, and the results are presented as the mean ± standard error of the mean. Wilcoxon signed-ranks test was used for nonnormal distribution data analysis, and the results are presented as the median (25% to 75%). P < 0.05 was considered statistically significant. Results Patient characteristics Table 1 describes the clinical and laboratory characteristics of the study patients. Plasma aldosterone and the aldosterone-to-renin ratio were higher and serum potassium and plasma renin activity were lower in patients with APA than in patients with EH (P < 0.05). Blood pressure and duration of hypertension were similar in both patients with APA and patients with EH. Creatinine in all patients is within normal range. Human adipose tissue Inflammatory burden in perirenal adipose tissue of patients with APA We investigated whether perirenal adipose tissue of patients with APA differed from that of patients with NT and EH and found that IL-6 and TNF-α messenger RNA (mRNA) levels were significantly higher in perirenal adipose tissue of patients with APA than in NT and EH patient tissues (Fig. 1A). Western blotting confirmed increased expression of IL-6 protein in perirenal adipose tissue of patients with APA (Fig. 1B). Immunohistochemistry analysis of perirenal adipose tissue showed that, compared with NT and EH individuals, TNF-α expression was significantly increased in patients with APA (Fig. 1D). Figure 1. View largeDownload slide Inflammatory and fibrosis burden in perirenal adipose tissue (peri-N) of patients with APA. (A) IL-6 and TNF-α mRNA expression levels in perirenal adipose tissues from different patient groups. +, vs NT; *, vs EH. ++P < 0.01; **P < 0.01. (B) Western blot for IL-6 protein in perirenal adipose tissue. (C) Expression of early growth response factor-1 (EGR1), FN, COLI, and transforming growth factor β1 (TGFβ1) mRNA in perirenal adipose tissue between groups. +P < 0.05; *P < 0.05; #P < 0.05. (D) Representative perirenal adipose tissue histological findings demonstrated by immunohistochemical and Masson trichrome staining (blue indicates positive staining) (×400). +, vs NT; *, vs EH; #, vs NT. Scale bar = 50 μm. Figure 1. View largeDownload slide Inflammatory and fibrosis burden in perirenal adipose tissue (peri-N) of patients with APA. (A) IL-6 and TNF-α mRNA expression levels in perirenal adipose tissues from different patient groups. +, vs NT; *, vs EH. ++P < 0.01; **P < 0.01. (B) Western blot for IL-6 protein in perirenal adipose tissue. (C) Expression of early growth response factor-1 (EGR1), FN, COLI, and transforming growth factor β1 (TGFβ1) mRNA in perirenal adipose tissue between groups. +P < 0.05; *P < 0.05; #P < 0.05. (D) Representative perirenal adipose tissue histological findings demonstrated by immunohistochemical and Masson trichrome staining (blue indicates positive staining) (×400). +, vs NT; *, vs EH; #, vs NT. Scale bar = 50 μm. Fibrotic changes in perirenal adipose tissue of patients with APA Immunohistochemistry analysis of perirenal adipose tissue showed that FN and COLI expression was significantly higher in tissues from patients with APA than in tissues from patients with NT and EH. Masson’s staining showed that perirenal adipose tissues from patients with APA developed more extensive collagen deposition compared with tissues from individuals with NT and EH (Fig. 1D). Moreover, we detected the mRNA expression of fibrosis-related genes, including FN, COLI, early growth response factor-1 (EGR1), and transforming growth factor-β1 (TGFβ1). The mRNA levels of these genes were significantly increased in perirenal adipose tissues of the APA group (Fig. 1C). Adipokine mRNA is upregulated in adipose tissues of patients with APA To assess the characteristics of perirenal adipose tissue, we examined the molecular phenotype of white and brown fat. The expression of peroxisome proliferator–activated receptor γ (PPARγ), CCAAT-enhancer-binding protein (C/EBP) α, C/EBPβ, UCP1, PPARγ coactivator-1α (PGC1α), and cell death–inducing DFFA-like effector A (CIDEA) in perirenal adipose tissue of patients with APA were significantly increased compared with that of patients with NT and EH (Fig. 2A and 2B). We also found that mRNA levels of C/EBPβ, PGC1α, and CIDEA in subcutaneous adipose tissues of patients with APA were dramatically increased compared with individuals with NT and EH (Fig. 2C and 2D). Figure 2. View largeDownload slide Adipokine mRNA is upregulated in adipose tissues of patients with APA. (A, B) Expression of adipokine mRNA in white fat and brown fat in perinephric adipose tissues from different patient groups. (C, D) Expression of adipokine mRNA in white fat and brown fat in subcutaneous adipose tissue from different patient groups. +, vs NT; *, vs EH; #, vs NT. +P < 0.05; ++P < 0.01; *P < 0.05; **P < 0.01; #P < 0.05; ##P < 0.01. peri-N, perirenal adipose tissue; sub-Q, subcutaneous adipose tissue. Figure 2. View largeDownload slide Adipokine mRNA is upregulated in adipose tissues of patients with APA. (A, B) Expression of adipokine mRNA in white fat and brown fat in perinephric adipose tissues from different patient groups. (C, D) Expression of adipokine mRNA in white fat and brown fat in subcutaneous adipose tissue from different patient groups. +, vs NT; *, vs EH; #, vs NT. +P < 0.05; ++P < 0.01; *P < 0.05; **P < 0.01; #P < 0.05; ##P < 0.01. peri-N, perirenal adipose tissue; sub-Q, subcutaneous adipose tissue. Human perirenal adipose tissue SVF cell isolation and adipocyte differentiation in vitro To investigate the effect of aldosterone on SVF cells’ characteristics and function, we isolated primary preadipocytes from human perirenal adipose tissue (Fig. 3A and 3B). In addition, we treated differentiated SVF cells with aldosterone (10 nM) for 24 hours to measure mRNA expression of genes related to adipokines, inflammation, and fibrosis. Aldosterone significantly induced IL-6, FN, COLI, and TGFβ1 mRNA expression in differentiated SVF cells and induced a marked upregulation of PPARγ, C/EBPα, and CIDEA mRNA expression (Fig. 3C and 3D). Figure 3. View largeDownload slide Effects of aldosterone (ALD) on mRNA expression related to adipokines, inflammation, and fibrosis in fully differentiated human perirenal adipose tissue SVF cells. (A) Culture of isolated cells from the SVF of human perirenal fat (×100). (B) Differentiated SVF cells from human perirenal adipose tissue. (C, D) Effects of aldosterone on mRNA levels of genes associated with adipokines, lipid metabolism and inflammation, and fibrosis in differentiated human perirenal adipose tissue SVF cells. Well-differentiated cells were treated with aldosterone (10–8 M) for 24 hours. *P < 0.05 vs CON (control). Scale bar = 200 μm. Figure 3. View largeDownload slide Effects of aldosterone (ALD) on mRNA expression related to adipokines, inflammation, and fibrosis in fully differentiated human perirenal adipose tissue SVF cells. (A) Culture of isolated cells from the SVF of human perirenal fat (×100). (B) Differentiated SVF cells from human perirenal adipose tissue. (C, D) Effects of aldosterone on mRNA levels of genes associated with adipokines, lipid metabolism and inflammation, and fibrosis in differentiated human perirenal adipose tissue SVF cells. Well-differentiated cells were treated with aldosterone (10–8 M) for 24 hours. *P < 0.05 vs CON (control). Scale bar = 200 μm. Effect of aldosterone on the expression of genes related to adipokines, inflammation, and fibrosis in cultured mouse adipocytes To determine whether elevation of inflammation and fibrosis in perirenal adipose tissue of patients with APA were induced by high aldosterone levels, we treated 3T3-L1 and brown preadipocytes with aldosterone during differentiation. Aldosterone stimulated differentiation of 3T3-L1 and brown preadipocytes into mature adipocytes (Fig. 4A and 4C). Moreover, aldosterone induced an increase in adipokine gene expression in mature 3T3-L1 and brown preadipocytes, which indicated that aldosterone influences mature adipocytes (Fig. 5A and 5C). According to the previous results, aldosterone significantly increased the expression of proinflammatory cytokines such as IL-6 and TNF-α. The mRNA levels of fibrosis genes including FN, COLI, TGFβ1, and EGR1 were higher after aldosterone treatment (Fig. 4B and 4D and Fig. 5B and 5D ). Figure 4. View largeDownload slide Effect of aldosterone (ALD) on mouse 3T3-L1 preadipocytes and brown preadipocytes during differentiation. (A–D) Cells were treated with aldosterone from day 0 to day 8. Aldosterone stimulated mouse 3T3-L1 and brown preadipocyte differentiation. Effect of aldosterone treatment on gene expression on day 8. *P < 0.05 vs CON (control). Figure 4. View largeDownload slide Effect of aldosterone (ALD) on mouse 3T3-L1 preadipocytes and brown preadipocytes during differentiation. (A–D) Cells were treated with aldosterone from day 0 to day 8. Aldosterone stimulated mouse 3T3-L1 and brown preadipocyte differentiation. Effect of aldosterone treatment on gene expression on day 8. *P < 0.05 vs CON (control). Figure 5. View largeDownload slide Effect of aldosterone (ALD) on mature 3T3-L1 and brown preadipocytes. Well-differentiated 3T3-L1 and brown preadipocytes were treated with aldosterone for 24 hours. (A–D) Effect of aldosterone on gene expression in mature 3T3-L1 and brown preadipocytes. *P < 0.05 vs CON (control). Figure 5. View largeDownload slide Effect of aldosterone (ALD) on mature 3T3-L1 and brown preadipocytes. Well-differentiated 3T3-L1 and brown preadipocytes were treated with aldosterone for 24 hours. (A–D) Effect of aldosterone on gene expression in mature 3T3-L1 and brown preadipocytes. *P < 0.05 vs CON (control). Macrophages in SVF cell and perirenal adipose tissue of patients with APA We analyzed the mRNA expression of macrophage cell surface marker (CD68, CD11b) and CD4+ in SVF cells. As observed in Fig. 6A, CD68 mRNA expression was similar in the control and aldosterone groups (P > 0.05). CD11b and CD4+ mRNA could barely be detected in SVF cells (cycle threshold values were ∼35; data not shown). We further analyzed the previous genes’ expression in perirenal adipose tissue in patients with NT, EH, and APA (Fig. 6B). The mRNA levels of CD68, CD11b, and CD4+ were similar in perirenal adipose tissue in NT, EH, and APA. Coimmunofluorescence of CD68 and TNF-α in perirenal adipose tissues of patients with NT, EH, and APA showed that only a very small number of CD68 immunopositive cells were detected (Fig. 6C). TNF-α immunofluorescence results showed that TNF-α protein was significantly higher in perirenal adipose tissue of patients with APA than in that of patients with NT and EH. Using immunofluorescence, we also found that these CD68+ cells coexpressed the TNF-α (Fig. 6C). Figure 6. View largeDownload slide Macrophages in SVF cell and perirenal adipose tissue (peri-N) of patients with APA. (A) Gene expression of macrophage cell marker (CD68) in fully differentiated human perirenal adipose tissue SVF cells. Well-differentiated cells were treated with aldosterone (ALD; 10–8 M) for 24 hours. (B) Perirenal adipose tissue gene expression of macrophage cell surface markers (CD68, CD11b) and CD4+ in patients with NT, EH, and APA. (C) Triple immunofluorescence of CD68 (green), TNF-α (red), and 4′,6-diamidino-2-phenylindole (DAPI; blue) in perirenal adipose tissues between groups (×400). Scale bar = 50 μm. Figure 6. View largeDownload slide Macrophages in SVF cell and perirenal adipose tissue (peri-N) of patients with APA. (A) Gene expression of macrophage cell marker (CD68) in fully differentiated human perirenal adipose tissue SVF cells. Well-differentiated cells were treated with aldosterone (ALD; 10–8 M) for 24 hours. (B) Perirenal adipose tissue gene expression of macrophage cell surface markers (CD68, CD11b) and CD4+ in patients with NT, EH, and APA. (C) Triple immunofluorescence of CD68 (green), TNF-α (red), and 4′,6-diamidino-2-phenylindole (DAPI; blue) in perirenal adipose tissues between groups (×400). Scale bar = 50 μm. Discussion In this study, we demonstrated inflammation and fibrosis in perirenal adipose tissues of patients with APA, and these factors may be involved in the incidence and development of cardiovascular events observed in patients with APA. Recent data show that perirenal fat has unique properties and that its function may be different from adipose tissue in other deposits. Specifically, Svensson et al. (21) and Mohsen-Kanson et al. (22) reported that perirenal adipose tissue in healthy subjects possesses the molecular characteristics of brown adipose tissue. A recent study showed that the expression of UCP1 protein and mRNA was lower in perirenal adipose tissues from hypertensive patients compared with normotensive patients, but there was no statistical significance in subcutaneous adipose tissue (23), which indicated that perirenal adipose tissue may have a distinct characteristic. However, available data on perirenal adipose tissue in patients with APA are limited. We observed that mRNA expression of fat-related genes such as PPARγ, C/EBPα, C/EBPβ, UCP1, PGC1α, and CIDEA in perirenal adipose tissue was significantly higher in the APA group than in the NT and EH groups. Recent studies have found that the prevalence rate of PA is much higher than was previously; the incidence of PA is 4.6% to 16.6% among patients with hypertension (24). A previous study found that high-sensitivity C-reactive protein was markedly higher in patients with PA compared with matched patients with EH (25). Another study found that the mRNA level of IL-6 and MCP-1 was similar in omental adipose tissue of patients with APA and patients with nonfunctioning adenoma (26). A previous study demonstrated that expression of IL-6, TNF-α, and PPARγ genes in visceral adipose tissues from patients with APA and patients with nonfunctioning adenoma was similar (26). As patients with PA present a high cardiovascular disease risk compared with EH, we used patients with EH as controls to compare inflammation, and all control patients with adrenal adenoma were excluded. Moreover, we studied perirenal adipose tissue rather than visceral adipose tissue. Our results show that expression of IL-6 and TNF-α protein and mRNA in perirenal adipose tissue was significantly higher in the APA group than in the NT and EH groups, suggesting that excess aldosterone in vivo may be related to increased perirenal adipose tissue inflammation. Eplerenone (compared with placebo) reduced the risk of death and hospitalization in patients with systolic heart failure (27), and aldosterone induced cardiac endothelial cell proliferation and perivascular fibrosis (28, 29). Compared with EH, myocardial fibrosis is more common in patients with PA (30). Adipose tissue fibrosis plays an important role in adipocyte dysfunction (31). And studies have demonstrated that the extracellular matrix is associated with white adipose tissue metabolic and endocrine dysfunction (32–34). However, there are no data available regarding perirenal adipose tissue fibrosis changes in patients with APA. Our study demonstrated that expression of genes and proteins related to fibrosis such as FN and COLI was higher in perirenal adipose tissue in patients with APA than in patients with NT and EH. Aldosterone classically acts in a genomic manner through induction and modulation of gene transcription in target tissues expressing the cytoplasmic/nuclear MR (35). Previous studies have demonstrated that in animal models, aldosterone-induced inflammation occurs via a genomic mechanism in which the expression and activation of NFκB are increased (36). Moreover, multiple studies have demonstrated that administration of eplerenone reduced mRNA levels of IL-6 and TNF-α in obese mice adipose tissue (36, 37). In addition, the study also found that in diet-induced obese mice, endothelial MR activation mediates endothelial dysfunction and eplerenone alleviates obesity-induced inflammation (37). Our results reveal that excess aldosterone in patients with APA may induce perirenal adipose tissue inflammation and that inflammatory factors such as IL-6 and TNF-α may cause vascular endothelium damage. In vitro, aldosterone not only enhanced the differentiation of white and brown preadipocytes but also affected the function of mature adipocytes. In accordance with a previous study (11), our results show that aldosterone increases expression of IL-6 and TNF-α in mature 3T3-L1 adipocytes. As mentioned before, the previous study reported that aldosterone promoted T37i cells differentiation via MR (9), but Viengchareun et al. (38) reported that MR inhibits UCP1 expression in brown adipocytes (T37i cells). The authors point out that MR both allows brown preadipocytes to enter into the differentiation program and inhibits UCP1 expression (promoting intracellular triglyceride accumulation) (38). Furthermore, a recent study reported that MR antagonist treatment induced brown adipocyte-specific markers in primary murine preadipocytes, 3T3-L1, and human primary preadipocytes (39, 40). In 3T3-L1 cells, aldosterone decreased apelin and adiponectin levels and increased plasminogen activator inhibitor-1 (PCI-1) expression via glucocorticoid receptor but not MR (41, 42). Furthermore, the impact of aldosterone on human perirenal adipocytes is unknown. SVF cells isolated from human perirenal adipose tissue was used to analyze the effect of aldosterone, confirming the effect of aldosterone on human adipose cells. Macrophage accumulation in adipose tissue plays a key role in inflammation (43, 44). Hence, we examined macrophage cell surface marker in SVF cells and perirenal adipose tissue. The results show that CD68, one of the macrophage cell surface markers, can also be detected in SVF cells. Coimmunofluorescence revealed a high density of TNF-α but detected only a very small number of CD68 immunopositive cells (Fig. 6C). We did find that most of these CD68 immunopositive cells coexpressed TNF-α. Because the majority of TNF-α immunopositive cells did not colocalize with CD68, the current results suggest that in addition to macrophages, adipocytes play an important role in the inflammation of perirenal adipose tissue in patients with APA. It has been reported that adipose tissue macrophage accumulation was associated with obesity, which contributes to TNF-α expression (43). They performed CD68 real-time PCR and immunohistochemical analysis on human subcutaneous adipose tissues [body mass index (BMI) ranged from 19.4 to 60.1 kg/m2]; the results showed that BMI and adipocyte area were predictors for CD68-positive cells. They also found that obese subjects have more CD68+ cells compared with lean subjects. Another study also reported that epididymal adipose tissue has more macrophage infiltration in obese mice than lean mice (45). However, whether or not adipose tissue macrophage accumulation occurs in patients with APA is unknown. A previous study reported that elperenone suppressed macrophage infiltration in adipose tissue of obese mice (7). In our study, patients’ BMIs are lower than 27 kg/m2, which may be one of the reasons that we only found very few CD68-positive cells along with inflammation burden in perirenal adipose tissue of patients with APA. As we all know, both adipocytes and macrophages can release several cytokines, such as IL-6 and TNF-α (46). Our results in 3T3-L1, brown preadipocytes, and SVF-derived adipocytes also confirmed that adipocytes could release IL-6 and TNF-α with aldosterone stimulation. In conclusion, high aldosterone in patients with APA may induce perirenal adipose tissue dysfunction and lead to inflammation and fibrosis. Future studies will need to increase the sample size and investigate the relationship of regional adipose tissue inflammation, circulating inflammatory factors, and cardiovascular events in patients with APA. Appendix. Antibody Table Peptide/Protein Target  Name of Antibody  Manufacturer, Catalog No.   Species Raised in; Monoclonal or Polyclonal  Dilution Used  RRID  TNF-α  Anti-TNF-α antibody  Abcam, ab6671  Rabbit; polyclonal to TNF-α  1:100  AB_305641  COLI  Anti-Collagen I antibody (COL-1)  Abcam, ab6308  Mouse; monoclonal (COL-1) to Collagen I  1:40  AB_305411  FN  Anti-Fibronectin antibody  Abcam, ab23751  Rabbit; polyclonal to Fibronectin  1:500  AB_447656  IL-6  IL-6 (D3K2N) rabbit mAb  Cell Signaling Technology, 12153  Rabbit; monoclonal to IL-6 (D3K2N)  1:500  AB_2687897  CD68  Anti-CD68 antibody (KP1)  Abcam, ab955  Mouse; monoclonal (KP1) to CD68  1:25  AB_307338  TNF-α (immunofluorescence)  Anti-TNF-α antibody  Abcam, ab6671  Rabbit; polyclonal to TNF-α  1:25  AB_305641  Peptide/Protein Target  Name of Antibody  Manufacturer, Catalog No.   Species Raised in; Monoclonal or Polyclonal  Dilution Used  RRID  TNF-α  Anti-TNF-α antibody  Abcam, ab6671  Rabbit; polyclonal to TNF-α  1:100  AB_305641  COLI  Anti-Collagen I antibody (COL-1)  Abcam, ab6308  Mouse; monoclonal (COL-1) to Collagen I  1:40  AB_305411  FN  Anti-Fibronectin antibody  Abcam, ab23751  Rabbit; polyclonal to Fibronectin  1:500  AB_447656  IL-6  IL-6 (D3K2N) rabbit mAb  Cell Signaling Technology, 12153  Rabbit; monoclonal to IL-6 (D3K2N)  1:500  AB_2687897  CD68  Anti-CD68 antibody (KP1)  Abcam, ab955  Mouse; monoclonal (KP1) to CD68  1:25  AB_307338  TNF-α (immunofluorescence)  Anti-TNF-α antibody  Abcam, ab6671  Rabbit; polyclonal to TNF-α  1:25  AB_305641  Abbreviation: mAb, monoclonal antibody. View Large Abbreviations: APA aldosterone-producing adenoma BMI body mass index C/EBP CCAAT-enhancer-binding protein CIDEA cell death–inducing DFFA-like effector A COLI collagen I DMEM Dulbecco’s modified Eagle medium EGR1 early growth response factor-1 EH essential hypertension FBS fetal bovine serum FN fibronectin IL-6 interleukin-6 MR mineralocorticoid receptor mRNA messenger RNA NT normotension PA primary aldosteronism PCR polymerase chain reaction PGC1α peroxisome proliferator-activated receptor γ coactivator-1α PPARγ peroxisome proliferator–activated receptor γ RRID Research Resource Identifier SVF stromal vascular fraction TGFβ1 transforming growth factor β1 TNF-α tumor necrosis factor-α UCP1 uncoupling protein 1. Acknowledgments We thank Yu-Hua Tseng (Joslin Diabetes Center, Harvard Medical School, Boston, MA) for providing the mouse brown preadipocytes and Dang Qiang and Tong Chen (Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou, China) for their help in obtaining human adipose tissue samples. Financial Support: This work was supported by National Natural Science Foundation of China Grants 81628004, 31400992, 81470047, and 81500656, and Guangdong Science and Technology Project 2013B022000061. Disclosure Summary: The authors have nothing to disclose. References 1. Funder JW, Carey RM, Mantero F, Murad MH, Reincke M, Shibata H, Stowasser M, Young WF, Jr. The management of primary aldosteronism: case detection, diagnosis, and treatment: an endocrine society clinical practice guideline. J Clin Endocrinol Metab . 2016; 101( 5): 1889– 1916. Google Scholar CrossRef Search ADS PubMed  2. Milliez P, Girerd X, Plouin PF, Blacher J, Safar ME, Mourad JJ. 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Published: Jan 1, 2018

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