Angiotensin type 1 receptor modulates macrophage polarization and renal injury in obesity

Abstract

The mechanisms for increased risk of chronic kidney disease (CKD) in obesity remain unclear. The renin-angiotensin system is implicated in the pathogenesis of both adiposity and CKD. We investigated whether the angiotensin type 1 (AT₁) receptor, composed of dominant AT_1a and less expressed AT_1b in wild-type (WT) mice, modulates development and progression of kidney injury in a high-fat diet (HFD)-induced obesity model. WT mice had increased body weight, body fat, and insulin levels and decreased adiponectin levels after 24 wk of a high-fat diet. Identically fed AT_1a knockout (AT1aKO) mice gained weight similarly to WT mice, but had lower body fat and higher plasma cholesterol. Both obese AT1aKO and obese WT mice had increased visceral fat and kidney macrophage infiltration, with more proinflammatory M1 macrophage markers as well as increased mesangial expansion and tubular vacuolization, compared with lean mice. These abnormalities were heightened in the obese AT1aKO mice, with downregulated M2 macrophage markers and increased macrophage AT_1b receptor. Treatment with an AT₁ receptor blocker, which affects both AT_1a and AT_1b, abolished renal macrophage infiltration with inhibition of renal M1 and upregulation of M2 macrophage markers in obese WT mice. Our data suggest obesity accelerates kidney injury, linked to augmented inflammation in adipose and kidney tissues and a proinflammatory shift in macrophage and M1/M2 balance.

obesity is increasing worldwide, with 66% of adults in the US overweight and 33% obese (12, 36). Obesity is associated with, and contributes to, development of type 2 diabetes mellitus, cardiovascular disease, and nondiabetic chronic kidney disease (CKD) ( 45). Adipose tissue is a complex endocrine organ that releases hormones and adipokines, including TNF-α, IL-1β, and monocyte chemoattractant protein-1 (MCP-1), all components of the renin-angiotensin system (RAS), with diverse functions that affect glucose homeostasis, lipid metabolism, and inflammation (13, 41, 51).

Macrophage infiltration is a common feature of obesity and many kidney diseases (5, 18). However, macrophages have significant heterogeneity in their functions (9). Macrophages have recently been classified into two polarization states, M1 and M2. M1 or “classically activated” macrophages are induced by classic immune pathways (such as LPS and interferon-γ), and enhance proinflammatory cytokine production, such as TNF-α, IL-1β, and IL-6. M2 or “alternatively activated” macrophages are generated by exposure to IL-4 and IL-13. M2 macrophages are important in resolution of inflammation and tissue repair through synthesis of anti-inflammatory cytokines IL-10 and IL-1 decoy receptor and endocytic clearance capacities (8). Inhibition of proinflammatory macrophages has beneficial effects in experimental chronic inflammatory renal injury (46). However, relevance to obesity-associated CKD is unknown.

RAS activation, via the AT₁ receptor on macrophages, directly promotes macrophage infiltration and activation (10, 37, 40, 42, 43). The RAS activation is implicated in pathogenesis of both adiposity and CKD (6). An angiotensin receptor blocker, which decreases AT₁ activity, attenuates obesity and metabolic abnormalities (20, 26).

Wild-type (WT) mice have two AT₁ receptors, the dominant AT_1a and less expressed AT_1b. Recently, Crowley et al. (3) reported that AT_1b mRNA is upregulated in podocytes in murine autoimmune nephritis when a systemic AT_1a receptor is absent, suggesting that AT_1b may have a compensating role in augmented kidney injury and inflammation. We hypothesize that the AT₁ receptor is activated in macrophages in obesity and affects obesity-induced kidney injury by modulating macrophage polarization. We investigated this hypothesis using AT1a-deficient (AT1aKO) mice and studied the impact of AT_1a vs. AT_1b and an AT₁ receptor blocker (ARB) on obesity-induced kidney injury.

MATERIALS AND METHODS

Mice.

Adult male (8-wk-old) AT1aKO mice on a C57BL/6J background and C57BL/6J WT mice were purchased from Jackson Laboratory (Bar Harbor, ME). Mice were housed in a pathogen-free barrier facility under normal conditions with a 12:12-h light-dark cycle at 21°C with 40% humidity and 12 air exchanges/h, with food and water ad libitum. All animal protocols were approved by the Vanderbilt University Institutional Animal Care and Use Committee according to National Institutes of Health guidelines.

Before the study, all mice were fed a standard normal chow diet (Purina Rodent “5001” meal, 23.4% protein, 4.5% fat, 6.0% fiber, 0.40% sodium, Tusculum Feed Center, Nashville, TN). Obesity was induced by placing mice on a high-fat diet (HFD; 58% of total calories from fat, F1850, Bioserv Industries, Frenchtown, NJ) as previously described (26). At age 10–12 wk, mice were divided into four groups and treated for 24 wk as follows: 1) WT on normal chow (lean WT; n = 10); 2) AT1aKO mice on normal chow (lean AT1aKO; n = 10); 3) WT mice on HFD (obese WT; n = 12); and 4) AT1aKO mice on HFD (obese AT1aKO; n = 10). To determine whether treatment with ARB alters the phenotype of the WT macrophages, mice were treated for 12 wk as follows: 1) WT mice on HFD (WT+HFD; n = 4) or 2) WT mice on HFD with ARB (losartan, 320 mg/l dry wt based on effects in CKD; WT+HFD+ARB; n = 5). The dose of losartan (320 mg/l dry wt) is comparable to the dose of 25–32 mg/kg previously used by our group and others to study CKD, suggesting that a higher dose of ARB affords superior renoprotection in chronic kidney disease independently of hemodynamic effects (7, 11, 50). Normal adult WT mice (age 10–12 wk) were studied as baseline control (n = 5). Food intake was weighed daily, and body weight was measured every 4 wk. Fasting blood glucose, plasma, and urine were collected every 8 wk. Metabolic studies were performed as indicated below. Mice were killed at 12 or 24 wk. White adipose tissue (epididymal fat pads), skeletal muscle, and kidneys were harvested for morphological and molecular analysis. To assess gene expression in macrophages, peritoneal macrophages were harvested from four mice in each group above at the end of the study (see below).

Blood measurements.

Blood glucose and plasma measurements were made 6 h after daytime food withdrawal. Fasting blood glucose levels were determined using an Accu-Check glucose monitor (Roche Diagnostics Boehringer Mannheim, Indianapolis, IN). Plasma insulin, adiponectin, and lipid were assessed at the Mouse Metabolic Phenotyping Center (MMPC) at Vanderbilt University (http://www.mc.vanderbilt.edu/root/vumc.php?site=mmpc). Plasma insulin was measured by RIA (Linco Research, St. Louis, MO). Plasma adiponectin was measured by Luminex in a single-plex format (Millipore, formerly Linco Research, St. Charles, MO). Plasma triglyceride, total cholesterol, and HDL cholesterol levels were determined using kits (Raichem, San Diego, CA) at the MMPC at Vanderbilt University.

Tail-cuff blood pressure measurement, urine albumin, and renal function.

Systolic blood pressure (SBP) was measured in trained, conscious mice using tail-cuff impedance plethysmography (BP-2000 Blood Pressure Analysis System, Visitech Systems, Apex, NC). Urine was collected from mice individually housed in polycarbonate metabolic cages (Nalgene, Rochester, NY). Excretion of urinary albumin was determined using albumin-to-creatinine ratio (ACR). Urinary albumin and creatinine were determined using a mouse Albuwell ELISA kit and a Creatinine Companion kit (Exocell, Philadelphia, PA). Plasma creatinine was measured with a Quantichrom Creatinine Assay kit, using a modified Jaffé method that accurately measures creatinine in mice, as previously described (Bioassay Systems) (16).

Analysis of body composition.

Whole-animal body composition was assessed using the minispec Live Mice Whole Body Composition Analyzer (LF50) based on TD-NMR. This method provides a precise method for measurement of lean tissue, fat, and fluid in live mice and rats (Bruker Optik, Ettlingen, Germany). Animals were fasted for 16 h before scanning to reduce the variability attributed to food in the digestive tract.

Structural analyses.

Epididymal fat pads and the kidney cortex were dissected and immersion-fixed in 4% paraformaldehyde/PBS solution overnight at 4°C and embedded in paraffin using standard techniques. Four-micrometer sections were dewaxed, rehydrated, stained with periodic acid-Schiff, and immunohistochemistry was performed as described below. A semiquantitative score (0–4) was used to evaluate the degree of mesangial expansion and/or glomerulosclerosis: 0 represents no lesion, 1+ represents mesangial expansion and/or glomerulosclerosis of <25% of the glomerulus, while 2+, 3+, and 4+ represent mesangial expansion and/or glomerulosclerosis of 25–50, >50–75, and >75% of the glomerulus (24). All glomeruli in one section were evaluated and scored. Tubulointerstitial fibrosis was assessed qualitatively. All sections were examined without the examiner's knowledge of the treatment protocol.

Immunohistochemistry.

Immunostaining was performed in paraformaldehyde-fixed, paraffin-embedded sections using the following specific antibodies: 1) rat anti-mouse F4/80 for macrophages (1:20, Serotec, Raleigh, NC) (25); 2) rabbit anti-phospho-c-Jun (Ser73, 1:100, Cell Signaling, Danvers, MA); 3) rabbit-anti-c-Jun (60A6, 1:100, Cell Signaling); 4) rabbit-anti-phospho-JNK (Thr183/Tyr185, 1:100, Cell Signaling); 5) rabbit anti-albumin (1:200, Nordic Immunology, Tilburg, The Netherlands); and 6) rabbit anti-megalin (HPA005980, Sigma). The intensity of albumin and megalin immunostaining was quantified using image J software (National Institutes of Health, Bethesda, MD) as described previously (30) and expressed as optical density (OD) units. F4/80-positive cells were counted in high-power fields, with the average expressed from a total of 6–8 high-power fields in fat and 10–14 high-power fields in the kidney cortex. Phospho-cJun-positive tubular cells and phospho-JNK-positive tubules in high-power fields were counted in the kidney cortex. Control slides treated with nonspecific antisera instead of primary antibodies showed no staining.

Peritoneal macrophages.

Peritoneal macrophages were collected from four mice in each group at 24 wk. Four days before euthanization, mice underwent peritoneal injection of 3% thioglycollate and macrophages were harvested as previously described (34).

Gene expression studies.

Total RNA was isolated from kidney cortex, epididymal fat pads, and peritoneal macrophages using the RNeasy Mini kit according to the manufacturer's instructions (Qiagen). RNA was reverse transcribed using the TaqMan reverse transcription kit (Applied Biosystems). Real-time PCR amplifications were performed on an ABI Prism 7000 Sequence Detection System (Applied Biosystems) using TaqMan Universal PCR Master Mix (Applied Biosystems). The following inventoried TaqMan assays IDs were used: Mm99999056_m1 for MCP-1, Mm00434228_m1 for IL-1β, Mm00443258_m1 for TNF-α, Mm01329362_m1 for the mannose receptor, Mm00657889_mH for Ym-1, Mm00439616_m1 for IL-10, Mm00441724 for TGF-β, Mm00439622_m1 for decoy IL-1R (type II), Mm01166161_m1 for AT1a, Mm01701115_m1 for AT1b, and Hs99999901_s1 for 18s rRNA. 18S rRNA was used as an internal control. The relative levels of mRNA expression for these genes were obtained as previously described (19). Kidney nephrin mRNA expression was assessed by quantitative real-time PCR using specific primers (5′-t g g a t c t g a t g t g a c c c t c a and 5′-c t g g g t c a c t g a c t t c a g c a) as described previously (23). The ratio of M1/M2 macrophages in adipose tissue was calculated by the mRNA expression ratio of M1 (IL-1β) vs. M2 markers (mannose receptor). The ratio of M1/M2 in peritoneal macrophages was calculated by the mRNA expression ratio of M1 (IL-1β+MCP-1) vs. M2 markers (mannose receptor+Ym-1+TGF-β+IL-10+decoy IL-1R).

SDS-PAGE, Western blot analysis.

Western blots for Ym-1 protein expression were performed as previously described (19) using primary rabbit anti-Ym-1 antibody (1:1,000 dilution, StemCell Technologies) followed by horseradish peroxidase-conjugated anti-rabbit secondary antibody (Amersham Biosciences, Buckinghamshire, UK). Images were visualized using an ECL Western Blotting Detection Reagent (Amersham Biosciences). Membranes were reprobed with mouse anti-α-tubulin antibody (Sigma) as a loading control, and expression was semiquantitatively assessed by SCION Image Beta 4.03 (Scion) as a densitometric ratio relative to each loading control. Western blotting for kidney megalin expression was performed after separating protein samples for 6 h on 4–20% gradient gels with rabbit anti-megalin antibody (1:500 dilution, Santa Cruz Biotechnology). Megalin is a very large protein (600 kDa). Therefore, two additional SDS-PAGE (10%) gels were run using the same samples, one probed with GAPDH and one stained with ponceau S (to show similar protein loading). Megalin expression was normalized to GAPDH.

Statistical analysis.

Data are expressed as means ± SE. Differences among groups were examined for statistical significance using one-way ANOVA followed by Tukey's test or Mann-Whitney-U test as appropriate. A P value <0.05 was considered significant.

RESULTS

Systemic metabolic and hemodynamic parameters.

Body weight was comparable between WT and AT1aKO mice at baseline [25.0 ± 0.4 vs. 25.8 ± 0.6 g, P not significant (pNS)]. WT and AT1aKO mice on HFD had similar food intake (2.28 ± 0.05 vs. 2.37 ± 0.05 g/day, pNS) and similar increases in body weight (60%) (Fig. 1A). However, obese AT1aKO mice had significantly less body fat (31%) than obese WT mice (39%) (Fig. 1B), confirmed by decreased epididymal fat pad mass (Fig. 1C). Interestingly, the whole body muscle percentage was significantly higher in AT1aKO mice on normal chow and HFD compared with WT mice (Fig. 1D). The liver-to-body weight ratio was similar in WT and AT1aKO mice (Fig. 1E).

Fig. 1.

AT_1a deficiency decreases adipose tissue mass. A: body weight of lean vs. obese wild-type (WT) and AT_1a knockout (AT1aKO) mice. B: total body fat percentage. C: epididymal fat mass at 24 wk. D: muscle weight/total body weight. E: liver weight/total body weight (n = 6–8/group). *P < 0.05. **P < 0.01. ***P < 0.001.

Fasting blood glucose levels were similarly increased over baseline in obese WT and obese AT1aKO mice after 24 wk of a high-fat diet (Table 1). Plasma insulin levels were significantly increased over baseline in obese WT and numerically lower in obese AT1aKO mice. Plasma adiponectin levels were significantly decreased in obese WT mice but remained similar to baseline levels in obese AT1aKO mice (Table 1). Although circulating triglycerides did not differ among the groups, plasma total cholesterol levels were significantly higher in lean and obese AT1aKO mice compared with lean and obese WT mice (Table 2). Plasma HDL was also higher in lean AT1aKO mice, with similarly high levels in obese WT and obese AT1aKO mice (Table 2).

Table 1.

Metabolic parameters in lean and obese WT and AT1aKO mice

	0 wk	24 wk
	Blood glucose, mg/dl
Lean WT	131.8 ± 4.9	133.0 ± 4.8
Lean AT1aKO	132.6 ± 2.7	132.6 ± 2.7
Obese WT	135.4 ± 6.0	188.6 ± 11.7*
Obese AT1aKO	132.9 ± 4.0	189.2 ± 9.3*
	Plasma insulin, ng/ml
Obese WT	0.6 ± 0.0	4.9 ± 3.9†
Obese AT1aKO	0.5 ± 0.1	3.9 ± 0.7†
	Plasma adiponectin, μg/ml
Obese WT	12.8 ± 0.6	9.6 ± 0.7*
Obese AT1aKO	11.3 ± 0.7	11.5 ± 0.3‡

Values are means ± SE. WT, wild-type; AT1aKO, ANG II type 1a deficient.

^*P < 0.01, 24 wk vs. 0 wk,

^†P < 0.001, 24 wk vs. 0 wk,

^‡P < 0.05, WT vs. AT1aKO at 24 wk.

Table 2.

Plasma lipids in lean and obese WT and AT1aKO mice

	Triglyceride, mg/dl	Total Cholesterol, mg/dl	HDL, mg/dl
Lean WT	79.3 ± 6.8	80.8 ± 0.8	47.5 ± 1.5
Lean AT1aKO	79.0 ± 5.3	116.6 ± 3.1*	58.6 ± 1.7*
Obese WT	76.9 ± 1.7	185.1 ± 7.3†	70.4 ± 1.6†
Obese AT1aKO	78.3 ± 3.5	213.3 ± 5.8‡§	71.7 ± 1.8§

Values are means ± SE.

^*P < 0.01 lean WT vs. lean AT1aKO.

^†P < 0.01 obese WT vs. lean WT.

^‡P < 0.01 obese WT vs. obese AT1aKO.

^§P < 0.01 obese AT1aKO vs. lean AT1aKO.

Obesity is associated with increased blood pressure (27). Our data confirmed mild but significantly higher SBP in obese WT vs. lean WT mice, but no hypertension in any AT1aKO mice (WT obese vs. lean 110 ± 2 vs. 96 ± 6 mmHg, P < 0.05; AT1aKO obese vs. lean 89 ± 5 vs. 90 ± 4 mmHg, pNS; P < 0.01 obese WT vs. obese AT1aKO).

Since rodents have two isoforms of the AT₁ receptor, namely, AT_1a and AT_1b, we next studied AT_1b receptor expression. AT_1b mRNA was ∼2.3-fold higher in visceral adipose tissue of obese AT1aKO vs. obese WT mice (fold change vs. lean WT: lean WT 1.00 ± 0.08, lean AT1aKO 1.21 ± 0.11, obese WT 0.90 ± 0.12, obese AT1aKO 2.26 ± 0.37, P < 0.01 obese AT1aKO vs. obese WT). In WT mice, obesity increased macrophage AT_1a, but not AT_1b, mRNA (Fig. 2A). In contrast, macrophage AT_1b mRNA was markedly increased in obese AT1aKO vs. obese WT mice (Fig. 2B). Thus deletion of AT_1a resulted in marked compensatory upregulation of AT_1b.

Fig. 2.

Increased AT_1b mRNA in macrophages of obese AT1aKO mice. A: macrophage AT_1a mRNA was increased in obese WT vs. lean WT mice. B: macrophage AT_1b mRNA was similar in lean AT1aKO vs. lean WT mice and remained unchanged in obese WT but was significantly upregulated in obese mice when AT_1a was deficient. *P < 0.05.

Renal injury.

Although plasma creatinine levels were normal in all mice at 24 wk (lean WT 0.49 ± 0.10, lean AT1aKO 0.52 ± 0.08, obese WT 0.44 ± 0.06, obese AT1aKO 0.41 ± 0.02 mg/dl; pNS), HFD feeding induced structural kidney injury in WT mice that included mesangial expansion, tubular vacuolization, flattening, and dilation (P < 0.05 vs. lean WT, Fig. 3, A–C). AT_1a receptor deficiency significantly potentiated these HFD-induced injuries (P < 0.05 vs. obese WT, Fig. 3, A–C). Electron microscopy revealed no or very limited foot process effacement, but segmental loss of glomerular endothelial fenestration in both obese WT and obese AT1aKO mice (data not shown). Glomerular basement membrane thickness was similar in obese WT and obese AT1aKO mice (206 ± 12 vs. 198 ± 10 nm, pNS). Compared with lean mice with normal kidneys, ACR rose comparably in both obese AT1aKO (4-fold increase) and obese WT mice (4.7-fold increase) (Fig. 3D). Renal nephrin mRNA expression, a corollary of podocyte differentiation, was variable but not different among groups (lean WT 1.3 ± 0.6 arbitrary units, lean AT1aKO 0.84 ± 0.3, obese WT 1.6 ± 0.4, obese AT1aKO 1.7 ± 0.3, pNS). Immunohistochemical analysis showed that obese AT1aKO mice had 47% increased tubular albumin expression vs. obese WT mice (Fig. 4A, B, P < 0.05), associated with 33% increased tubular megalin expression vs. obese WT mice (P < 0.05) (Fig. 4, E and F). To further quantitatively assess megalin expression, we performed Western blotting on whole kidney samples. Lean AT1aKO mice showed 1.5-fold higher megalin expression vs. lean WT mice (P = 0.06, Fig. 4, G and H), although immunohistochemical staining did not detect such change (Fig. 4, C and D). After HFD feeding, Western blotting confirmed that obese AT1aKO mice had 2.2-fold higher megalin expression vs. obese WT mice (P < 0.05, Fig. 4, G and H), suggesting that the ACR difference may be contributed to by alterations in tubular function.

Fig. 3.

Kidney structural injury and albuminuria in obesity. A–C: obese WT mice had increased mesangial expansion (arrows) and tubular vacuolization (arrowheads). Obese AT1aKO mice had worsened mesangial expansion (arrows) and tubular vacuolization (arrowheads; A, periodic acid-Schiff, ×200). D: albumin-creatinine ratio (ACR) was significantly increased in both obese WT and obese AT1aKO vs. the corresponding lean mice. *P < 0.05. **P < 0.01.

Fig. 4.

Expression of albumin and megalin in kidney of WT and AT1aKO mice. A and B: immunohistochemical analysis showed obese AT1aKO mice had 47% increased tubular albumin expression vs. obese WT (P < 0.05). C–F: immunohistochemical analysis showed that lean WT and lean AT1aKO mice had comparable megalin expression in proximal tubules. Obese AT1aKO mice had 33% higher tubular megalin expression vs. obese WT mice (P < 0.05). G and H: Western blots showed lean AT1aKO mice had 1.5-fold higher kidney megalin expression vs. lean WT. Obese AT1aKO mice had 2.2-fold higher megalin expression vs. obese WT mice (P < 0.05). Ponceau S-stained SDS-PAGE further confirmed the similar loading for megalin Western blotting. Immunostaining, ×400 for A–F. *P < 0.05.

Scattered and similar renal interstitial infiltration of macrophages was observed in both lean WT and AT1aKO mice (Fig. 5, A and B). Obesity increased renal F4/80⁺ macrophage infiltration in the interstitium, especially in obese AT1aKO mice (nearly 3-fold higher vs. obese WT, Fig. 5, A and B). We next examined expression of M1 macrophage markers (MCP-1, TNF-α) and the M2 macrophage marker (Ym-1). Kidney MCP-1 mRNA levels increased 3.5-fold in obese vs. lean WT mice (P < 0.05), but increased 21-fold in obese AT1aKO mice (P < 0.001 vs. lean WT and obese WT, Fig. 5C). Kidney TNF-α mRNA expression was similar in lean and obese WT but increased almost 2-fold in obese AT1aKO mice (P < 0.01, Fig. 5D). Kidney Ym-1 protein expression did not differ between lean WT and lean AT1aKO mice (Fig. 5, E and F). In contrast, kidney Ym-1 expression was decreased 48% in obese AT1aKO vs. obese WT mice (Fig. 5, G and H).

Fig. 5.

Macrophage infiltration and expression of M1 (MCP-1, TNF-α) and M2 (Ym-1) markers in the kidney. A and B: obese WT mice had increased interstitial F4/80⁺ macrophage infiltration. Obese AT1aKO mice had greater renal macrophage infiltration (anti-F4/80 immunostaining, ×400; A). B: immunostaining quantification (means ± SE, 6–8 animals/group). C and D: obese WT mice had increased renal MCP-1 mRNA but not TNF-α mRNA expression. Obese mice with AT_1a deficiency had higher renal MCP-1 and also TNF-α mRNA levels. E and F: kidney Ym-1 expression did not differ in lean WT vs. lean AT1aKO mice. G and H: obese AT1aKO vs. obese WT mice showed significantly decreased renal Ym-1 expression by Western blotting. *P < 0.05. **P < 0.01. ***P < 0.001.

Systemic and adipose inflammatory parameters.

Various stresses, including inflammation, activate c-Jun NH2-terminal kinase1 (JNK1) and transcription factor activator protein-1 (AP-1), which in turn upregulate proinflammatory and profibrogenic genes (29). We therefore assessed renal phosphorylated-c-Jun, a component of AP-1. Only rare immunostaining for phosphorylated c-Jun was seen in tubular cells in lean WT and AT1aKO mice (Fig. 6A). Obesity significantly increased expression of phosphorylated-c-Jun, 3.9-fold in WT and 10-fold in AT1aKO mice, in both proximal and collecting tubules (P < 0.01 obese AT1aKO vs. obese WT) (Fig. 6, A and B). Interestingly, obesity also induced higher renal c-Jun expression (data not shown) and phosphorylated JNK expression in tubules in obese AT1aKO vs. obese WT mice (P < 0.01, Fig. 6, C and D).

Fig. 6.

Phospho-cJun and phospho-JNK immunostaining. A: obese WT mice had activated p-cJun staining in proximal and collecting tubules, with even more staining in obese AT1aKO mice (anti-p-cJun immunostaining, ×400). B: immunostaining quantification (means ± SE, 6–8 animals/group). C: obese AT1aKO mice had greater p-JNK staining in tubular epithelial cells vs. obese WT mice (×400). D: immunostaining quantification (means ± SE). *P < 0.05. **P < 0.01.

We next assessed whether adipose tissue macrophage infiltration was also altered. Lean WT or AT1aKO mice had no detectable F4/80⁺ macrophages in epididymal fat tissue (Fig. 7, A and B). Obese WT mice had significantly increased adipose F4/80⁺ macrophage infiltration, with even greater infiltration in obese AT1aKO mice (Fig. 7, A and B). We next examined adipose tissue expression of M1 and M2 macrophage markers. Adipose tissue of obese WT mice had 6.7-fold higher mRNA expression of MCP-1 and 6.4-fold higher mRNA expression of IL-1β vs. lean WT mice (both P < 0.05 vs. lean WT, Fig. 8, A and B). Obese AT1aKO mice also had 5.9- and 6.4-fold higher adipose tissue MCP-1 and IL-1β vs. lean WT, respectively (both P < 0.05, Fig. 8, A and B). Adipose tissue TNF-α mRNA expression was not significantly different among groups (data not shown). In contrast, adipose expression of M2 markers, the mannose receptor and Ym-1, was significantly downregulated (31 and 35%, respectively) in obese AT1aKO vs. obese WT mice (Fig. 8, C, F, and G) (although no difference between lean mice, Fig. 8, C–E), leading to a higher adipose tissue M1/M2 ratio (expressed as IL-1β/mannose receptor, 8.6 ± 1.9 vs. 5.9 ± 1.0, Fig. 8H). Of note, despite an absence of macrophages in adipose tissues of lean AT1aKO mice, mRNA expression of MCP-1 and IL-1β was numerically (3.3- and 1.8-fold, respectively) higher in this tissue vs. lean WT (Fig. 8, A and B). These data suggest other cells, such as adipocytes or capillary endothelial cells, contribute to the adipose tissue inflammatory activation and that AT_1a influences cytokine production in other cell types.

Fig. 7.

Macrophages in adipose tissue. A: obese WT mice had increased F4/80⁺ macrophage infiltration in adipose tissue. A: obese AT1aKO mice had even greater macrophage infiltration (anti-F4/80 immunostaining, ×400). B: immunostaining quantification (means ± SE, 6–8 animals/group). *P < 0.05.

Fig. 8.

Expression of M1 (MCP-1, IL-1β) and M2 (mannose receptor, Ym-1) markers in adipose tissue. A and B: increased expression of M1 markers MCP-1 and IL-1β in adipose tissue of obese WT and obese AT1aKO mice. C: lower M2 marker mannose receptor mRNA in adipose tissue of obese AT1aKO vs. obese WT mice. D and E: there was no significant difference of fat Ym-1 in lean WT vs. lean AT1aKO mice. F and G: obese AT1aKO vs. obese WT mice showed significantly decreased expression of the M2 marker Ym-1 in adipose tissue. H: M1/M2 ratio in adipose tissue was increased in obese AT1aKO vs. WT mice. *P < 0.05.

To further explore the impact of the AT_1a receptor on macrophage polarization, we analyzed expression of M1 and M2 markers in isolated peritoneal macrophages. Macrophages from obese WT mice had increased MCP-1 and IL-1β mRNA vs. lean WT (2.0- and 3.5-fold, respectively). This M1 activation of macrophages was markedly enhanced in obese AT1aKO mice, with nearly 9-fold higher MCP-1 and 13-fold higher IL-1β mRNA levels vs. lean WT mice (P < 0.05 vs. lean and obese WT, Fig. 9, A and B). Macrophage M2 marker mannose receptor mRNA increased similarly in obese WT and obese AT1akO mice compared with lean levels (2.3- and 3.1-fold, respectively, Fig. 9C). Macrophage M2 marker Ym-1 mRNA was significantly decreased in obese WT but maintained in obese AT1aKO mice (Fig. 9D). Macrophage M2 marker TGF-β mRNA was increased in response to obesity in WT, but not in AT1aKO mice (Fig. 9E). Macrophage IL-10 and decoy IL-1 receptor mRNA expressions, M2 markers, were not significantly different among groups (Fig. 9, F and G). Thus the M1/M2 ratio was much higher in macrophages of obese AT1aKO vs. obese WT mice (Fig. 9H).

Fig. 9.

Expression of M1 (MCP-1, IL-1β) and M2 (mannose receptor, Ym-1, TGF-β, IL-10, decoy IL-1R) markers in peritoneal macrophages. A and B: increased expression of MCP-1 and IL-1β proinflammatory genes in peritoneal macrophages of obese AT1aKO vs. obese WT mice. C: expression of mannose receptor mRNA was similarly increased in obese WT and obese AT1aKO vs. lean mice. D: expression of Ym-1 mRNA was significantly decreased in macrophages of obese WT but not in obese AT1aKO mice. E: expression of TGF-β mRNA was increased in obese WT vs. lean WT mice but remained at similar lower levels in obese as lean AT1aKO mice. F and G: expression of IL-10 (F) and decoy IL-1R mRNA (G) was not significantly different among the groups. H: M1/M2 ratio in peritoneal macrophages was increased in obese AT1aKO vs. obese WT mice (n = 4/group for peritoneal macrophages).*P < 0.05. **P < 0.01. ***P < 0.001.

To determine whether pharmacological blockade of the AT₁ receptor (ARB), which blocks both AT_1a and AT_1b, alters the macrophage phenotype response to obesity, we studied WT mice on HFD with or without ARB treatment for 12 wk. ARB treatment did not affect body weight but decreased blood glucose levels and epididymal fat mass (Table 3). ARB abolished the obesity-induced increase in renal macrophages (Fig. 10, A and B). ARB decreased obesity-induced upregulation of TNF-α, MCP-1, and IL-1β mRNA in the kidney and restored the obesity-induced decrease in mannose receptor and Ym-1 mRNA (Fig. 10, C–G).

Table 3.

Metabolic parameters in ARB-treated obese mice

	0 wk	12 wk
Blood glucose, mg/dl
WT+HFD	115.1 ± 3.1	216.6 ± 8.8*
WT+HFD+ARB	111.1 ± 5.6	142.4 ± 6.7*†
Body weight, g
WT+HFD	24.0 ± 0.2	44.8 ± 1.5*
WT+HFD+ARB	25.9 ± 0.7	42.3 ± 0.9*
Epididymal fat mass, g
WT+HFD		1.2 ± 0.1
WT+HFD+ARB		0.9 ± 0.1‡

Values are means ± SE. ARB, angiotensin receptor blocker; HFD, high-fat diet.

^*P < 0.01, 12 wk vs. 0 wk.

^†P < 0.01, WT+HFD+ARB vs. WT+HFD at 12 wk.

^‡P < 0.05, WT+HFD+ARB vs. WT+HFD at 12 wk.

Fig. 10.

Blockade of AT₁ receptor (ARB) suppressed infiltrated macrophages in the kidney of obese WT mice. A and B: treatment with ARB for 12 wk in obese WT mice abolished F4/80(+) macrophage infiltration in the kidney (anti-F4/80 immunostaining, ×400; A). C–G: WT mice fed HFD for 12 wk had increased renal TNF-α, MCP-1, and IL-1β mRNA expression, but decreased mannose receptor and Ym-1 mRNA expression. ARB treatment in obese WT downregulated TNF-α, MCP-1, and IL-1β mRNA, but increased mannose receptor and Ym-1 mRNA expression. B: immunostaining quantification (means ± SE, n = 4–5 animals/group).

DISCUSSION

In the present study, we investigated whether the AT₁ receptor affects inflammation and kidney injury induced by obesity. Obesity accelerated kidney injury in WT mice. Unexpectedly, obesity-related kidney injury was even worse with genetic deletion of the AT_1a receptor. Although obese WT mice had higher SBP than lean mice, SBP remained ≤110 mmHg in all groups. These data support the notion that the amplified structural kidney injury in obese AT1aKO mice is not primarily mediated by hypertension. Rather, our findings suggest that amplified structural kidney damage in obese AT1aKO mice was associated with augmented inflammation in kidneys and adipose tissue.

Our finding that obesity-related kidney injury is enhanced in the AT1aKO mice is consistent with a growing number of reports that AT_1a deficiency can amplify tissue injury (2, 3, 33). We have previously found that unilateral ureteral obstruction (UUO)-induced renal injury was worsened by repletion with AT1aKO bone marrow (33). A recent study of chronic ANG II infusion in chimeric mice repleted with AT1aKO bone marrow showed greater hypertension, exaggerated renal expression of MCP-1, and persistent renal macrophage accumulation than in mice with AT_1a intact bone marrow, suggesting that bone marrow-derived AT₁ receptors may have a role in limiting mononuclear cell accumulation in the kidney (2). Furthermore, a lack of the AT_1a receptor augmented kidney injury and inflammation in MRL-fas^lpr/lpr (lpr) mice, a model of lupus (3).

Macrophage infiltration in the kidney has an important role in the development and progression of renal diseases. Macrophage phenotypes and function are critical determinants of the balance of promoting tissue injury vs. resolution of injury (17). Activation of the AT₁ receptor has a direct influence on monocyte/macrophage infiltration (28). Our previous data demonstrate that systemic ANG II amplifies macrophage-driven atherosclerosis (34). However, the specific role of the AT₁ receptor on macrophages has been controversial. The absence of AT₁ receptors on macrophages has deleterious effects in pathological conditions including atherosclerosis or kidney fibrosis (15, 33). We therefore considered the possibility that macrophage functions may be modulated by systemic AT_1a deficiency in this obesity model. We found that obese WT mice had enhanced renal macrophage infiltration and that this response was even greater in obese AT1aKO mice, perhaps related to increased AT_1b. To characterize the impact of the AT₁ receptor on macrophage polarization in obesity-related kidney injury, we analyzed expression of a panel of M1 and M2 macrophage markers in the kidney, adipose tissue, and isolated peritoneal macrophages. Obesity increased renal M1 markers in WT mice, a response that was further amplified in kidneys of AT1aKO mice, suggesting that abnormally activated inflammation may contribute to the more severe kidney injury in obese mice. Interestingly, kidney Ym-1 expression, a marker specific for the M2 macrophage phenotype, was significantly suppressed in obese AT1aKO compared with obese WT mice. Thus lack of the AT₁ receptor, with an increase in AT_1b, shifted the M1/M2 balance in the kidney, promoting renal inflammation.

Our finding of augmented AT_1b in macrophages in AT1aKO mice in response to obesity supports a role for AT_1b in this obesity-related kidney injury when AT_1a is deleted. The improvement of renal injury and inflammation in HFD with ARB, which blocks both receptors, further emphasizes the importance of AT₁ in mediating injury in response to obesity. Recently, Aki et al. (1) demonstrated that, in a model of antiglomerular basement membrane, renal infiltration of M1 and M2 macrophages can be modulated by ARB. We also found that the anti-inflammatory action of ARB in this obesity-related kidney injury model was associated with downregulation of M1 markers and upregulation of M2 markers in the kidney. These data also show that AT1aKO mice do not mirror the impact of blockade or absence of AT₁, as disease-related compensatory upregulation of AT_1b in AT1aKO mice clearly mediates some of the RAS-dependent injury.

To further explore the downstream mechanisms activated with this enhanced kidney inflammation induced by obesity, we assessed phosphorylated JNK and phosphorylated c-Jun, a component of the transcription factor AP-1. AP-1 regulates many genes involved in the progression of kidney disease. The transcriptional activity of AP-1 is regulated by phosphorylation of Ser63 and 73 through SAPK/JNK (4). Macrophages can be activated through JNK to induce TNF-α production. MCP-1 induces inflammatory responses of tubular epithelial cells via AP-1 activation (29, 44). Our data suggest that AP-1 is activated in the renal tubular epithelial cells of obese AT1aKO mice and thus could contribute to the more severe kidney injury in these mice. Compared with obese WT, obese AT1aKO mice had less proteinuria that was associated with preserved nephrin mRNA and increased tubular albumin and megalin expression. Interestingly, we previously showed that alterations in albumin and magalin correlate with tubule function (31). Thus double transgenic mice generated by crossing megalin knockout mosaic mice (lacking megalin expression in 60% of proximal tubule cells) with NEP25 mice (a transgenic line expressing human CD25 in the podocyte), and injuring these mice with immunotoxin caused massive nonselective proteinuria and mild glomerular and tubular injury. Comparison of megalin-containing to megalin-deficient proximal tubule cells showed that albumin, immunoglobulin light chain IgA and IgG preferentially accumulated in proximal tubule cells expressing megalin. On the other hand, increased megalin has been reported to be associated with reduced proteinuria in a diabetic nephropathy model (39). These results show that megalin plays a pivotal role in reabsorption of small to large molecular size proteins and provides direct in vivo evidence that reabsorption of filtered proteins triggers events leading to tubule injury (31). Our observations also support the possibility that AT_1a deficiency, with AT_1b upregulation, regulated megalin expression and increased tubular protein reabsorption in obese AT1aKO mice. Further studies will be needed to explore the mechanisms by which the AT₁ receptor regulates megalin expression.

Emerging data suggest that macrophages recruited into adipose tissue of obese individuals are proinflammatory and contribute to insulin resistance (32, 48). Obesity also induces a macrophage phenotypic switch toward proinflammatory M1 macrophages in adipose tissue (8, 21, 22, 35). Consistent with previous studies (47, 49), we found that obesity increased macrophage infiltration in adipose tissue and markedly increased M1 markers. In the absence of the AT_1a receptor, with increased AT1b, this response in adipose tissue was augmented. Interestingly, although expression of M1 markers in visceral fat tissue was not different between obese WT vs. obese AT1aKO mice, expression of the potentially beneficial M2 markers was markedly downregulated by AT₁ receptor deficiency with concurrently increased AT_1b. A similar shift in the M1/M2 ratio in peritoneal macrophages was seen in obese AT1aKO mice due to a robust increase in M1 markers.

Obesity not only induced greater inflammation in kidney and adipose tissue in obese vs. lean AT1aKO mice, it also increased body weight and blood glucose levels, without affecting SBP. These results complement findings by Rong et al. (38), who showed that high fat- vs. normal chow-fed AT1aKO mice developed increased body weight, epididymal adipose tissue mass, and blood glucose levels, also without an effect on SBP (38). The higher SBP, as well as body weight in our study, likely reflect the later start point (10–12 vs. 8 wk) and longer duration of the follow-up in our study (24 vs. 12 wk). Not surprisingly, administration of ARB, which blocks both AT_1a and AT1b receptors, improved all the metabolic abnormalities in obese AT1aKO mice (38).

In summary, obesity aggravates kidney injury, contributed to by augmented inflammation in adipose tissue and the kidney. Our data suggest that the AT₁ receptor modulates macrophage phenotypes and functions in obesity. Lack of AT_1a receptors with AT_1b upregulation on macrophages thus not only promotes macrophage infiltration into adipose tissue and kidney but also adversely shifts the M1/M2 balance in response to obesity. Pharmacological inhibition of the AT₁ receptor with ARB in obese WT mice abolished obesity-induced renal macrophage infiltration, further suggesting a role of the AT₁ receptor in obesity-induced kidney injury. We speculate that direct infiltration of inflammatory M1 macrophages into the kidney as well as macrophage infiltration into adipose tissue and adipocyte-specific metabolites, including free fatty acids, leptin, and adiponectin, contribute to obesity-related kidney injury (14).

GRANTS

These studies were supported in part by ADA research award 7-04-RA-69 (L.-J. Ma) and National Institute of Diabetes and Digestive and Kidney Diseases Grant P50 DK044757-18 (A. B. Fogo and V. Kon). This work was also supported in part by National Heart, Lung, and Blood Institute Grant HL087061 (V. Kon and S. Fazio).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.