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Comparative Study
. 2015 Sep;242(1):56-64.
doi: 10.1016/j.atherosclerosis.2015.06.055. Epub 2015 Jul 4.

Atherosclerosis following renal injury is ameliorated by pioglitazone and losartan via macrophage phenotype

Suguru Yamamoto  1 , Jiayong Zhong  2 , Patricia G Yancey  3 , Yiqin Zuo  4 , MacRae F Linton  5 , Sergio Fazio  6 , Haichun Yang  4 , Ichiei Narita  7 , Valentina Kon  8
Affiliations
Comparative Study

Atherosclerosis following renal injury is ameliorated by pioglitazone and losartan via macrophage phenotype

Suguru Yamamoto et al. Atherosclerosis. 2015 Sep.

Abstract

Objective: Chronic kidney disease (CKD) amplifies atherosclerosis, which involves renin-angiotensin system (RAS) regulation of macrophages. RAS influences peroxisome proliferator-activated receptor-γ (PPARγ), a modulator of atherogenic functions of macrophages, however, little is known about its effects in CKD. We examined the impact of combined therapy with a PPARγ agonist and angiotensin receptor blocker on atherogenesis in a murine uninephrectomy model.

Methods: Apolipoprotein E knockout mice underwent uninephrectomy (UNx) and treatment with pioglitazone (UNx + Pio), losartan (UNx + Los), or both (UNx + Pio/Los) for 10 weeks. Extent and characteristics of atherosclerotic lesions and macrophage phenotypes were assessed; RAW264.7 and primary peritoneal mouse cells were used to examine pioglitazone and losartan effects on macrophage phenotype and inflammatory response.

Results: UNx significantly increased atherosclerosis. Pioglitazone and losartan each significantly reduced the atherosclerotic burden by 29.6% and 33.5%, respectively; although the benefit was dramatically augmented by combination treatment which lessened atherosclerosis by 55.7%. Assessment of plaques revealed significantly greater macrophage area in UNx + Pio/Los (80.7 ± 11.4% vs. 50.3 ± 4.2% in UNx + Pio and 57.2 ± 6.5% in UNx + Los) with more apoptotic cells. The expanded macrophage-rich lesions of UNx + Pio/Los had more alternatively activated, Ym-1 and arginine 1-positive M2 phenotypes (Ym-1: 33.6 ± 8.2%, p < 0.05 vs. 12.0 ± 1.1% in UNx; arginase 1: 27.8 ± 0.9%, p < 0.05 vs. 11.8 ± 1.3% in UNx). In vitro, pioglitazone alone and together with losartan was more effective than losartan alone in dampening lipopolysaccharide-induced cytokine production, suppressing M1 phenotypic change while enhancing M2 phenotypic change.

Conclusion: Combination of pioglitazone and losartan is more effective in reducing renal injury-induced atherosclerosis than either treatment alone. This benefit reflects mitigation in macrophage cytokine production, enhanced apoptosis, and a shift toward an anti-inflammatory phenotype.

Keywords: Atherosclerosis; Chronic kidney disease; Losartan; Macrophage phenotype; PPARγ; Pioglitazone.

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Figures

Fig. 1
Fig. 1
Synergistic effects of pioglitazone and losartan, on renal injury-induced acceleration in atherosclerosis. Apolipoprotein E knockout mice underwent sham operation (Sham, n = 10) or uninephrectomy (UNx, n = 40) and were divided into four groups: no treatment (UNx, n = 10), pioglitazone (UNx + Pio, n = 10), losartan (UNx + Los, n = 10), and pioglitazone together with losartan (UNx + Pio/Los, n = 10). Atherosclerosis was assessed on cryosections from the proximal aorta and stained with Oil-Red-O. **p < 0.05 vs. UNx, and #p < 0.05 vs. UNx + Pio/Los.
Fig. 2
Fig. 2
Pioglitazone and losartan combination lessens necrotic areas in proximal atherosclerotic lesions and increases macrophage content. Apolipoprotein E knockout mice underwent sham operation (Sham, n = 10) or uninephrectomy (UNx, n = 40) and were divided into four groups: no treatment (UNx, n = 10), pioglitazone (UNx + Pio, n = 10), losartan (UNx + Los, n = 10), and pioglitazone together with losartan (UNx + Pio/Los, n = 10). Necrotic area assessed by acellular area in Harris H&E sections (A) and macrophages content assessed by MOMA-2 staining (B), calculated as ratio with atherosclerotic lesion, respectively. **p < 0.05 vs. UNx.
Fig. 3
Fig. 3
Pioglitazone and losartan modulate renal damage-induced macrophage phenotype. Immunofluorescent staining for CCR7 (A), iNOS (B), Ym-1 (C) and arginase 1 (D) assessed as fractions of total macrophages stained with CD68 in atherosclerotic lesions of mice with sham-operation or uninephrectomy with no treatment (UNx), pioglitazone (UNx + Pio), losartan (UNx + Los), and pioglitazone together with losartan (UNx + Pio/Los). **p < 0.05 vs. UNx, and #p < 0.05 vs. UNx + Pio/Los.
Fig. 4
Fig. 4
Combination treatment with pioglitazone and losartan increased apoptotic macrophages in proximal atherosclerotic lesions. Apoptoric macrophage in the atherosclerotic lesion assessed by staining with TUNEL, CD68 and DAPI in atherosclerotic lesions of mice with sham-operation or uninephrectomy with no treatment (UNx), pioglitazone (UNx + Pio), losartan (UNx + Los), and pioglitazone together with losartan (UNx + Pio/Los).
Fig. 5
Fig. 5
Pioglitazone and losartan modulate LPS-induced macrophage M1 phenotypic change and inflammatory reaction. RAW264.7 macrophages were reacted with LPS (50 ng/ml, n = 4), LPS and pioglitazone (10 μM) (n = 4), LPS and losartan (10 μM) (n = 4), and LPS and both pioglitazone and losartan (n = 4) for 24 h. Quantitative real-time PCR for iNOS (A), CCR7 (B), TNF-α (C), MCP-1 (D), and arginase-1 (E) was performed. GAPDH was used as an internal control. **p < 0.05 vs. UNx, and #p < 0.05 vs. UNx + Pio/Los.
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