Objective Chronic kidney disease (CKD) amplifies atherosclerosis which involves renin-angiotensin system

Objective Chronic kidney disease (CKD) amplifies atherosclerosis which involves renin-angiotensin system (RAS) regulation of macrophages. 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; Natural264.7 and main peritoneal mouse cells were used to examine pioglitazone and losartan effects on macrophage phenotype and inflammatory response. Results UNx significantly improved atherosclerosis. Pioglitazone and losartan each significantly reduced LSD1-C76 the atherosclerotic burden by 29.6% and 33.5% respectively; LSD1-C76 although the benefit was dramatically augmented by combination treatment which lessened atherosclerosis by 55.7%. Assessment of plaques exposed significantly higher macrophage area in UNx + Pio/Los (80.7 ± 11.4% < 0.05 < 0.05 < 0.05 was considered to be significant. 3 Results 3.1 Systemic guidelines Table 1 shows the systemic guidelines. There were no variations in body weight or blood glucose among the organizations. In agreement with previous reports [11 37 UNx caused a moderate but significant increase in serum creatinine and this was not revised by pioglitazone or losartan. BP decreased in mice treated with losartan only and in combination with pioglitazone. Pioglitazone treatment alone did not impact BP however total cholesterol and triglycerides levels improved both in mice treated with pioglitazone alone and in combination with losartan. Table 1 Systemic guidelines. 3.2 Atherosclerotic lesions and necrotic area UNx significantly increased atherosclerotic lesion area as assessed by Oil-Red-O staining of aortic cross-sections by 67.7% compared to sham (331 385 ± 25 20 μm2 in UNx < 0.05). These results are in agreement with previous findings with this model [11 37 (Fig. 1). Pioglitazone and losartan each significantly reduced UNx-dependent atherosclerosis by 29.6% and 33.5% respectively (233 408 ± 17 116 μm2 in UNx + Pio and 220 335 LSD1-C76 ± 24 382 μm2 in UNx + Los both < 0.05 < 0.05 < 0.05). Compared to untreated UNx all treatment regimens decreased the necrotic area with the Pio/Los combination causing the greatest reduction. (4.67 ± 1.00% in UNx + Pio 5.03 ± 0.97% in UNx + Los and 2.98 ± 0.89% in UNx + Pio/Los < 0.05 < < 0.05 for each comparison Fig. 2B). The macrophage phenotype within the atherosclerotic lesions was also affected by treatment. UNx significantly improved the subtype of macrophages expressing markers of the M1 phenotype including CCR7 (75.2 ± 4.8% < 0.05) and ActRIB iNOS (61.9 ± 4.8% < 0.05) (Fig. 3A and B). The lesions of UNx mice also experienced fewer cells with markers of the M2 phenotype including Ym-1 (12.0 ± 1.1% < 0.05) and arginase 1 (11.8 ± 1.3% < 0.05) (Fig. 3C and D). In contrast pioglitazone and losartan treatment reduced M1 phenotype prevalence (CCR7: 40.3 ± 4.3% in UNx + Pio and 29.1 ± 6.0% in UNx + Los < 0.05 < 0.05 < 0.05 < 0.05 vs. UNx) (Fig. 3C and D). The percent apoptotic macrophages assessed by TUNEL staining was significantly improved in UNx + Pio/Los mice compared to untreated UNx UNx + Los and UNx + Pio (23.50 ± 1.32% vs 3.82 ± 1.63% 9.62 ± 0.92% 9.91 ± 1.89% Fig. 4) 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 … Fig. 4 Combination treatment with pioglitazone and losartan improved 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 … 3.4 Macrophage inflammation and phenotype modulation in vitro Pioglitazone alone and together with losartan modulated the LPS-induced response of iNOS CCR7 TNF-α and MCP-1 expression in Natural264.7 macrophages (Fig. 5A-D) and thioglycollate-elicited peritoneal macrophages from C57BL/6 mice (Supplemental Fig. 2A-D). Losartan only experienced a smaller effect on cytokine activation iNOS production (Fig. 5A and Supplemental Fig. 2A) and manifestation of additional inflammatory cytokines in both cell types (Fig. 5B-D and Supplemental Fig. 2B-D). By contrast pioglitazone alone or with losartan improved macrophage arginase1 mRNA manifestation in both cell types (Fig. 5E and Supplemental Fig. 2E). Fig. 5 Pioglitazone and losartan modulate LPS-induced LSD1-C76 macrophage M1 phenotypic switch and inflammatory reaction. Natural264.7 macrophages were reacted with LPS.