Deletion of bone marrow myeloperoxidase attenuates chronic kidney disease accelerated atherosclerosis

Abstract

Increased myeloperoxidase (MPO) expression and activity are associated with atherosclerotic disease in patients with chronic kidney disease (CKD). However, the causal relationship between MPO and the development and progression of atherosclerosis in patients with CKD is unknown. Eight-week-old male low-density-lipoprotein-receptor-deficient mice were subjected to 5/6 nephrectomy, irradiated, and transplanted with bone marrow from MPO-deficient mice to induce bone marrow MPO deletion (CKD-bMPOKO) or bone marrow from WT mice as a control to maintain preserved bone marrow MPO(CKD-bMPOWT). The mice were maintained on a high-fat/high-cholesterol diet for 16 weeks. As anticipated, both groups of mice exhibited all features of moderate CKD, including elevated plasma creatinine, lower hematocrit, and increased intact parathyroid hormone but did not demonstrate any differences between the groups. Irradiation and bone marrow transplantation did not further affect body weight, blood pressure, creatinine, or hematocrit in either group. The absence of MPO expression in the bone marrow and atherosclerotic lesions of the aorta in the CKD-bMPOKO mice was confirmed by immunoblot and immunohistochemistry, respectively. Decreased MPO activity was substantiated by the absence of 3-chlorotyrosine, a specific by-product of MPO, in aortic atherosclerotic lesions as determined by both immunohistochemistry and highly sensitive LC-MS. Quantification of the aortic lesional area stained with oil red O revealed that CKD-bMPOKO mice had significantly decreased aortic plaque area as compared with CKD-bMPOWT mice. This study demonstrates the reduction of atherosclerosis in CKD mice with the deletion of MPO in bone marrow cells, strongly implicating bone-marrow-derived MPO in the pathogenesis of CKD atherosclerosis.

Keywords: atherosclerosis; chronic kidney disease; macrophages; myeloperoxidase; oxidized amino acids.

Figure 1

Bone marrow MPO deficiency in 5/6 nephrectomized mice does not alter lipid profiles. Deletion of bone marrow MPO (bMPOKO) does not alter. A, total cholesterol, B, high-density lipoprotein (HDL), or C, triglycerides in 5/6 nephrectomized (CKD) mice as compared with CKD mice with preserved bone marrow MPO (CKD-bMPOWT) after 16 weeks on a high-fat/high-cholesterol diet. All p values >0.05.

Figure 2

Bone marrow deletion of MPO in mice with 5/6 nephrectomy on a high-fat diet does not affect vascular reactivity. Response relaxation curve of response to acetylcholine of aortic rings precontracted with phenylephrine from 5/6 nephrectomized mice with bone marrow MPO deficiency (CKD-bMPOKO) or WT bone marrow expression of MPO (CKD-bMPOWT) on high-fat diet/high-cholesterol diet for 16 weeks n = 5/group. All p values >0.05.

Figure 3

MPO and its oxidation products colocalize with macrophages and are decreased in the vascular wall of 5/6 nephrectomized mice with bone marrow deletion of MPO. Representative immunofluorescence and double labeling in the aortic cross sections from male LDLr^−/− 5/6 nephrectomized mice after 16 weeks on a high-fat/high-cholesterol diet with bone marrow deletion of MPO (CKD-bMPOKO) or intact bone marrow MPO expression (CKD-bMPOWT). Staining for Mac-2 (macrophage marker, green) and red staining for (A) MPO, (B) 3-chlorotyrosine, and (C) 3-nitrotyrosine, are shown. Yellow staining (MERGE) indicates the colocalization of MPO and oxidation markers with Mac-2. Also represented are the negative controls with DAPI (blue) staining only. Scale bars represent 20 μm.

Figure 4

Bone marrow MPO deficiency in 5/6 nephrectomized mice on a high-fat/high-cholesterol diet decreases MPO-dependent oxidation in atherosclerotic lesions. Mass spectrometric quantification of oxidized amino acids in aortic proteins from irradiated 5/6 nephrectomized LDLr ^−/− mice (CKD) mice with WT bone marrow (CKD-bMPOWT) and with bone marrow MPO deficiency (CKD-bMPOKO) after 16 weeks on a high-fat/high-cholesterol diet. A, 3-chlorotyrosine and B, 3-nitrotyrosine expressed as ratios to the precursor amino acid, tyrosine, in μmol/mol (n= 12 each). ∗p < 0.05.

Figure 5

Atherosclerosis is reduced in 5/6 nephrectomized mice with bone marrow MPO deficiency in proportion to the MPO-dependent oxidation in atherosclerotic lesions. Oil red staining and morphometry of en face aorta from irradiated 5/6 nephrectomized LDLr ^−/− mice (CKD) after 16 weeks on a high-fat/high-cholesterol diet. A, CKD mice with WT bone marrow (CKD-bMPOWT). B, CKD mice with bone marrow MPO deficiency (CKD-bMPOKO). Scale bar represents 2 mm. C, analysis of total atherosclerotic lesion area as a ratio (ratio of Oil red O stained area to the total surface area of the en-face section of the aortic tree expressed as a percentage) reveals decreases in the area occupied by atherosclerotic lesions in the CKD-bMPOKO mice as compared with the CKD-bMPOWT mice. Panels D and E show correlation of oxidized amino acids in aortic proteins and stained atherosclerotic area (n = 14).∗p < 0.05.

Figure 6

Serum Amyloid A levels are increased in 5/6 nephrectomized mice with bone marrow MPO deficiency. Serum amyloid A (SAA) protein levels are increased with bone marrow MPO deficiency in irradiated 5/6 nephrectomy mice (CKD) LDLr ^−/− mice after 16 weeks on a high-fat/high-cholesterol diet (n= 9 each group; ∗p < 0.05).