Elastin degradation and vascular smooth muscle cell phenotype change precede cell loss and arterial medial calcification in a uremic mouse model of chronic kidney disease

Abstract

Arterial medial calcification (AMC), a hallmark of vascular disease in uremic patients, is highly correlated with serum phosphate levels and cardiovascular mortality. To determine the mechanisms of AMC, mice were made uremic by partial right-side renal ablation (week 0), followed by left-side nephrectomy at week 2. At 3 weeks, mice were switched to a high-phosphate diet, and various parameters of disease progression were examined over time. Serum phosphate, calcium, and fibroblast growth factor 23 (FGF-23) were up-regulated as early as week 4. Whereas serum phosphate and calcium levels declined to normal by 10 weeks, FGF-23 levels remained elevated through 16 weeks, consistent with an increased phosphate load. Elastin turnover and vascular smooth muscle cell (VSMC) phenotype change were early events, detected by week 4 and before AMC. Both AMC and VSMC loss were significantly elevated by week 8. Matrix metalloprotease 2 (MMP-2) and cathepsin S were present at baseline and were significantly elevated at weeks 8 and 12. In contrast, MMP-9 was not up-regulated until week 12. These findings over time suggest that VSMC phenotype change and VSMC loss (early phosphate-dependent events) may be necessary and sufficient to promote AMC in uremic mice fed a high-phosphate diet, whereas elastin degradation might be necessary but is not sufficient to induce AMC (because elastin degradation occurred also in uremic mice on a normal-phosphate diet, but they did not develop AMC).

Figure 1

Serum chemistry profiles over time: blood urea nitrogen (BUN) (A), phosphate (B), calcium (C), and FGF-23 (D) at baseline (n = 48), week 4 (n = 9), week 6/7 (n = 26), week 8 (n = 10), week 10/11 (n = 17), week 12 (n = 8), and week 16 (n = 8). Data are reported as means ± SEM. *P < 0.05, compared with baseline. All mice in the study were used for blood draws at the baseline, week 6/7, and week 10/11 time points.

Figure 2

Incidence of arterial medial calcification in CKD. Progression of calcification normalized to dry weight of aortic arch (A) at baseline (n = 8), week 4 (n = 7), week 8 (n = 10), and week 12 (n = 9), and of abdominal aorta (B) at baseline (n = 8), week 4 (n = 9), week 8 (n = 10), and week 12 (n = 8). Data are reported as means ± SEM. *P < 0.05, compared with baseline.

Figure 3

Representative histochemical micrographs of von Kossa staining for baseline (A), week 8 (B), week 12 (C), and week 16 (D) time points, with corresponding H&E-stained slides from adjacent sections (E–H). All sections were of the abdominal aorta region, and nuclei were counterstained with Harris hematoxylin. Scale bars = 30 μm throughout, except scale bar = 100 μm for D and H.

Figure 4

Representative micrographs of immunohistochemical staining. Baseline staining of SM22α (A) and Runx2/Cbfa1 (B), week 16 staining of SM22α (C), and week 4 staining of Runx2/Cbfa1 (D), arrows indicate positively stained nuclei. L, lumen. Inset shows nonspecific IgG staining as a control. All sections were of the abdominal aorta region, and SM22α nuclei were counterstained with methyl green. Scale bars = 30 μm. Immunohistochemistry scores for SM22α (E) and Runx2/Cbfa1 (F) were determined as described in the Materials and Methods section. Data are reported as means ± SEM. *P < 0.05, compared with baseline. **P < 0.05, compared with week 4 and week 8.

Figure 5

Progression of elastinolysis and calcification in CKD abdominal aortic media in von Kossa images at baseline and at weeks 4, 8, 12, and 16 (A–E, respectively), with corresponding eosin fluorescence images in adjacent sections (F–J). Scale bars = 30 μm.

Figure 6

Comparison of progression of aortic desmosine content in CKD at baseline (n = 6) and at week 4 (n = 7), week 8 (n = 5), and week 12 (n = 8). Data are reported as means ± SEM. *P < 0.05, compared with baseline.

Figure 7

Progression of MMP activation in CKD. Quantitative analysis of MMP-2 (A) and MMP-9 (B) immunostaining. Insets: Week 16 micrograph. All sections were of the abdominal aorta region; nuclei were counterstained with methyl green. Arrows indicate positively stained cells. L, lumen. Scale bars = 30 μm. (C) Cathepsin SWestern blot quantitation normalized to internal protein control. Data are reported as means ± SEM. *P < 0.05, compared with baseline.

Figure 8

Vascular smooth muscle cell loss in CKD. Comparison of number of VSMC/unit area at baseline (n = 7) and at week 4 (n = 8), week 8 (n = 6), week 12 (n = 9), and week 16 (n = 8). Data are reported as means ± SEM. *P < 0.05, compared with baseline.

Figure 9

Timeline of serum and tissue biochemical changes occurring in HP-fed CKD mice from week 0 to week 16. Increased and decreased levels are relative to baseline. Ca, calcium; Pi, phosphate; HP, high phosphate.