Uremic serum-induced calcification of human aortic smooth muscle cells is a regulated process involving Klotho and RUNX2

Abstract

Vascular calcification (VC) is common in subjects with chronic kidney disease (CKD) and is associated with increased cardiovascular risk. It is an active process involving transdifferentiation of arterial smooth muscle cells (SMCs) into osteogenic phenotype. We investigated the ability of serum from CKD subjects to induce calcification in human SMCs in vitro (calcific potential of sera: CP), and associated changes in expression of Runt-related transcription factor 2 (RUNX2), SM22α, and Klotho. Sera from subjects with CKD (18 stage 3, 17 stage 4/5, and 29 stage 5D) and 20 controls were added to human cultured SMCs and CP quantified. The CP of CKD sera was greater (P<0.01) than that of controls, though not influenced by CKD stage. Modification of diet in renal disease estimated glomerular filtration rate (MDRD-4 eGFR) (P<0.001), serum phosphate (P=0.042), receptor activator of nuclear factor κappa-B ligand (RANKL) (P=0.001), parathyroid hormone (PTH) (P=0.014), and high-density lipoprotein (HDL)/cholesterol ratio (P=0.026) were independent predictors of CP accounting for 45% of variation. Adding calcification buffer (CB: calcium chloride [7 mM] and β-glycerophosphate [7 mM]) increased the CP of control sera to approximate that of CKD sera. CP of CKD sera was unchanged. CKD sera increased RUNX2 expression (P<0.01) in human SMCs and decreased SM22α expression (P<0.05). Co-incubating control but not CKD serum with CB further increased RUNX2 expression (P<0.01). Both SM22α and Klotho expression decreased significantly (P<0.01) in the presence of CKD serum, and were virtually abolished with stage 5D sera. These findings support active regulation by CKD serum of in vitro VC by induction of RUNX2 and suppression of SM22α and Klotho.

Keywords: Chronic kidney disease; Human aortic smooth muscle Cells; Klotho; RUNX2; Uraemic serum; Vascular calcification.

Figure 1. Induction and time course of calcification in human SMCs

Human SMCs cultured to ∼60% confluence were incubated in complete culture medium alone (Control; open bar), media containing 7 mM CaCl₂, 7 mM β-GP (solid bars) or a combination of both (A). The time course of calcification using CaCl₂ and β-GP combined is shown in (B). Calcification was quantified and normalised for total cell protein as described in the ‘Materials and methods’ section. The data are presented as means ± S.E.M. of three independent experiments with five replicates in each.

Figure 2. Effects of serum on calcification of human SMCs

Human SMCs cultured to ∼60% confluence were incubated for 5 days in complete culture medium with 10% serum from controls (open bars, (A)) or from CKD3, CKD4/5, or HD cohorts (solid bars, (A)). In parallel, cells were also incubated with serum and CB together (hatched bars, (B)). Calcification was quantified and normalised for total cell protein as described in the ‘Materials and methods’ section. The data are presented as means ± S.E.M. of three independent experiments with five replicates in each.

Figure 3. Expression of RUNX2 and Klotho in human SMCs in the presence of calcification inducers

Human SMCs cultured to ∼60% confluence were incubated in complete culture medium alone (Control; open bar), media containing 7 mM CaCl₂, 7 mM β-GP, or a combination of both (solid bars). Cell lysates were collected and probed for RUNX2 (A) or Klotho (B) using Western blotting as described in the ‘Materials and methods’ section. The data are presented as means ± S.E.M. of at least three independent experiments and expressed as percentages relative to maximal expression in cells incubated with CB for RUNX2 or control for Klotho expression.

Figure 4. Effects of serum on the expression of RUNX2, SM22α, and Klotho in human SMCs

Human SMCs cultured to ∼60% confluence were incubated for 5 days in complete culture medium with serum in the absence (open and black bars) and presence of CB (7 mM CaCl₂ plus 7 mM β-GP) (hatched bars). Cell lysates were collected and probed for RUNX2 (A), SM22α (B), or Klotho (C) using Western blotting as described in the ‘Materials and methods’ section. The data are presented as means ± S.E.M. of at least three independent experiments and expressed in percentages relative to maximal expression in cells incubated with HD serum for RUNX2 or control serum for SM22α and Klotho expression.