Vitamin D Metabolism and Action in Human Marrow Stromal Cells: Effects of Chronic Kidney Disease

Abstract

Human marrow stromal cells (hMSCs) are targets of 1! ,25-dihydroxyvitamin D [1! ,25(OH)₂D₃] action to promote their differentiation to osteoblasts, but they also participate in vitamin D metabolism by converting 25-dihydroxyvitamin D₃ [25(OH)D₃] to 1! ,25(OH)₂D₃ by 1α-hydroxylase (CYP27B1). Chronic kidney disease (CKD) is associated with impaired renal biosynthesis of 1! ,25(OH)₂D, low bone mass, and increased fracture risk. We tested whether CKD influences hMSCs' responses to vitamin D₃ metabolites. The hMSCs were obtained from tissues discarded during arthroplasty for hip osteoarthrosis, including a subject who had been undergoing hemodialysis for 2+ years. There was a significant positive correlation between in vitro stimulation of osteoblastogenesis (alkaline phosphatase activity) by 1! ,25(OH)₂D₃ and subjects' estimated glomerular filtration rate (eGFR, r=0.47, p=0.015, n=26, 56–83 years of age). Osteoblastogenesis was stimulated in hMSCs from both the hemodialysis and control subjects by 1! ,25(OH)₂D₃ (10 ! M), 25(OH)D₃ (100 ! M), or D₃ (1000 ! M). Thus, vitamin D metabolism may play an autocrine/paracrine role in osteoblast differentiation of hMSCs. These findings suggest that in CKD patients 25(OH)D-sufficiency may play an important role in skeletal health; osteoblastic bone formation in CKD patients may not be optimal unless there is sufficient serum 25(OH)D substrate for the MSCs to synthesize and respond to local 1! ,25(OH)₂D.

1. Introduction

Chronic kidney disease (CKD) is associated with impaired renal biosynthesis of 1! ,25(OH)₂D, low bone mass, and increased fracture risk [1]. Osteoblastogenesis of human marrow stromal/mesenchymal stem cells (hMSCs) is stimulated by both 1! ,25(OH)₂D₃ and 25-hydroxyvitamin D₃ [25(OH)D₃] [2], the later effect requiring conversion to 1,25(OH)₂D₃ by 25-hydroxyvitamin D₃ 1α-hydroxylase (CYP27B1) [3]. CYP27B1 in hMSCs is upregulated by 25(OH)D [2] and PTH [4] and is downregulated by 1! ,25(OH)₂D [2], similar to regulation of renal CYP27B1. These findings suggest an autocrine/paracrine role of vitamin D metabolism in osteoblastogenesis of hMSCs. Because nothing is known about the effect of CKD on this process, we tested whether responsiveness of hMSCs to vitamin D metabolites was influenced by renal status of the subjects from whom the cells were obtained.

2. Materials and methods

Bone marrow samples from subjects older than 55-years were obtained with IRB approval as femoral tissue discarded during primary hip arthroplasty for osteoarthritis, with exclusion criteria, isolation methods [2] and eGFR [5] as described. This series included 26 men (n=10) and women (n=16) whose MSCs were used for in vitro osteoblast differentiation. The group mean values (assay normal range) were age 68.2 ± 1.4 years, serum 25(OH)D 30.6 ± 1.7 ng/mL (20–57), 1,25(OH)₂D 44.2 ± 2.8 pg/mL (18– 62), and PTH 38.2 ± 3.7 pg/mL (10–65). We also obtained deidentified discarded marrow from a 57-year old male orthopedic patient with end-stage renal disease (ESRD) who had been undergoing hemodialysis for 2+ years; although serum 25(OH)D level was not available, this subject had secondary hyperparathyroidism and was being treated with cinacalcet, calcium acetate, and active D, commonly used meds in the ESRD population. Reagents were obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise specified. The hMSCs were maintained in phenol red-free ! -MEM, 10% fetal bovine serumheat inactivated (FBS-HI), 100 u/mL penicillin, and 100 ! g/mL streptomycin (Invitrogen, Carlsbad, CA). Constitutive gene expression was determined by RT-PCR, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal control [2]. Upon confluence of passage 2 hMSCs, medium was changed to osteoblastogenic medium (α-MEM with 1% FBS-HI, 100 U/ml penicillin, 100 µg/ml streptomycin, 10⁻⁸ M dexamethasone, 5 mM β-glycerophosphate, 50 µg/ml ascorbate-2-phosphate) ± treatments for 7 days. As an index of osteoblast differentiation, alkaline phosphatase (ALP) enzyme activity was measured spectrophotometrically and expressed as a ratio of treated-to-control [2]. Quantitative data were analyzed with non-parametric tools; if data allowed, parametric tools were used.

3. Results

3.1 Effect of eGFR on responsiveness of human MSCs to 1! 25(OH)₂D₃

There was stimulation of in vitro osteoblast differentiation by 10 nM 1! ,25(OH)₂D₃ in the majority of hMSC specimens. There was a significant correlation between magnitude of stimulation and eGFR of the subjects from whom the hMSCs were obtained (Pearson r=0.47, p=0.015, Fig. 1).

Figure 1.

Correlation between magnitude of stimulation of Alkaline Phosphatase (ALP) activity by 1! ,25(OH)₂D₃ in hMSCs and subjects' eGFR. Line indicates Pearson r= 0.47, p=0.015 for all specimens. Units for estimated glomerular filtration rate (eGFR) are mL/min/1.73 m². Adapted from [5].

3.2 Effects of D₃, 25(OH)D₃, and 1! ,25(OH)₂D₃ on osteoblastogenesis in human MSCs from a subject undergoing hemodialysis and an age/gender-matched control subject

There was downregulated constitutive mRNA expression of CYP27B1 (1! -hydroxylase) and upregulated CYP24A1 (24-hydroxylase) in hMSCs from a 57-year-old male orthopedic subject who had been undergoing hemodialysis for more than 2 years, compared with MSCs from an age/gender-matched control subject (eGFR 101 mL/min/1.73 m²) (Fig 2). These data are consistent with the dialysis subject being treated with active D.

Figure 2.

Effects of D₃, 25(OH)D₃, and 1! ,25(OH)₂D₃ to stimulate osteoblastogenesis with hMSCs from a subject undergoing hemodialysis and control subject with normal eGFR. A) Gel electrophoretogram shows downregulated constitutive mRNA for CYP27B1 (1! -hydroxylase) and CYP27A1 (25-hydroxylase), upregulated CYP24A1 (24-hydroxylase), and similar CYP2R1 (25-hydroxylase) and VDR (vitamin D receptor) in MSCs from hemodialysis subject, compared with control. B) Bars indicate Alkaline Phosphatase (ALP) activity as mean ± SEM for 4 replicate dishes with each treatment.

The effects of vitamin D₃ metabolites on osteoblast differentiation were assessed with these specimens (Fig. 2). All three metabolites, 1000 mM D₃, 100 mM 25(OH)D₃, and 10 mM 1! ,25(OH)₂D₃, stimulated ALP activity in hMSCs from both subjects (ANOVA, p<0.001).

4. Discussion

These findings support the hypotheses that vitamin D metabolism serves an autocrine/paracrine role in human osteoblast differentiation and that 25(OH)D-sufficiency may be important for skeletal health in CKD. Detecting that 1! ,25(OH)₂D₃ stimulated osteoblast differentiation in hMSCs is consistent with other reports [2, 3, 4, 7]. We previously showed that stimulation of osteoblastogenesis by 25(OH)D₃ depended upon CYP27B1 mRNA in hMSCs [3]. This is the first report of stimulation by vitamin D₃ (cholecalciferol) - a finding that is consistent with mRNA expression in hMSCs of 25-hydroxylases [2]. The regulation of 25-hydroxylase activity of CYP2R1 and CYP27A1 in hMSCs and other extra-hepatic cells requires further investigation. In these experiments, we controlled for age and cell numbers because other studies showed the influence of subject age on intrinsic properties of hMSCs including proliferation rates [8], osteoblast differentiation potential [4, 8], CYP27B1 [4] and PTH receptor [9] gene expression, and responsiveness to PTH [9] and to 25(OH)D [4].

This study is limited by the unavailability of additional clinical information for this cohort such as serum FGF-23, which is known to be elevated in CKD [10]. It is also known that FGF-23 upregulates vitamin D- 24-hydroxylase in the kidney [11]. If that upregulation also occurs with hMSCs in subjects with low eGFR (and it may because hMSCs express mRNA for the FGF-23 receptor pair klotho and FGFR1, data not shown), we could propose elevated 24-hydroxylase as a testable mechanism to account for the observed in vitro resistance to 1! ,25(OH)₂D₃. There are many examples of the enduring influence of the clinical environment on isolated hMSCs, including age [4,8,9], estrogen status [12], vitamin D status [2], and medications such as estrogen replacement therapy [12] or alendronate [13]. In addition, it will be necessary to test cells from more subjects receiving hemodialysis to compare dose-responsiveness to vitamin D metabolites.

There is controversy whether to monitor/correct serum 25(OH)D in CKD; according to clinical practice guidelines set forth by the National Kidney Foundation, treatment with vitamin D is recommended but is graded as “opinion” level. This reflects the paucity of high quality evidence [6]. Finding stimulation of osteoblastogenesis by cholecalciferol or 25(OH)D in this pilot study suggests that treatment of CKD with active D may be inadequate in the setting of 25(OH)D deficiency because of an autocrine/paracrine role of vitamin D metabolism in osteoblast differentiation of hMSCs (Fig. 3). Thus, in CKD patients, osteoblastic bone formation may not be optimal unless there is sufficient serum 25(OH)D substrate for the hMSCs to synthesize and respond to local 1! ,25(OH)₂D₃.

Figure 3.

Autocrine/paracrine hypothesis of the significance of marrow synthesis of 1! ,25(OH)₂D. In CKD, serum 25(OH)D may serve as a substrate for synthesis of 1! ,25(OH)₂D and local stimulation of osteoblast differentiation with marrow stromal cells (MSCs).

Highlights.

There was a correlation between stimulation of in vitro osteoblast differentiation of human marrow stromal cells and the eGFR of the subject from whom the cells were obtained.

Cholecalciferol, calcitriol, and calcitriol stimulated in vitro osteoblast differentiation of marrow stromal cells from control and CKD subjects.

Osteoblastic bone formation in CKD patients may not be optimal unless there is sufficient serum 25(OH)D substrate for marrow stromal cells to synthesize and respond to local 1,25(OH)₂D.

Abbreviations

1!, 25(OH)₂D₃

1!, 25-dihydroxyvitamin D₃

25(OH)D₃

25-dihydroxyvitamin D₃

ALP

alkaline phosphatase

CKD

Chronic kidney disease

eGFR

estimated glomerular filtration rate

hMSCs

human Marrow Stromal Cells

VDR

vitamin D receptor