Vitamin D receptor agonists increase klotho and osteopontin while decreasing aortic calcification in mice with chronic kidney disease fed a high phosphate diet - PubMed (original) (raw)

Vitamin D receptor agonists increase klotho and osteopontin while decreasing aortic calcification in mice with chronic kidney disease fed a high phosphate diet

Wei Ling Lau et al. Kidney Int. 2012 Dec.

Abstract

Vascular calcification is common in chronic kidney disease, where cardiovascular mortality remains the leading cause of death. Patients with kidney disease are often prescribed vitamin D receptor agonists (VDRAs) that confer a survival benefit, but the underlying mechanisms remain unclear. Here we tested two VDRAs in a mouse chronic kidney disease model where dietary phosphate loading induced aortic medial calcification. Mice were given intraperitoneal calcitriol or paricalcitol three times per week for 3 weeks. These treatments were associated with half of the aortic calcification compared to no therapy, and there was no difference between the two agents. In the setting of a high-phosphate diet, serum parathyroid hormone and calcium levels were not significantly altered by treatment. VDRA therapy was associated with increased serum and urine klotho levels, increased phosphaturia, correction of hyperphosphatemia, and lowering of serum fibroblast growth factor-23. There was no effect on elastin remodeling or inflammation; however, the expression of the anticalcification factor, osteopontin, in aortic medial cells was increased. Paricalcitol upregulated osteopontin secretion from mouse vascular smooth muscle cells in culture. Thus, klotho and osteopontin were upregulated by VDRA therapy in chronic kidney disease, independent of changes in serum parathyroid hormone and calcium.

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Figures

Figure 1

Figure 1

Experimental design. CKD was induced by partial renal ablation: the right kidney was exposed, decapsulated, and electrocauterized (surgery 1), followed by left total nephrectomy two weeks later (surgery 2). Non-CKD control (NC) or CKD mice were placed on normal 0.5% phosphate diet (NP) or high 1.5% phosphate diet (HP) for three weeks. Concurrently, mice were given no treatment, calcitriol 30 ng/kg (C30), paricalcitol 100 ng/kg (P100) or paricalcitol 300 ng/kg (P300). The vitamin D receptor agonists (VDRAs) were given via intraperitoneal injections three times a week for three weeks.

Figure 2

Figure 2

(A) CKD mice on high phosphate diet (CKD+HP) developed vascular calcification that was significantly decreased by VDRA therapy. Aortic arch calcium content expressed as μg calcium normalized to mg dry weight (mean ± s.e.m.). *P<0.01 and **P<0.001 compared to CKD+HP group. Calcification was not different between the NC+NP, NC+HP and CKD+NP subgroups (_P_=1 for all posthoc Tukey analyses). Aortic calcium content was analyzed from all mice in the study (refer to Table 1 for n per group). (B) Thoracic aorta with Alizarin Red-S stain showing marked medial calcification in CKD+HP animal (ii) compared to NC+NP animal (i), and significantly less calcification in an animal treated with paricalcitol (CKD+HP+P300, iii). Scale bars are 150 μm and objective is 10X. C30 = calcitriol 30 ng/kg i.p. three times per week × three weeks, HP = high 1.5% phosphate diet, NC = non-CKD control mice, NP = normal 0.5% phosphate diet, P100 = paricalcitol 100 ng/kg, P300 = paricalcitol 300 ng/kg, CKD = chronic kidney disease mice.

Figure 2

Figure 2

(A) CKD mice on high phosphate diet (CKD+HP) developed vascular calcification that was significantly decreased by VDRA therapy. Aortic arch calcium content expressed as μg calcium normalized to mg dry weight (mean ± s.e.m.). *P<0.01 and **P<0.001 compared to CKD+HP group. Calcification was not different between the NC+NP, NC+HP and CKD+NP subgroups (_P_=1 for all posthoc Tukey analyses). Aortic calcium content was analyzed from all mice in the study (refer to Table 1 for n per group). (B) Thoracic aorta with Alizarin Red-S stain showing marked medial calcification in CKD+HP animal (ii) compared to NC+NP animal (i), and significantly less calcification in an animal treated with paricalcitol (CKD+HP+P300, iii). Scale bars are 150 μm and objective is 10X. C30 = calcitriol 30 ng/kg i.p. three times per week × three weeks, HP = high 1.5% phosphate diet, NC = non-CKD control mice, NP = normal 0.5% phosphate diet, P100 = paricalcitol 100 ng/kg, P300 = paricalcitol 300 ng/kg, CKD = chronic kidney disease mice.

Figure 3

Figure 3

(A) Correction of hyperphosphatemia correlated with increased fractional excretion of phosphate in VDRA-treated CKD+HP animals (FEphos calculated from 24-hour urine collections expressed as mean percentage ± s.e.m., _n_=6 for NC+NP and NC+HP groups, _n_=4 for CKD+HP group, _n_=5 for CKD+HP+C30 and CKD+HP+P300 groups). (B) Total urinary phosphate from 24-hour urine collections was significantly increased in CKD+HP animals compared to the NC+NP group. There was a trend for increased total phosphate excretion in VDRA-treated CKD+HP mice (_P_=0.06 and _P_=0.9 for CKD+HP+C30 and CKD+HP+P300 groups respectively, compared to CKD+HP animals). (C) Representative blot of serum klotho protein in individual mice from select groups (upper panel, 130 kDa band). Same blot was stripped and reprobed for immunoglobulin G heavy chain (IgG-HC) as loading control (lower panel). (D) Serum klotho levels were decreased in CKD, and were increased by VDRA therapy in high phosphate-fed CKD mice to levels that were significantly higher than in control mice. VDRAs did not significantly raise serum klotho in NC+HP and CKD+NP groups (posthoc Tukey P values shown on chart). Levels expressed as arbitrary units normalized to IgG-HC using densitometric analyses (mean ± s.e.m.), _n_=5 for NC+NP group, _n_=6 for NC+HP, CKD+NP and CKD+HP groups, _n_=3 in remaining groups. *P<0.01 and **P<0.001 compared to CKD+HP group, #P<0.05 and ##P<0.001 compared to NC+NP group.

Figure 3

Figure 3

(A) Correction of hyperphosphatemia correlated with increased fractional excretion of phosphate in VDRA-treated CKD+HP animals (FEphos calculated from 24-hour urine collections expressed as mean percentage ± s.e.m., _n_=6 for NC+NP and NC+HP groups, _n_=4 for CKD+HP group, _n_=5 for CKD+HP+C30 and CKD+HP+P300 groups). (B) Total urinary phosphate from 24-hour urine collections was significantly increased in CKD+HP animals compared to the NC+NP group. There was a trend for increased total phosphate excretion in VDRA-treated CKD+HP mice (_P_=0.06 and _P_=0.9 for CKD+HP+C30 and CKD+HP+P300 groups respectively, compared to CKD+HP animals). (C) Representative blot of serum klotho protein in individual mice from select groups (upper panel, 130 kDa band). Same blot was stripped and reprobed for immunoglobulin G heavy chain (IgG-HC) as loading control (lower panel). (D) Serum klotho levels were decreased in CKD, and were increased by VDRA therapy in high phosphate-fed CKD mice to levels that were significantly higher than in control mice. VDRAs did not significantly raise serum klotho in NC+HP and CKD+NP groups (posthoc Tukey P values shown on chart). Levels expressed as arbitrary units normalized to IgG-HC using densitometric analyses (mean ± s.e.m.), _n_=5 for NC+NP group, _n_=6 for NC+HP, CKD+NP and CKD+HP groups, _n_=3 in remaining groups. *P<0.01 and **P<0.001 compared to CKD+HP group, #P<0.05 and ##P<0.001 compared to NC+NP group.

Figure 3

Figure 3

(A) Correction of hyperphosphatemia correlated with increased fractional excretion of phosphate in VDRA-treated CKD+HP animals (FEphos calculated from 24-hour urine collections expressed as mean percentage ± s.e.m., _n_=6 for NC+NP and NC+HP groups, _n_=4 for CKD+HP group, _n_=5 for CKD+HP+C30 and CKD+HP+P300 groups). (B) Total urinary phosphate from 24-hour urine collections was significantly increased in CKD+HP animals compared to the NC+NP group. There was a trend for increased total phosphate excretion in VDRA-treated CKD+HP mice (_P_=0.06 and _P_=0.9 for CKD+HP+C30 and CKD+HP+P300 groups respectively, compared to CKD+HP animals). (C) Representative blot of serum klotho protein in individual mice from select groups (upper panel, 130 kDa band). Same blot was stripped and reprobed for immunoglobulin G heavy chain (IgG-HC) as loading control (lower panel). (D) Serum klotho levels were decreased in CKD, and were increased by VDRA therapy in high phosphate-fed CKD mice to levels that were significantly higher than in control mice. VDRAs did not significantly raise serum klotho in NC+HP and CKD+NP groups (posthoc Tukey P values shown on chart). Levels expressed as arbitrary units normalized to IgG-HC using densitometric analyses (mean ± s.e.m.), _n_=5 for NC+NP group, _n_=6 for NC+HP, CKD+NP and CKD+HP groups, _n_=3 in remaining groups. *P<0.01 and **P<0.001 compared to CKD+HP group, #P<0.05 and ##P<0.001 compared to NC+NP group.

Figure 3

Figure 3

(A) Correction of hyperphosphatemia correlated with increased fractional excretion of phosphate in VDRA-treated CKD+HP animals (FEphos calculated from 24-hour urine collections expressed as mean percentage ± s.e.m., _n_=6 for NC+NP and NC+HP groups, _n_=4 for CKD+HP group, _n_=5 for CKD+HP+C30 and CKD+HP+P300 groups). (B) Total urinary phosphate from 24-hour urine collections was significantly increased in CKD+HP animals compared to the NC+NP group. There was a trend for increased total phosphate excretion in VDRA-treated CKD+HP mice (_P_=0.06 and _P_=0.9 for CKD+HP+C30 and CKD+HP+P300 groups respectively, compared to CKD+HP animals). (C) Representative blot of serum klotho protein in individual mice from select groups (upper panel, 130 kDa band). Same blot was stripped and reprobed for immunoglobulin G heavy chain (IgG-HC) as loading control (lower panel). (D) Serum klotho levels were decreased in CKD, and were increased by VDRA therapy in high phosphate-fed CKD mice to levels that were significantly higher than in control mice. VDRAs did not significantly raise serum klotho in NC+HP and CKD+NP groups (posthoc Tukey P values shown on chart). Levels expressed as arbitrary units normalized to IgG-HC using densitometric analyses (mean ± s.e.m.), _n_=5 for NC+NP group, _n_=6 for NC+HP, CKD+NP and CKD+HP groups, _n_=3 in remaining groups. *P<0.01 and **P<0.001 compared to CKD+HP group, #P<0.05 and ##P<0.001 compared to NC+NP group.

Figure 4

Figure 4

(A) Representative Western blot showing klotho protein (130 kDa band) from whole kidney lysates (upper panel). Same blot was stripped and reprobed for beta-actin as loading control (lower panel). (B) Klotho protein in the kidney was significantly decreased by high phosphate diet (in both non-CKD control and CKD mice, ##P<0.001 compared to NC+NP group). VDRA treatment did not increase expression of kidney klotho in CKD mice. Levels expressed as arbitrary units normalized to beta-actin using densitometric analyses (mean ± s.e.m., _n_=3 for all groups). (C) Decreased klotho immunostaining in kidneys from NC+HP and CKD mice; i) NC+NP animal, ii) NC+HP animal, iii) CKD+HP animal, iv) CKD+HP+P300 animal. Scale bars are 50 μm and objective is 20X.

Figure 4

Figure 4

(A) Representative Western blot showing klotho protein (130 kDa band) from whole kidney lysates (upper panel). Same blot was stripped and reprobed for beta-actin as loading control (lower panel). (B) Klotho protein in the kidney was significantly decreased by high phosphate diet (in both non-CKD control and CKD mice, ##P<0.001 compared to NC+NP group). VDRA treatment did not increase expression of kidney klotho in CKD mice. Levels expressed as arbitrary units normalized to beta-actin using densitometric analyses (mean ± s.e.m., _n_=3 for all groups). (C) Decreased klotho immunostaining in kidneys from NC+HP and CKD mice; i) NC+NP animal, ii) NC+HP animal, iii) CKD+HP animal, iv) CKD+HP+P300 animal. Scale bars are 50 μm and objective is 20X.

Figure 4

Figure 4

(A) Representative Western blot showing klotho protein (130 kDa band) from whole kidney lysates (upper panel). Same blot was stripped and reprobed for beta-actin as loading control (lower panel). (B) Klotho protein in the kidney was significantly decreased by high phosphate diet (in both non-CKD control and CKD mice, ##P<0.001 compared to NC+NP group). VDRA treatment did not increase expression of kidney klotho in CKD mice. Levels expressed as arbitrary units normalized to beta-actin using densitometric analyses (mean ± s.e.m., _n_=3 for all groups). (C) Decreased klotho immunostaining in kidneys from NC+HP and CKD mice; i) NC+NP animal, ii) NC+HP animal, iii) CKD+HP animal, iv) CKD+HP+P300 animal. Scale bars are 50 μm and objective is 20X.

Figure 5

Figure 5

(A) Arterial medial osteopontin (OPN) levels were increased by VDRA treatment. (i) OPN expression was low but detectable in the aortic media of CKD mice fed a high phosphate diet (CKD+HP). VDRA treatment increased smooth muscle cell expression of OPN in the aortic media in (ii) CKD+HP+C30 animal; and (iii) CKD+HP+P300 animal. Arrows point to aortic medial cells expressing OPN. Scale bars are 30 μm and objective is 40X. (B) Quantitation of OPN immunostaining showed no OPN expression in aortas from non-CKD controls, weak staining in CKD high phosphate-fed mice, and increased levels in VDRA-treated CKD mice (mean ± s.e.m., _n_=3 for all except CKD+HP+paricalcitol, _n_=5 where P100 and P300 samples were grouped). (C) Treatment of cultured VSMCs with 50 nM paricalcitol increased OPN levels in the media (significantly higher levels by ELISA at 48hr compared to time zero, **P<0.001). Klotho (2 ng/mL) with/without FGF23 (2 ng/mL) did not upregulate OPN secretion. Three wells were sampled per time-point per treatment group, data are mean ± s.e.m. EtOH = ethanol control, KL = klotho, P50 = paricalcitol 50 nM.

Figure 5

Figure 5

(A) Arterial medial osteopontin (OPN) levels were increased by VDRA treatment. (i) OPN expression was low but detectable in the aortic media of CKD mice fed a high phosphate diet (CKD+HP). VDRA treatment increased smooth muscle cell expression of OPN in the aortic media in (ii) CKD+HP+C30 animal; and (iii) CKD+HP+P300 animal. Arrows point to aortic medial cells expressing OPN. Scale bars are 30 μm and objective is 40X. (B) Quantitation of OPN immunostaining showed no OPN expression in aortas from non-CKD controls, weak staining in CKD high phosphate-fed mice, and increased levels in VDRA-treated CKD mice (mean ± s.e.m., _n_=3 for all except CKD+HP+paricalcitol, _n_=5 where P100 and P300 samples were grouped). (C) Treatment of cultured VSMCs with 50 nM paricalcitol increased OPN levels in the media (significantly higher levels by ELISA at 48hr compared to time zero, **P<0.001). Klotho (2 ng/mL) with/without FGF23 (2 ng/mL) did not upregulate OPN secretion. Three wells were sampled per time-point per treatment group, data are mean ± s.e.m. EtOH = ethanol control, KL = klotho, P50 = paricalcitol 50 nM.

Figure 5

Figure 5

(A) Arterial medial osteopontin (OPN) levels were increased by VDRA treatment. (i) OPN expression was low but detectable in the aortic media of CKD mice fed a high phosphate diet (CKD+HP). VDRA treatment increased smooth muscle cell expression of OPN in the aortic media in (ii) CKD+HP+C30 animal; and (iii) CKD+HP+P300 animal. Arrows point to aortic medial cells expressing OPN. Scale bars are 30 μm and objective is 40X. (B) Quantitation of OPN immunostaining showed no OPN expression in aortas from non-CKD controls, weak staining in CKD high phosphate-fed mice, and increased levels in VDRA-treated CKD mice (mean ± s.e.m., _n_=3 for all except CKD+HP+paricalcitol, _n_=5 where P100 and P300 samples were grouped). (C) Treatment of cultured VSMCs with 50 nM paricalcitol increased OPN levels in the media (significantly higher levels by ELISA at 48hr compared to time zero, **P<0.001). Klotho (2 ng/mL) with/without FGF23 (2 ng/mL) did not upregulate OPN secretion. Three wells were sampled per time-point per treatment group, data are mean ± s.e.m. EtOH = ethanol control, KL = klotho, P50 = paricalcitol 50 nM.

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