Correction of Vascular Calcification and Hyperphosphatemia in CKD Rats Treated with ASARM Peptide

Abstract

Background: Abnormalities in calcium, phosphorus, PTH, vitamin D metabolism, bone, and vascular calcification occur in chronic kidney disease mineral bone disorder (CKD-MBD). Calciphylaxis, involving painful, ulcerative skin lesions, is also a major problem associated with CKD-MBD. There are no quality medical interventions to address these clinical issues. Bone ASARM peptides are strong inhibitors of mineralization and induce hypophosphatemia by inhibiting phosphate uptake from the gut. We hypothesize treatment of CKD-MBD rats with ASARM peptides will reverse hyperphosphatemia, reduce soft-tissue calcification, and prevent calciphylaxis.

Methods: To test our hypothesis, we assessed the effects of synthetic ASARM peptide in rats that had undergone a subtotal 5/6th nephrectomy (56NEPHREX), a rodent model of CKD-MBD. All rats were fed a high phosphate diet (2% Pi) to worsen mineral metabolism defects. Changes in serum potassium, phosphate, BUN, creatinine, PTH, FGF23, and calcium were assessed in response to 28 days of ASARM peptide infusion. Also, changes in bone quality, soft-tissue calcification, and expression of gut Npt2b (Slc34a2) were studied following ASARM peptide treatment.

Results: Rats that had undergone 56NEPHREX treated with ASARM peptide showed major improvements in hyperphosphatemia, blood urea nitrogen (BUN), and bone quality compared with vehicle controls. Also, ASARM-infused 56NEPHREX rats displayed improved renal, brain, and cardiovascular calcification. Notably, ASARM peptide infusion prevented the genesis of subdermal medial blood vessel calcification and calciphylaxis-like lesions in 56NEPHREX rats compared with vehicle controls.

Conclusions: ASARM peptide infusion corrects hyperphosphatemia and improves vascular calcification, renal calcification, brain calcification, bone quality, renal function, and skin mineralization abnormalities in 56NEPHREX rats. These findings confirm our hypothesis and support the utility of ASARM peptide treatment in patients with CKD-MBD.

Keywords: ASARM; CKD-MBD; DMP1; FGF23; MEPE; basic science; calciphylaxis; calciprotein particles; chronic kidney disease; matrix vesicles; osteopontin; vascular calcification.

Graphical abstract

Figure 1.

Experimental design. (A) Protocol and rat numbers for osmotic pump infusion of ASARM peptide. (B) Timeline of experiment. Rats were first maintained on a control diet (NPO4; 0.33% P, 2000 IU vitamin D, and 0.8% Ca; #170497; Teklad). Baseline sera was collected 2 days before switching to a high phosphate diet, and osmotic pumps were implanted 3 days after switching to a high phosphate diet (HPO4; 2% P, 2000 IU vitamin D, and 0.8% Ca; #170496; Teklad). The subcutaneous implantation of Alzet pumps was carried out 2 weeks post surgery. Pump infusion with ASARM peptide or vehicle was then carried out for 28 days, and rats were maintained on the high phosphate diet. The timing and number of serum and urine collections are highlighted with arrows and text. Male rats were used for the experiment (weight 285 g; aged 60–63 days at start of sham and 56NEPHREX surgeries). Note: two ASARM peptide 56NEPHREX rats did not recover from anesthesia following ASARM pump implantation. 56NEPHREX, subtotal 5/6th nephrectomy.

Figure 2.

ASARM infusion corrects key serum chemistries in subtotal 56NX rats fed a high phosphate diet (HPO4; 2% P, 2000 IU vitamin D, and 0.8% Ca; #170496; Teklad). Measurements were taken at the start and end of the experiment (28 days infusion). See Figure 1 for experimental design and rat numbers per group (n). For the changes between groups over time, the data were analyzed using two-way ANOVA with Tukey’s multiple comparison test (α=0.05)—see narrative below. For statistical analyses and comparisons at the start and end of the experiment, a one-way ANOVA and Tukey’s multi comparison test were carried out; ns, not significant; *P<0.01, significant. (A) Serum PO4: Error bars represent SD. For 56NX VEH versus 56NX ASARM, ASARM treatment has a significant effect at all values of time, with the interaction accounting for approximately 30% of the total variance (F=34.09, DFn=1, and Dfd=43; P<0.001). For SHAM VEH versus 56NX ASARM, time has no significant effect, with the interaction accounting for approximately <0.1% of the total variance (F=0.03, DFn=1, and Dfd=30; P=0.87). For SHAM VEH versus 56NX VEH, time has a significant effect with the interaction, accounting for approximately 13% of the total variance (F=24.03, DFn=1, and Dfd=31; P<0.001). (B) Serum Ca²⁺: Error bars represent SD. Interactions show significant serum Ca²⁺ changes over time for all groups (P<0.001). (C) Serum BUN: Error bars represent SEM. For 56NX VEH versus 56NX ASARM, ASARM treatment has a significant effect at all values of time, with the interaction accounting for approximately 23% of the total variance (F=32.32, DFn=1, and Dfd=42; P<0.001). For SHAM VEH versus 56NX ASARM, treatment time has no significant effect, with the interaction accounting for approximately 2% of the total variance (F=1.22, DFn=1, and Dfd=32; P=0.28). For SHAM VEH versus 56NX VEH, treatment time has a significant effect with the interaction, accounting for approximately 25% of the total variance (F=41.14, DFn=1, and Dfd=32; P<0.001). (D) Serum creatinine: Error bars represent SD. Interactions show significant serum creatinine changes over time for all groups (P<0.001). (E) Serum K⁺: Error bars represent SEM. Interactions show significant serum K⁺ changes over time for all groups (P<0.001). (F) Serum Na⁺: Error bars represent SEM. (G) Serum HCO₃: Error bars represent SD. (H) Serum albumin: Error bars represent SEM. SHAM VEH, sham-operated vehicle; 56NX VEH, subtotal 5/6th nephrectomy vehicle; 56NX ASARM, subtotal 5/6th nephrectomy ASARM peptide.

Figure 3.

ASARM infusion corrects key serum chemistries in subtotal 56NX rats fed a high phosphate diet (HPO4; 2% P, 2000 IU vitamin D, and 0.8% Ca; #170496; Teklad). Measurements were taken at the start and end of the experiment (28 days infusion). See Figure 1 for experimental design and rat numbers per group (n). For the changes between groups over time, the data were analyzed using two-way ANOVA with Tukey’s multiple comparison test (α=0.05)—see narrative below. For statistical analyses and comparisons at the start and end of the experiment, a one-way ANOVA and Tukey’s multiple comparison test were carried out; ns, not significant; *P<0.01, significant. (A) Serum protein: Error bars represent SEM. For SHAM VEH versus 56NX ASARM, treatment time has no significant effect, with the interaction accounting for approximately 2% of the total variance (F=0.79, DFn=1, and Dfd=25; P=0.38). For SHAM VEH versus 56NX VEH, treatment time has no significant effect, with the interaction accounting for approximately 7% of the total variance (F=2.18, DFn=1, and Dfd=24; P=0.15). For 56NX VEH versus 56NX ASARM, ASARM treatment time has a significant effect, with the interaction accounting for approximately 12% of the total variance (F=4.19, DFn=1, and Dfd=29; P=0.05). (B) Serum PTH: Error bars represent SEM. Treatment time (vehicle or ASARM) has a significant effect for all groups, with the interaction accounting for approximately 55% of the total variance (F=57.12, DFn=1, and Dfd=45; P<0.001. However, no significant treatment effects occurred between the three groups. (C) SERUM ALT: Error bars represent SD. No significant treatment or temporal effects occurred between the three groups. (D) Serum glucose: Error bars represent SD. For SHAM VEH versus 56NX VEH, there is a significant effect at all values of time, with the interaction accounting for approximately 21% of the total variance (F=20.69, DFn=1, and Dfd=23; P<0.001). For SHAM VEH versus 56NX ASARM, there is no significant effect at all values of time, with the interaction accounting for <0.1% of the total variance (F=0.01, DFn=1, and Dfd=22; P=0.91). For 56NX VEH versus 56NX ASARM, ASARM treatment has a significant effect at all values of time, with the interaction accounting for approximately 19% of the total variance (F=30.91, DFn=1, and Dfd= 25; P<0.001). (E) Serum FGF23: Error bars represent SEM. For SHAM VEH versus 56NX ASARM, there is a significant effect at all values of time, with the interaction accounting for approximately 14% of the total variance (F=12.81, DFn=1, and Dfd=28; P=0.001). For SHAM VEH versus 56NX VEH, there is no significant effect at all values of time, with the interaction accounting for approximately 9% of the total variance (F=4.21, DFn=1, and Dfd=27; P=0.05). For 56NX VEH versus 56NX ASARM, ASARM treatment no significant effect at all values of time, with the interaction accounting for 0.57% of the total variance (F=0.36, DFn=1, and Dfd=35; P=0.55). (F) Serum ALK PHOSPHATASE: Error bars represent SEM. For SHAM VEH versus 56NX VEH, there is a significant effect at all values of time, with the interaction accounting for approximately 38% of the total variance (F=30.37, DFn=1, and Dfd=23; P<0.001). For SHAM VEH versus 56NX ASARM, there is a significant effect at all values of time, with the interaction accounting for approximately 33% of the total variance (F=43.49, DFn=1, and Dfd=30; P<0.001). For 56NX VEH versus 56NX ASARM, ASARM treatment has a significant effect at all values of time, with the interaction accounting for approximately 31% of the total variance (F=34.6, DFn=1, and Dfd=33; P<0.001). (G) Serum uric acid: Error bars represent SEM. Interactions show no significant serum Ca²⁺ changes at all values of time for all groups. (H) Serum cholesterol: Error bars represent SEM. For SHAM VEH versus 56NX VEH, there is a significant effect at all values of time, with the interaction accounting for approximately 46% of the total variance (F=32.2, DFn=1, and Dfd=23; P<0.001). For SHAM VEH versus 56NX ASARM, there is a significant effect at all values of time, with the interaction accounting for approximately 27% of the total variance (F=12.96, DFn=1, and Dfd=27; P=0.001). For 56NX VEH versus 56NX ASARM, ASARM treatment has a significant effect at all values of time, with the interaction accounting for approximately 11% of the total variance (F=6.87, DFn=1, and Dfd=30; P=0.01). ALK-PHOSPHATASE, serum alkaline phosphatase; SERUM ALT, alanine aminotransferase. Note, results for serum aspartate aminotransferase (AST) were similar to serum alanine aminotransferase (ALT) results (C), with no significant difference between groups (data not shown for ALT).

Figure 4.

μCT scans of rat hearts, showing significantly reduced heart calcification in ASARM infused subtotal 56NEPHREX rats fed a high phosphate diet (HPO4; 2% P, 2000 IU vitamin D, and 0.8% Ca; #170496; Teklad). (A) Representative heart μCT scans. (B) Graphical output of heart mineral volume over total tissue volume (MV/TV). No measurable calcification occurred in sham vehicle rats (data not shown). SHAMX, sham operated. One-way ANOVA with Tukey multiple comparison; *P=0.003; ns, not significant—as shown on the graph. Note with an unpaired t test (two-tailed), the SHAM group and 56NEPHREX ASARM group were significantly different (P=0.03). Also, the 56NEPHREX vehicle versus 56NEPHREX ASARM group remained significantly different with an unpaired t test (two-tailed; P=0.004). μCT, microcomputed tomography.

Figure 5.

ASARM infusion prevents medial calcification of the aorta. (A) Representative aorta photos and corresponding μCT scans of vehicle and ASARM-treated subtotal 56NEPHREX rats. (B) Aorta histology sections (7 μM) stained with Von Kossa (black color, mineral). Note extensive medial calcification is prevented in rats infused with ASARM peptide. (C) Graph showing aorta MV/TV calculated using μCT analysis is reduced in rats infused with ASARM. No measurable calcification occurred in SHAM vehicle rats (data not shown). Unpaired t test (two-tailed). The 56NEPHREX vehicle versus 56NEPHREX ASARM were significantly different P=0.02. No detectable mineral deposition occurred with the SHAM-treated rats (not shown). Rats were fed the high phosphate diet (HPO4; 2% P, 2000 IU vitamin D, and 0.8% Ca; #170496; Teklad).

Figure 6.

ASARM peptide infusion decreases renal mineralization in 56NEPHREX rats fed a high phosphate diet ((HPO4; 2% P, 2000 IU Vitamin D and 0.8% Ca; Teklad #170496)). (A) Representative μCT scans of SHAM-operated and 56NEPHREX rats. (B) Graphical presentation of μCT quantification of renal total MV/TV. (C) Renal histology section stained with Von Kossa showing mineral deposition (black color) in 7-μM section of 56NEPHREX rat vehicle-treated kidney (×20). Note significant decreased calcification in ASARM-treated 56NEPHREX rats. Results for the one-way ANOVA with Tukey multiple comparison test were: 56NEPHREX vehicle versus 56NEPHREX ASARM P<0.001 and 56NEPHREX vehicle versus SHAM P<0.001. Statistics shown on the graph represent unpaired t test (two tailed). No significant difference occurred between 56NEPHREX ASARM versus SHAM groups for both tests.

Figure 7.

Skin subdermal VC is significantly reduced in 56NEPHREX rats infused with ASARM—calciphylaxis-like lesions are absent. (A) μCT skin scan showing subdermal calcified blood vessels. (B) Corresponding photograph showing “under skin” surface and blood vessels scanned in (A). (C) Photo showing upper skin surface with “peau d’orange” lesions that resemble calciphylaxis lesions. (D) Cross-sectional μCT x-ray “dicom” image showing evidence of medial VCs. (E and F) Corresponding histology—skin cross-section (7 μm) stained for mineral with Von Kossa (black color, mineral) ×20. (G) Graphical presentation of μCT quantification of skin total MV/TV. Results for the one-way ANOVA with Tukey multiple comparison test were: 56NEPHREX vehicle versus 56NEPHREX ASARM P=0.02 and SHAM versus 56NEPHREX vehicle P=0.04. Unpaired t test (two tailed) for the 56NEPHREX vehicle versus 56NEPHREX ASARM were also significantly different (P=0.02). No significant difference between SHAM and 56NEPHREX ASARM groups were measured for both tests. Rats were fed the high phosphate diet (HPO4; 2% P, 2000 IU vitamin D, and 0.8% Ca; #170496; Teklad). VC, vascular calcification.

Figure 8.

Role of ASARM peptides in CKD. CKD causes progressive hyperphosphatemia, predisposing to nanocrystal or calciprotein particle (CPP) formation. CPPs and high Pi interact with vascular smooth muscle cells (VSMC), inducing proliferation and transition to an osteoprogenitor phenotype. Osteogenic transition continues with the genesis of matrix vesicles and microcrystalline nidi of hydroxyapatite. VC proceeds through intimal and medial mineral deposition and growth. ASARM peptides inhibit renal and intestinal Pi uptake, reducing serum Pi (A), bind to CPPs (B), inhibit osteogenesis and VSMC phenotypic transition (C), and binding to and preventing the growth of hydroxyapatite and mineral (D). In CKD, defective expression of SIBLING proteins, altered proteolytic processing to ASARM peptides, altered phosphorylation and/or altered degradation (colored arrow) results in reduced levels of active ASARM peptides, thus promoting VC. For example, ASARM peptide is the only known physiologic substrate for PHEX (32,38,40,41,47,48,76,77) and PHEX is highly expressed in CKD (75). The reduced levels of ASARM peptides in CKD predisposes to VC because of the reduced inhibition of VC pathways.