Diphtheria Toxin- and GFP-Based Mouse Models of Acquired Hypoparathyroidism and Treatment With a Long-Acting Parathyroid Hormone Analog

Abstract

Hypoparathyroidism (HP) arises most commonly from parathyroid (PT) gland damage associated with neck surgery, and is typically treated with oral calcium and active vitamin D. Such treatment effectively increases levels of serum calcium (sCa), but also brings risk of hypercalciuria and renal damage. There is thus considerable interest in using PTH or PTH analogs to treat HP. To facilitate study of this disease and the assessment of new treatment options, we developed two mouse models of acquired HP, and used them to assess efficacy of PTH(1-34) as well as a long-acting PTH analog (LA-PTH) in regulating blood calcium levels. In one model, we used PTHcre-iDTR mice in which the diphtheria toxin (DT) receptor (DTR) is selectively expressed in PT glands, such that systemic DT administration selectively ablates parathyroid cells. For the second model, we generated GFP-PT mice in which green fluorescent protein (GFP) is selectively expressed in PT cells, such that parathyroidectomy (PTX) is facilitated by green fluorescence of the PT glands. In the PTHcre-iDTR mice, DT injection (2 × 5 μg/kg, i.p.) resulted in moderate yet consistent reductions in serum PTH and sCa levels. The more severe hypoparathyroid phenotype was observed in GFP-PT mice following GFP-guided PTX surgery. In each model, a single subcutaneous injection of LA-PTH increased sCa levels more effectively and for a longer duration (>24 hours) than did a 10-fold higher dose of PTH(1-34), without causing excessive urinary calcium excretion. These new mouse models thus faithfully replicate two degrees of acquired HP, moderate and severe, and may be useful for assessing potential new modes of therapy. © 2015 American Society for Bone and Mineral Research.

Keywords: ANIMAL MODELS; CELL/TISSUE SIGNALING-ENDOCRINE PATHWAYS; DISORDERS OF CALCIUM/PHOSPHATE METABOLISM; THERAPEUTICS.

Figure 1

Dose optimization of DT injection in PTHcre-iDTR mice. (A-B) Blood Ca⁺⁺ and serum PTH after a single i.p. injection of DT at 5 μg/kg (N=8-12, *p<0.05, **p<0.01, ***p<0.001). (C-D) Blood Ca⁺⁺ and serum PTH after one to five (as indicated) repetitive doses of DT, each one three days apart, at 5 μg/kg (N=6-12, *p<0.05, **p<0.01, ***p<0.001). (E) Correlation between Ca⁺⁺ and PTH in hypoparathyroid vs. parathyroid-intact mice. Blood for Ca⁺⁺ and PTH measurements was collected from PTHcre-iDTR mice 3 days after the last DT injection (2 i.p. injection 3 days apart at 5 μg/kg) on normal chow. The sigmoidal iCa-PTH correlation was obtained from the values in parathyroid-intact, non-DT injected PTHcre-iDTR mice fed a normal or low calcium diet for 2 weeks (N=7-8).

Figure 2

Characterization of PTHcre-iDTR mice after DT injection. (A-N) Representative parathyroid gland with surrounding thyroid gland. (A-C): Before DT injection. Intact parathyroid gland with robust PTH staining and no TUNEL positive cells in the parathyroid region. (D-F): 3 days after DT injection (2 i.p. injections at 5 μg/kg 3 days apart). In the parathyroid glands, increased vascularity is observed (D), dramatic decrease in PTH immunoreactivity (E), and abundant apoptotic TUNEL positive cells (F). (G-N): 1 month after vehicle or DT injection (2 i.p. injections at 5 μg/kg 3 days apart). In vehicle control, parathyroid glands (G) shows strong GFP (H) and PTH (I) staining (using Alexa Fluor 488 for GFP and 633 Far-red secondary antibody for PTH). In DT injected mice, the normal structure of the parathyroid glands is lost (K), and most GFP and PTH immunoreactivity no longer detectable. However, a few GFP and PTH double positive cells (L-N) remained. (O) Serum Pi levels in PTHcre-iDTR mice three days after DT or vehicle injection (N=12, ***p<0.001). (P) Urinary calcium excretion in PTHcre-iDTR mice three days after DT or vehicle injection (N=12). (Q) Serum PTH and blood ionized calcium levels in PTHcre-iDTR mice after DT vs. vehicle injection over 3 months. (N=6-8, **p<0.01, ***p<0.001).

Figure 3

Hypoparathyroid phenotype of GFP-PTX mice. (A-F) Photographs of the neck region of PTH-Cre;ROSA^mT/mG mice before and after parathyroidectomy. (A) Under halogen light, mouse parathyroid glands cannot easily be distinguished from the surrounding tissue. (B, C) Under UV illumination, GFP-positive parathyroid glands are easily identified. (D-F) Using fine scissors and forceps, green parathyroid glands were selectively removed. (F) Location of thyroid and parathyroids are outlined in red and white, respectively. (G) Survival rate of GFP-PTX mice compared to animals that underwent sham surgery (N=20). (H-K) Blood Ca⁺⁺, serum PTH, serum Pi, and urinary calcium excretion (uCa/uCr) in GFP-PTX mice 3 days after surgery (N=10-12, ***p<0.001). (L) Serum PTH and blood ionized calcium levels in GFP-PTX vs. sham-operated mice over 3 months (N=6-8, ***p<0.001).

Figure 4

LA-PTH injection in two mouse models of acquired hypoparathyroidism. Blood Ca⁺⁺ levels, blood Pi levels and urinary calcium excretion in GFP-PTX (A, C, E) and DT-treated PTHcre-iDTR mice (B, D, F) after a single s.c. injection of different doses of LA-PTH, as indicated, or PTH(1-34). (N=5-7, * p<0.05, **p<0.01, ***p<0.001). Color-shaded curves represent the reference range (mean ± SD) of control animals.

Figure 5

LA-PTH injection in GFP-PTX mice. Urinary cAMP excretion and serum 1,25(OH)₂D₃ levels at baseline (A, C) and after a single s.c. injection of different LA-PTH doses as indicated, and PTH(1-34) (B, D). (N=5-8, * p<0.05, **p<0.01, ***p<0.001).