Introduction

Glucocorticoids induce clinically relevant recalcitrant hypocalcemia in people with hypoparathyroidism. However, this relationship has not been documented in dogs. Glucocorticoids reduce intestinal calcium absorption, renal calcium resorption, bone remodeling, and increases protein bound calcium through corticosteroid-induced alkalosis.^1–4 In this report, we describe a dog with occult and subsequently confirmed primary hypoparathyroidism (PH) who experienced clinically relevant refractory hypocalcemia while being treated with glucocorticoids for immune-mediated hemolytic anemia (IMHA) and immune-mediated thrombocytopenia (IMTP).

Case Reports

A 7 yr old neutered male Pomeranian weighing 4.6 kg (10.1 lb), was presented to Lauderdale Veterinary Specialists with a 24 hr history of pigmenturia, melena, ventral abdominal petechiation, lethargy, and anorexia (day of initial examination was designated as day 1). There was no known toxin ingestion, pharmaceutical administration, or recent vaccinations. Pertinent physical examination findings included a rectal temperature of 37.5°C; heart rate of 110 beats per minute; respiratory rate of 32 breaths per minute; blood pressure of 120 mmHg; body condition score 4/9; mental dullness; petechiation of the ventral abdomen, pinnae, and prepuce; and melena was noted during rectal examination.

Serum biochemical parameters outside of the reference interval were blood urea nitrogen (40.3 mg/dL; reference interval 6–36 mg/dL), total calcium (5.3 mg/dL; reference interval 8.5–12.0mg/dL), ionized calcium (0.68 mg/dL; reference interval 1.13–1.42 mg/dL), total phosphorus (8.3 mg/dL; reference interval 2.2–6.6 mg/dL), total bilirubin (1.8 mg/dL; reference interval 0.0–0.4 mg/dL), albumin (2.4 g/dL; reference interval 2.5–4.3 g/dL), and total protein (4.8 g/dL; reference interval 5.5–8.1 g/dL). Ionized magnesium was within the reference interval (0.70 mg/dL; reference interval 0.62–1.5 mg/dL). A urinalysis revealed a urine specific gravity of 1.035 and moderate bilirubinuria and hematuria. Clinically important findings on complete blood count were leukocytosis (24.9 × 10³/μL; reference interval 6.0–13.5 × 10³/μL), neutrophilia (14.94 × 10³/μL; reference interval 3.0–11.50 × 10³/μL), band neutrophils (1.74 × 10³/μL; reference interval 0–1.0 × 10³/μL), anemia (16.3%; reference interval 37–55%), absolute reticulocytosis (140,000/μL; reference interval >60,000/μL), increased numbers of circulating nucleated red blood cells (11/100 WBC; reference interval 0–1/100 WBC), spherocytosis, erythrocytic microagglutination, and marked thrombocytopenia (15 × 10³/μL; reference interval 150–550 × 10³/μL). Thoracic radiographs and abdominal ultrasonography were unremarkable. Serum antigen for Dirofilaria immitis and serum antibodies directed against Borrelia burgdorferi, Ehrlichia canis, Ehrlichia ewingii, Anaplasma phagocytophilum, and Anaplasma platys were not detected.

Based on the findings of a regenerative anemia, spherocytosis, and spontaneous erythrocytic agglutination with concurrent thrombocytopenia, the dog was presumed to have IMHA and IMTP (i.e., Evan’s syndrome). The severity of the anemia was worsened by blood loss through the gastrointestinal and urinary systems secondary to the IMTP. The cause of hypocalcemia was not obvious at this time, but was suspected to be a result of pancreatitis, hypovitaminosis D, acquired or relative hypoparathyroidism, or primary hypoparathyroidism.

Initial management consisted of prednisone (1.1 mg/kg, per os [PO] q 12 hr), sucralfate (108 mg/kg, PO q 2 hr), doxycycline (6.5 mg/kg, PO q 12 hr), famotidine (1.1 mg/kg, PO q 24 hr), aspirin (1.0 mg/kg, PO q 24 hr), and calcium gluconate 10% (3 mg/kg/hr elemental calcium, diluted 1:2 with 0.9% saline, IV, constant rate infusion) until ionized calcium normalized ∼8 hr later (1.28 mg/dL). The following day, the anemia (packed cell volume [PCV] 11%) and hypoproteinemia (4.0 g/dL) worsened. The dog was blood typed DEA 1 positive and was administered 10.8 mL/kg of DEA 1 positive packed red blood cells IV over 4 hr without incident. Two hours after transfusion, the PCV and total solids increased to 20% and 5.0 g/dL, respectively. Following the blood transfusion, mycophenolate mofetil^a (10 mg/kg, IV q 12 hr) was added to the treatment protocol.

In the evening following the blood transfusion (day 2 from initial presentation), the dog developed a dull mentation, generalized ataxia, intermittent focal facial tremors, and facial pruritis. The ionized calcium at that time was 0.51 mg/dL. Chelation of calcium with citrate from the transfused blood was the suspected cause of the progressive hypocalcemia. A bolus of calcium gluconate 10% (7.5 mg/kg elemental calcium IV once) was administered over 1 hr. A continuous electrocardiogram was used to monitor heart rate and rhythm during the administration of calcium gluconate. No abnormalities were noted. This was followed by a calcium gluconate 10% (3 mg/kg/hr, elemental calcium, diluted 1:2 with 0.9% saline, IV constant rate infusion) until ionized calcium normalized 6 hr later (1.31 mg/dL). The diagnosis of PH was made (day 5) based on ionized hypocalcemia (0.58 mg/dL), paired with an inappropriately low serum parathyroid hormone (PTH) concentration (0.5 pmol/L; reference interval 0.5–5.8). Following this diagnosis, calcitriol^b (22 ng/kg, PO q 12 hr) was added to the treatment regimen. Over the next 72 hr, the daily ionized calcium concentrations were 0.89, 0.76, and 0.77 mg/dL. However, when parenteral calcium was not provided, the dog swiftly displayed clinical signs of hypocalcemia with ionized calcium concentrations that ranged from 0.56 to 0.64 mg/dL.

Eleven days after initial presentation, the dog was discharged from the hospital with resolution of thrombocytopenia (176 × 10³/μL) and clinical signs associated with hypocalcemia (tremors, focal twitches, facial pruritis) although anemia (16%) and ionized hypocalcemia (0.78 mg/dL) persisted. Medical management at the time of discharge included famotidine (1.1 mg/kg, PO q 24 hr), aspirin (0.5 mg/kg, PO q 24 hr), prednisone (1.1 mg/kg, PO q 12 hr), mycophenolate mofetil (7.6 mg/kg, PO q 12 hr), doxycycline (6.5 mg/kg, PO q 12 hr × 28 days), calcium carbonate (81.1mg/kg, PO q 8 hr), calcitriol (33 ng/kg, PO q 12 hr), and calcium gluconate 10% (30.3 mg/kg, elemental calcium, diluted 1:2 with 0.9% saline, subcutaneously [SC], 8 hr). The dose of calcitriol was increased at discharge because of the lack of expected clinical improvement with the initial dose. Subcutaneous calcium gluconate administration was recommended as a short-term measure to prevent clinical signs of hypocalcemia while oral calcitriol took effect.

Fourteen days after initial presentation, the dog presented for re-evaluation, and serum ionized hypocalcemia persisted (0.62 mg/dL). During the subsequent 2 wk, attempts were made to taper the amount of SC calcium gluconate without success. Each attempt to reduce the volume of calcium gluconate was met with episodes of clinically relevant hypocalcemia. It was recommended that the dose of calcitriol be increased but the owner declined and instead ergocalciferol (5434 IU/kg, PO q 24 hr) was started in addition to calcitriol.

Twenty-four days after initial presentation, the dog was evaluated for lichenification, erythema, and black discoloration of the skin over the intrascapular region, as well as lichenification of the preputial, right tarsus, left inguinal, and left caudal thigh areas. Multiple areas of cutaneous calcification were noted radiographically. Serum ionized hypocalcemia (0.59 mg/dL) persisted but the hyperphosphatemia had resolved (5.3 mg/dL), and the total magnesium concentration remained unremarkable. The following day, the dog underwent placement of a vascular access port^c in the right jugular vein for administration of calcium gluconate and resection of the intrascapular region of necrosis with primary closure. Four days after placement of the vascular access port, the dog remained asymptomatic for hypocalcemia, and over the next 14 days, the volume of calcium gluconate given IV was titrated to the lowest effective dose. The intrascapular surgery site healed uneventfully.

The dog also remained persistently anemic (PCV 22%; total solids of 6.8 g/dL). Because there was no clinical evidence of hemorrhage noted and the anemia was strongly regenerative, ongoing immune mediated erythrocyte destruction was suspected. Thus, cyclosporine^d (10.8 mg/kg PO q 24 hr) was added and mycophenolate mofetil was tapered until its cessation on day 31. After a transient improvement in hematocrit following the addition of cyclosporine, the anemia returned (PCV: 17%, total solids of 7.0 g/dL). Again, there was no clinical evidence of hemorrhage and the anemia was regenerative, making uncontrolled immune-mediated destruction likely. It was recommended that the dog have serum concentrations of cyclosporine measured to evaluate if plasma cyclosporine concentrations were within the therapeutic range. However, the owner declined and in lieu of this recommendation, leflunomide^e (2.1 mg/kg, PO q 24 hr) was added to the immunosuppressive therapy.

The dog was presented 42 days from initial presentation for evaluation of a firm, gritty, ulcerated, exudative, necrotic cutaneous lesion that extended from the caudal prepuce into the left inguinal region. The hypocalcemia persisted in the dog with total calcium (7.3 mg/dL) and ionized calcium (0.9 mg/dL). The preputial and inguinal wounds were surgically debrided. Nine hours following the procedure, the dog suffered cardiopulmonary arrest.

On postmortem gross examination, the thoracic cavity was filled with an abundant white opaque fluid consistent with a chylous effusion. The left atrioventricular valve was mildly thickened by multiple coalescing white smoothly marginated nodules consistent with endocardiosis. Within the tail of the spleen was a 1 cm well-delineated, dark red triangular infarct. Fixed tissues including thyroid and adrenal glands, lung, heart, spleen, liver, kidney, pancreas, intestine, and haired skin were submitted to Bronson Animal Disease Diagnostic Laboratory for histopathologic examination.

Microscopic examination of the thyroid glands revealed lymphoplasmacytic inflammation and fibrosis adjacent to the thyroid gland along with a lack of parathyroid tissue in all sections examined (Figure 1). These changes were interpreted to be consistent with degeneration of the parathyroid glands as a result of an immune-mediated mechanism. Multiple sections of haired skin were examined with multifocal to coalescing effacement of the dermis and subcutis by various sizes of mineralization islands as seen with calcinosis cutis (CC). Multifocal, mild mineralization was noted within the parenchyma of multiple other organs including the thyroid gland, lung, heart, and kidney. The death of this dog could not be attributed to a single factor, and various disease pathways may have contributed such as decreased function of multiple organs from calcification, chronic hypocalcemia, chylous effusion, and valvular insufficiency caused by endocardiosis.

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Discussion

Naturally occurring primary hypoparathyroidism is a rare endocrine disorder in dogs that results in the inadequate synthesis and secretion of parathyroid hormone. Parathyroid hormone increases blood calcium and decreases phosphate ion blood concentrations by altering calcium and phosphorous handling in the kidney, bone, and small intestine.⁵ A deficiency in parathyroid hormone manifests as hypocalcemia and hyperphosphatemia.⁵ Histologic findings in most dogs with naturally occurring primary hypoparathyroidism consist of lymphocytic and/or plasmacytic inflammation or fibrous tissue within the parathyroid glands, similar to what was seen histologically in this dog.⁵

Maintenance therapy for dogs with hypoparathyroidism consists of oral administration of a vitamin D analogue (calcitriol, ergocalciferol, or alfacalcidol) and calcium supplementation.⁵ The dog in this report was treated with intravenous and subcutaneous calcium gluconate as well as oral calcitriol, ergocalciferol, and calcium carbonate.

There is limited published information on the treatment of hypoparathyroidism and a concurrent immune-mediated disease with prolonged immunosuppressive doses of glucocorticoids. Three dogs have been reported in the literature to have hypoparathyroidism and a concurrent immune mediated disease, one with IMHA and two with IMTP.^6,7 The dog with concurrent IMHA was euthanized 33 days following discharge due to the development of acute kidney failure.⁶ One of the dogs with concurrent IMTP survived to the last known follow-up. Details of immunosuppressive therapy for that dog were not provided. The other dog treated for concurrent IMTP experienced resolution of both its IMTP and hypoparathyroidism.⁷ A presumptive diagnosis of PH was made in the aforementioned dog, although histopathology was not obtained to confirm lymphocyte-mediated destruction of the parathyroid glands. One possible explanation for transient hypoparathyroidism in that dog is the development of anti-calcium sensing receptor (CaSR) antibodies. In humans with anti-CaSR–mediated hypoparathyroidism, anti-CaSR antibodies activate CaSR, leading to inhibition of PTH release from the parathyroid glands.⁸ This phenomenon results in a functional hypoparathyroidism not from irreversible parathyroid damage typically seen in PH.⁸ Long-term management in that dog with cyclosporine may have facilitated resolution of hypoparathyroidism by reducing production of anti-CaSR antibodies.

The treatment of a dog with hypoparathyroidism and a concurrent immune-mediated disease with immunosuppressive doses of glucocorticoids presents a clinical challenge because of the negative impact glucocorticoids has on calcium homeostasis. Glucocorticoids reduce intestinal calcium absorption, renal calcium resorption, bone remodeling, and increase calcium binding to protein through corticosteroid-induced alkalosis.^1–4 Healthy animals given glucocorticoids maintain normal serum calcium concentrations because the calcium-lowering effect of glucocorticoids is negated by an immediate release of PTH.³ In healthy humans, increased plasma PTH concentrations were identified 15 min after initiation of an intravenous infusion of cortisol.⁹

Clinically relevant hypocalcemia is a documented complication of glucocorticoid administration in people with hypoparathyroidism.^1–4,10,11 The long-term use of glucocorticoids in people with hypoparathyroidism is avoided as it has been shown to antagonize the effects of vitamin D metabolites.^12,13 In people with hypoparathyroidism clinical signs related to hypocalcemia have been reported to occur 10 hr to 2 days following initiation of glucocorticoid administration and persist despite appropriate calcium supplementation (oral and intravenous) and oral vitamin D administration.^2–4 The onset of clinically relevant, refractory hypocalcemia is believed to be related to glucocorticoid administration because discontinuation results in resolution of clinical signs and improved plasma calcium concentrations.^2–4,11

We believe the dog in this report had latent hypoparathyroidism that was unmasked following initiation of therapy for IMHA and IMTP with glucocorticoids. Similar to humans given glucocorticoids with hypoparathyroidism, this dog developed severe hypocalcemia related clinical signs within 2 days of starting glucocorticoids and a refractory hypocalcemia despite appropriate oral vitamin D analogs and calcium administration. Glucocorticoids were not tapered in the dog reported here because of persistent erythrocyte destruction.

Corticosteroids are considered the cornerstone of treatment of immune-mediated disease in dogs.¹⁴ However, in cases of PH and a concurrent immune-mediated disease, it may be prudent to use the lowest effective dose of glucorticoid in combination with an adjunctive immunosuppressive (i.e., cyclosporine, azathioprine, mycophenolate mofetil, leflunomide, etc). Furthermore, if glucocorticoid therapy is required in a patient with PH, clinically relevant hypocalcemia should be expected and higher than normal initial doses of oral vitamin D and calcium should be used.

The dog in this report represents a unique case of PH in that the dog was dependent on parenteral calcium gluconate to prevent clinical signs related to hypocalcemia despite being treated with high doses calcitriol and ergocalciferol. The complications associated with the development of CC following subcutaneous calcium gluconate administration in dogs has previously been investigated.¹⁵ In normal skin, calcium and phosphate ions are in equilibrium within extracellular fluid.¹⁶ The introduction of excess calcium within the subcutaneous space disrupts calcium/phosphate balance and promotes formation of crystal nuclei that are deposited on organic matrixes.¹⁷ Additionally, the dysregulation of mitochondrial calcium/phosphate ion concentrations and crystal deposition can induce cell death.¹⁷ The concurrent administration of an immunosuppressive dose of glucocorticoids may have enhanced the development of CC in this dog. The role of hypercortisolemia in the development of CC is believed to be through the formation of an organic matrix that attracts and binds calcium.¹⁸ There was no long-term intention to treat the dog with SC calcium gluconate to circumvent vitamin D supplementation. The use of SC calcium gluconate was intended as a short-term support measure until the dog responded to vitamin D therapy.

It is believed the cause for this dog’s refractory hypocalcemia is related to administration of glucocorticoids; however, another potential cause may be related to oral bioavailability. In humans, pharmacokinetic evaluation of commercial oral preparations of calcitriol show substantial limitations. The most important hindrance is that calcitriol area under the curve and Cmax do not increase proportionally with increasing calcitriol dose.¹⁹ Pharmacokinetics of commercially available oral calcitriol in dogs has not been investigated. However, the oral bioavailability of DN101, a concentrated formulation of calcitriol, showed a bioavailability that ranged from 17 to 126%.¹⁹ Proposed reasons for the observed variability included polymorphic breed differences CYP24A1 (the main calcitriol catabolizing enzyme), breed differences in serum concentrations of vitamin D-binding proteins, and/or vitamin D-binding protein polymorphisms resulting in variable binding affinities.¹⁹ In people, one way to circumvent oral bioavailability issues is the use of parenteral calcitriol.⁵ However, there are no pharmacokinetics, pharmacodynamics, or safety data available for parenteral calcitriol in the dog.

Conclusion

In conclusion, refractory hypocalcemia resistant to oral calcium, ergocalciferol, and calcitriol supplementation is a possible outcome when glucocorticoids are administered to dogs with PH. Therefore, the use of glucocorticoids in dogs with PH should be used with caution and careful evaluation of calcium homeostasis undertaken.