Parathyroid Hormone-related Protein Research Articles

Detection of renal tubular transporter proteins in canine urinary extracellular vesicles using liquid chromatography-tandem mass spectrometry.

Urinary extracellular vesicles (UEVs) are membranous particles that carry renal tubular transporter proteins. Here, we evaluate whether selected renal tubular transporter proteins can be detected in UEVs isolated from small volume (1-5 mL) canine urine samples of healthy dogs and canine patients with elevated circulating parathyroid hormone (PTH)/PTH-related peptide (PTHrp) concentrations, hypercortisolism, and primary hypoadrenocorticism using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The total creatinine content of each urine sample was calculated from urine volume and creatinine concentration. UEVs were isolated by size exclusion chromatography prior to quantification by nanoparticle tracking analysis and proteomic analysis by LC-MS/MS. Group comparisons were made using non-parametric statistics. Aquaporin-2 (AQP2) and the renal sodium/phosphate co-transporter (NPT2A) were detected in UEVs isolated from small volume samples of almost all healthy dogs but were not detected in most dogs with elevated circulating PTH/PTH related peptide (PTHrp) concentrations, hypercortisolism and primary hypoadrenocorticism. Total creatinine content of the urine sample was strongly positively correlated with the number of UEVs (rs = .84, P < .001); thus, total creatinine was used as a surrogate marker of UEV number. In healthy dogs, AQP2 and NPT2A were both detected in samples containing at least 1.7 × 109 UEVs or 24 μmol creatinine, however in non-healthy dogs, AQP2 and NPT2A were not detected in most samples containing up to 6.3 × 109 UEVs or 32 μmol creatinine.

7880 The Role Of PTHrP In Glucose Metabolism And Insulin Secretion

Abstract Disclosure: I.M. Max-Harry: None. N. Kantake: None. C.S. Nunemaker: None. T.J. Rosol: None. Insulin and glucagon are the two most important hormones for blood glucose homeostasis. Identifying the key players in their regulation will contribute greatly towards combatting types 1 and 2 diabetes. Parathyroid Hormone-related protein (PTHrP), a protein expressed by pancreatic beta cells, has been implicated in various developmental and physiological roles, but particularly, PTHrP increases beta-cell proliferation and insulin secretion in pancreatic islets. Incubating mouse pancreatic islets and Min6 cells with 10nM exogenous PTHrP showed a significant increase in both insulin secretion (for islets, PTHrP: 865.4ng/ml ± 77.66, con: 498.5ng/ml ± 40.63, p=0.024, n=4) and glucagon secretion (PTHrP: 20.87pmol/l ± 3.776, con: 8.798pmol/l ± 1.616, p= 0.068, n=3) after 24hours. In order to further investigate the effects observed, min6 cells were transiently transfected to overexpress PTHrP, and a glucose-stimulated insulin secretion (GSIS) assay was carried out with 2mM and 20mM glucose. The results showed a reduction in basal insulin secretion at 2mM glucose and an increase in insulin secretion at 20mM glucose for the PTHrP transfected cells. PTHrP improved the stimulation index of Min6 cells (PTHrP: 2.279 ± 0.2056, con: 1.659 ± 0.1386, p= 0.03, n=6) and enhanced their sensitivity to glucose. Preliminary proliferation assays showed that cells transfected with PTHrP have increased proliferation and a protection from cell death when incubated in serum-free media. This study, in addition to our previously published research showing that a lack of intact PTHrP leads to islet dysfunction, revealed that PTHrP may be crucial to appropriate glucose sensitivity of beta cells. Further studies are being carried out to understand the mechanism behind these effects and the genes/proteins involved in the regulation of insulin secretion by PTHrP. Presentation: 6/1/2024

5117 Unusual Sarcoid Presentation as a Case of Hypercalcemia

Abstract Disclosure: M. Salim: None. A. Chauhan: None. S. Avula: None. A. Ahmed: None. K. Zekarias: None. Background: Hypercalcemia may arise from a variety of causes and can pose challenging diagnostic dilemma. Clinical Case: An 89-year-old male presented with worsening malaise related to persistent hypercalcemia. He reported productive cough, polyuria, polydipsia, generalized muscle weakness and weight loss. Physical exam was remarkable for upper and lower extremities weakness. Laboratory tests showed calcium 11.6 (8.8-10.2 mg/dL), phosphorus 2.8 mg/dL (2.5-4.5 mg/dL), albumin 4.2 g/dL (3.5-5.2 g/dL), parathyroid hormone (PTH) 10 pg/mL (15-65 pg/mL), parathyroid hormone-related protein (PTH-rp) 4.5 pmol/L (0.0-2.3 pmol/L), TSH 6.24 (0.30 - 4.20 uIU/mL), free T4 1.10 ng/dL (0.90-1.70 ng/dL), GFR 61 mL/min/1.73m2, 25 OH Vit D 31 ng/mL (20-75 ng/mL), vit D 1,25-dihydroxy 134 pg/mL (19.9-79.3 pg/mL), calcium urine/creatinine 0.54. Hypercalcemia was first noted 11 months before the visit with a level of 14.0 mg/dL (8.8-10.2 mg/dL), phosphorus 3.5 mg/dL, PTH 11, PTH-rp 1.4 pmol/L, 25 OH Vit D 62 ng/mL, Vit D 1,25-dihydroxy 90 pg/mL, angiotensin-1-converting enzyme 12 U/L (16-85 U/L), serum and urine electrophoresis showed no monoclonal gammopathy present, and tuberculosis interferon-gamma test negative. Autoimmune workup was unremarkable, except for weak positive anti-signal recognition particle (SRP) antibodies and anti-Mi-2 antibodies. CT chest/pelvis/abdomen demonstrated peripheral predominant reticular opacities suggestive of interstitial lung disease and no malignant findings. Treatment history included zoledronic acid 11 months before the visit. The patient was referred for a granulomatous disease workup.Upon one month follow-up, the hypercalcemia symptoms were worsening. Repeat calcium was 12.3 mg/dL and fungal serologies were negative. The patient was started on prednisone empirically. PET CT was ordered and revealed patchy inflamed musculature of extremities suspicious of dermatomyositis/polymyositis. Further labs showed unremarkable myomarker panel, u3-Hydroxy-3-Methylglutaryl Coenzyme A reductase (HMGCR) antibody, Anti-cN-1A antibody, ACE 53 U/L, creatine kinase 32 U/L (39-309 U/L), aldolase 5.4 U/L (1.2-7.6 U/L), soluble IL-2R 1,021 U/mL (137-838 U/mL), and lysozyme 3.89 ug/mL (&amp;lt;2.75 ug/mL). A muscle biopsy was done, which showed benign skeletal muscle with patchy myofiber atrophy, patchy interstitial fibrosis, and focal interstitial mild chronic inflammation with no evidence of sarcoid granulomas in the sample. One week lab follow-up post initiation of steroid showed calcium of 10.1 mg/dL and subsequently improvement in his overall labs. Working diagnosis of sarcoid myositis was made. Conclusion: This case report highlights the approach of diagnosing a rare case of hypercalcemia due to sarcoid myositis. In a highly clinical suspicious patient, PET CT scan might be a necessary modality for diagnosis. Presentation: 6/2/2024

6634 Dual-Mediated PTH And Osteolytic Hypercalcemia In Metastatic Endometrial Carcinoma

Abstract Disclosure: C.K. Persaud: None. D.J. Beckman: None. Background: Hypercalcemia of malignancy occurs in 20-30% of advanced stage cancer patients. Etiologies include parathyroid hormone-related peptides (PTHrP)-mediated, osteolytic metastases-mediated, overproduction of 1,25-dihydroxycholecalciferol, and rarely parathyroid hormone (PTH)-mediated. PTH-mediated hypercalcemia of malignancy is rare and occurs through ectopic PTH production or primary hyperparathyroidism. We report a rare case of both PTH-mediated and osteolytic-mediated hypercalcemia in the setting of malignancy. Clinical Case: A 57-year-old female with history of endometrial cancer and multiple solid organ metastasis, presented with two weeks of progressive poor oral intake, nausea, emesis, and right flank pain. On hospital admission, she had a serum calcium of &amp;gt;20.1 mg/dL (8.6-10.2), PTH of 203 pg/mL (15.0-65.0), and creatinine of 1.7 mg/dL (0.7-1.2, baseline 1.3). Non-contrasted CT chest/abdomen/pelvis noted increased size of known right adrenal metastasis, new left adrenal metastasis, periaortic lymphadenopathy, and scattered nodules in the subcutaneous tissues of the abdomen and chest, however bone metastasis were not noted. Patient’s hypercalcemia was treated with zoledronic acid, calcitonin, cinacalcet, and eventually denosumab due to difficulty controlling hypercalcemia. Additional labs obtained during evaluation included ionized calcium &amp;gt;2.5 nmol/L (1.12-1.32), PTHrP &amp;lt;2.0 pmol/L (&amp;lt;2.0), 1,25-dihydroxycholecalciferol &amp;lt;10 pg/mL (21-65). Patient’s right adrenal metastasis biopsy one month prior to admission was subsequently stained for PTH and was negative. Technetium-99m sestamibi scan was obtained for localization of parathyroid adenoma or carcinoma. The scan did not localize, however did show numerous new lytic lesions in the skull, spine, sternum, rib cage, and right humerus. Patient’s hospital course was complicated by progressively worsening renal function, anemia, and thrombocytopenia. Patient was discharged to a long-term acute rehab facility off hypercalcemia treatment with a corrected calcium of 10.0 mg/dL. Conclusion: PTH-mediated hypercalcemia is a rare cause of hypercalcemia of malignancy. PTH levels are typically low or low-normal in osteolytic mediated hypercalcemia. Generally, if PTH is elevated in hypercalcemia, an alternate etiology is not evaluated. This case highlights a rare dual-mediated hypercalcemia of malignancy. The patient’s PTH level elevation was discordant with her high degree of hypercalcemia and prolonged treatment course. She was subsequently found to have widespread bone metastases contributing to hypercalcemia. While multiple etiologies of hypercalcemia can be rare, it is important to evaluate for all contributing causes, especially with additional clinical conditions, such as malignancy. Presentation: 6/3/2024

8126 PTH-Independent, Calcitriol-Mediated Hypercalcemia In Renal Cell Carcinoma

Abstract Disclosure: G.K. Rai: None. A. De Rosairo: None. K. Madani: None. D. Sacoto: None. A.A. Franco-Akel, MD: None. R. Belokovskaya: None. Hypercalcemia of malignancy is a known complication of advanced cancer. It occurs through various mechanisms, one of which is via 1,25-dihydroxyvitamin D (calcitriol). Calcitriol-mediated hypercalcemia accounts for approximately 1% of cases of hypercalcemia in the setting of malignancy and is usually seen in lymphomatous solid tumors. We present a gentleman with clear cell renal cell carcinoma associated with hypercalcemia with normal levels of intact parathyroid hormone (PTH), PTH-related protein (PTHrP), and 25-hydroxyvitamin D but elevated calcitriol levels. A 48 year-old-male with no significant past medical history with complaints of fatigue, hematuria, and weight loss of 38 pounds in 3 months was following urology for an elective right-sided nephrectomy. MRI of the abdomen and pelvis found a 16.1 cm renal mass with necrotic areas, causing mass effects on the liver, pancreas, and IVC. Preoperative laboratory showed hypercalcemia of 13.7 mg/dL (corrected 13.9 mg/dL) with microcytic anemia (hemoglobin 8.8 g/dL, MCV 72 fl). Workup showed low iPTH (3 pg/dL), 25-hydroxyvitamin D (16.9 ng/dL), and phosphorus level (1.7mg/dL) with elevated calcitriol (94.3 pg/dL) and a normal PTHrP. CT chest showed multiple pulmonary nodules on bilateral lobes, with the largest nodule ∼ 4.5 mm. Hypercalcemia was managed with IV fluids, calcitonin 300 units Q12 for two days, Denosumab IV infusion, prednisone 60 mg for three days, and oral ergocalciferol 50,000 units weekly. Calcium levels normalized, after which the patient underwent nephrectomy. Pathology revealed renal cell carcinoma grade G4 with mixed features of clear cell carcinoma with sarcomatoid and rhabdoid features. We report an incidental finding of hypercalcemia in a patient with renal mass. The elevated calcitriol levels can be attributed to ectopic calcitriol production through the renal mass. Post nephrectomy calcium levels remained within normal range for consecutive weeks. With this, we concur that hypercalcemia most likely originated from the renal mass that had ectopic production of calcitriol. Dysregulated calcitriol production is a well-known complication of lymphomas and granulomatous disease but is seldom reported with solid tumors. Usually, the etiology for hypercalcemia in patients with renal cell carcinoma is the overproduction of PTHrP, which was not the case with our patient. The ultimate resolution of hypercalcemia of malignancy was attained by treating the underlying malignancy. Presentation: 6/1/2024

8126 PTH-independent, Calcitriol-mediated Hypercalcemia in Renal Cell Carcinoma

Abstract Disclosure: G.K. Rai: None. A. De Rosairo: None. K. Madani: None. D. Sacoto: None. A.A. Franco-Akel, MD: None. R. Belokovskaya: None. Hypercalcemia of malignancy is a known complication of advanced cancer. It occurs through various mechanisms, one of which is via 1,25-dihydroxyvitamin D (calcitriol). Calcitriol-mediated hypercalcemia accounts for approximately 1% of cases of hypercalcemia in the setting of malignancy and is usually seen in lymphomatous solid tumors. We present a gentleman with clear cell renal cell carcinoma associated with hypercalcemia with normal levels of intact parathyroid hormone (PTH), PTH-related protein (PTHrP), and 25-hydroxyvitamin D but elevated calcitriol levels. A 48 year-old-male with no significant past medical history with complaints of fatigue, hematuria, and weight loss of 38 pounds in 3 months was following urology for an elective right-sided nephrectomy. MRI of the abdomen and pelvis found a 16.1 cm renal mass with necrotic areas, causing mass effects on the liver, pancreas, and IVC. Preoperative laboratory showed hypercalcemia of 13.7 mg/dL (corrected 13.9 mg/dL) with microcytic anemia (hemoglobin 8.8 g/dL, MCV 72 fl). Workup showed low iPTH (3 pg/dL), 25-hydroxyvitamin D (16.9 ng/dL), and phosphorus level (1.7mg/dL) with elevated calcitriol (94.3 pg/dL) and a normal PTHrP. CT chest showed multiple pulmonary nodules on bilateral lobes, with the largest nodule ∼ 4.5 mm. Hypercalcemia was managed with IV fluids, calcitonin 300 units Q12 for two days, Denosumab IV infusion, prednisone 60 mg for three days, and oral ergocalciferol 50,000 units weekly. Calcium levels normalized, after which the patient underwent nephrectomy. Pathology revealed renal cell carcinoma grade G4 with mixed features of clear cell carcinoma with sarcomatoid and rhabdoid features. We report an incidental finding of hypercalcemia in a patient with renal mass. The elevated calcitriol levels can be attributed to ectopic calcitriol production through the renal mass. Post nephrectomy calcium levels remained within normal range for consecutive weeks. With this, we concur that hypercalcemia most likely originated from the renal mass that had ectopic production of calcitriol. Dysregulated calcitriol production is a well-known complication of lymphomas and granulomatous disease but is seldom reported with solid tumors. Usually, the etiology for hypercalcemia in patients with renal cell carcinoma is the overproduction of PTHrP, which was not the case with our patient. The ultimate resolution of hypercalcemia of malignancy was attained by treating the underlying malignancy. Presentation: 6/1/2024

7928 A "PTH-Mediated" Hypercalcemia Of Malignancy

Abstract Disclosure: N. Sekhon: None. R.J. Bingham: None. Background: We present a challenging diagnostic case of a 73-year-old female with PTH-mediated hypercalcemia in the presence of new metastatic uterine cancer. Case: A 73-year-old female with a past medical history of hypertension and abdominal aortic aneurysm was seen in the hospital for hypercalcemia. She endorsed constipation, fatigue, and generalized weakness for the past few weeks. This was the second hospitalization related to hypercalcemia over the period of 10 days. During the previous hospitalization, her admission calcium was 17 mg/dL and was treated with IV fluids, calcitonin, and zoledronic acid. There was a history of intentional weight loss of about 30-40 lbs in the last 3 months by reducing carbohydrate intake. The patient had a 40-pack years of smoking history. There was no family history of calcium disorders or cancers. Home medications included amlodipine 10 mg QD, docusate sodium 100 mg BID, senna 1 tab QHS, and Vit D 5000 U QD. On physical examination, she was hemodynamically stable but appeared confused and lethargic. The rest of the examination was unremarkable. Laboratory results were remarkable for hypercalcemia (Ca: 13.3 mg/dL), hypophosphatemia (Phosphorus: 2.3 mg/dL), and elevated PTH of 418.3 pg/mL (Ref. Range: 18.4-80.1 pg/mL). CT abdomen/ pelvis revealed innumerable ill-defined hypodense lesions throughout the liver concerning metastatic disease and a 5 cm mass in the uterine fundus. The patient was worked up with the view of hypercalcemia related to malignancy. However, PTHrP (PTH-related peptide) came back normal, which was checked twice during both hospitalizations. Liver biopsy showed metastatic high-grade carcinoma highly suspicious for GYN malignancy with negative PTH staining. Imaging studies for localization including CT soft tissue neck and CT 4D parathyroid with and without contrast failed to show any parathyroid lesions. Endometrial biopsy obtained during subsequent hospitalization showed high-grade adenocarcinoma with focal squamous differentiation. Immunohistochemical staining identified scattered positivity for PTH. The patient was managed as per hypercalcemia of malignancy guidelines with IV fluids and denosumab. Discussion: Hypercalcemia of malignancy due to ectopic PTH secretion has been reported in &amp;lt; 1% of cases. Only two endometrial cancer cases with ectopic PTH have been reported in the literature. PTH-mediated hypercalcemia is seen in primary hyperparathyroidism, parathyroid cancers, or ectopic PTH. In all cases of hypercalcemia of malignancy, primary hyperparathyroidism should be ruled out in the presence of elevated PTH. Diagnosing ectopic PTH secretion by tumor can be challenging due to constant changes in cells with metastasis. Conclusion: This is a rare case of endometrial cancer with liver metastasis and ectopic PTH secretion by primary tumor leading to recurrent hospitalizations related to hypercalcemia. Presentation: 6/2/2024

8165 A Case Of Rhabdomyolysis-Induced Hypocalcemia And Hypercalcemia

Abstract Disclosure: A. Desai: None. A.L. Izzi: None. B. Flores Chang: None. A. Sajan: None. Introduction: Rhabdomyolysis is a syndrome characterized by muscle breakdown and the release of intracellular muscle contents into circulation. It is associated with electrolyte abnormalities and acute renal failure that can lead to prolonged hospitalization. We report a case of persistent hypercalcemia from recovery of acute renal failure and severe rhabdomyolysis. Case Description: A 47-year-old man with past medical history of polysubstance use initially presented with opiate-induced acute encephalopathy and respiratory failure. He developed severe rhabdomyolysis during the hospital course leading to acute renal failure requiring hemodialysis with hypocalcemia. The initial labs showed corrected calcium 6.2 mg/dL [9.2-11.0], ionized calcium 3.48 mg/dL [4.65-5.28], creatinine kinase level was &amp;gt;72,600 U/L [38-174], BUN 110 mg/dL [8-23], creatinine 7.8 mg/dL [0.6-1.2], and GFR 7 ml/min/BSA. His renal function improved over the next several days in the hospital. The serum creatinine improved to 4 mg/dL, BUN 49 mg/dL and GFR 20 ml/min/BSA respectively after couple of intermittent dialysis sessions. Six days after the last hemodialysis session, patient developed severe hypercalcemia with corrected calcium values ranging from 13.5 to 14.7 mg/dL and ionized calcium 7.41 mg/dL. Labs showed PTH 10 pg/mL [15-65], 1,25-dihydroxyvitamin D &amp;lt;10.0 pg/dL [21-65] and 25-OH Vitamin D levels 15.3 ng/ mL [30-96], which is consistent non-PTH medicated hypercalcemia. The 24-hour urine calcium was 696 mg [0-300 mg/24 hrs, 2900 cc]. PTH-related peptide was not collected. He was treated with intermittent doses of calcitonin and a single dose of pamidronate when the calcium levels were &amp;gt;13 mg/dL. Hypercalcemia resolved in eleven days after initially noted to have hypercalcemia. The corrected calcium improved to 10.1 mg/dL on the day of discharge. Discussion:Severe rhabdomyolysis is associated with acute kidney injury. The initial hypocalcemic phase during rhabdomyolysis is due to entry of calcium into damaged myocytes and deposition of calcium salts in damaged muscle. This is seen in the oliguric phase of acute kidney injury. After the recovery of rhabdomyolysis, the polyuric phase persists during renal recovery. During this time, up to one third of patients may have rebound hypercalcemia due to massive muscle calcium release. As per case reports the hypercalcemia phase lasts from 8 days to 3.5 months. In our case, the patient initially developed hypocalcemia in the oliguric phase of acute kidney injury from rhabdomyolysis. He developed hypercalcemia in the polyuric phase of renal recovery. The hypercalcemia resolved with intravenous hydration, calcitonin and pamidronate. Rhabdomyolysis is a rare cause for both hypocalcemia and hypercalcemia. Our case highlights the biphasic calcium response associated with rhabdomyolysis in the setting of renal impairment and recovery. Presentation: 6/1/2024

7388 Rare Case of Mediastinal Parathyroid Carcinoma

Abstract Disclosure: Y. Esaki: None. J. Moalem: None. A. Israel: None. I. Shafiq: None. Introduction: Parathyroid carcinoma accounts for 1% of primary hyperparathyroidism with an estimated prevalence of 0.005% of all cancers. Ectopic locations of parathyroid tumors have been found in 6-16% of cases, most commonly in the thyroid, the thymus, or behind the esophagus. Parathyroid carcinoma is rarely found in the mediastinum and only 30 cases have been reported in the literature. Clinical Case: A 68-year-old male without significant past medical history was evaluated by his primary care physician for constipation, polyuria and 20 lb weight loss over the past year. He presented to the hospital after blood work revealed serum calcium of 13.5 mg/dl (8.6-10.2 mg/dl). Initial hospital workup demonstrated serum calcium 12.9 mg/dl, phosphorus 2.6 mg/dl (2.7-4.5 mg/dl), 25-OH vitamin D 21 ng/ml (30-60 ng/ml), 1,25-(OH)2 vitamin D 143.0 pg/ml (19.9-79.3 pg/ml), PTH-related protein &amp;lt; 2.0 pmol/L (0.0-2.3 pmol/L) and PTH 1,250.0 pg/ml (15.0-65.0 pg/ml). CT scan revealed a 3.5 cm x 2.6 cm heterogeneous right paratracheal soft tissue mass with areas of calcifications with mass effect on SVC. Thyroid ultrasound showed multinodular thyroid tissues with a left 1.1 cm heterogeneous solid nodule along the mid posterior aspect as well as a 0.5 cm hypoechoic lesion inferior to the left lobe. Sestamibi scan demonstrated a large area of increased uptake corresponding to the mass seen in the superior mediastinum on CT imaging. He underwent neck exploration and left hemithyroidectomy as well as median sternotomy with resection of the paratracheal mass. Intraoperative PTH decreased from the baseline of 1,177.0 pg/ml to 65.6pg/ml following the removal of the paratracheal mass. The final pathology of right anterior mediastinal mass was consistent with a 5 cm parathyroid carcinoma with angioinvasion, intermediate lymphatic invasion, invasion into adjacent structures, and Ki67 proliferative index focally &amp;gt; 5%. His post-operative course was complicated by hypocalcemia, which improved with treatment with calcitriol and calcium supplementation. He declined the genetic testing. His calcium and parathyroid hormone have remained in the normal range after 1 year. Conclusion: This case illustrates a rare case of parathyroid carcinoma found in the mediastinum. It is important to recognize that parathyroid carcinoma can present in ectopic locations including the mediastinum, as this can affect the preoperative investigation and management. Fine needle biopsy (FNA) prior to the initial operation is not recommended as it is difficult to differentiate the parathyroid carcinoma from benign disease based on FNA, and it may cause tumor rupture and seeding. Also, in the mediastinal parathyroid carcinoma, localization of the lesions including the size and invasiveness is critical in order to allow for radical surgery. Presentation: 6/3/2024

8041 Eneboparatide, A Potent Stimulator Of Urinary Calcium Reabsorption, Induces Prolonged Renal Cortex Retention And PTH1 Receptor Signaling

Abstract Disclosure: G. Ravel: Employee; Self; Amolyt Pharma. R. Datta: Consulting Fee; Self; Amolyt Pharma. S. Milano: Employee; Self; Amolyt Pharma. M. Ovize: Employee; Self; Amolyt Pharma. S. Allas: Employee; Self; Amolyt Pharma. M. Aouadi: None. M.D. Culler: Employee; Self; Amolyt Pharma. Natural parathyroid hormone (PTH) replacement therapy for chronic hypoparathyroidism (cHP) is not ideal due to both short circulating half-life and brief receptor signaling, making the effect too transient. Following agonist binding to the PTH 1 receptor (PTH1R), the magnitude and duration of the receptor signal, and consequently the biological response, appears to depend on the affinity of the ligand for a specific receptor conformation termed R0, which determines the duration of ligand binding. Eneboparatide (Enebo) is a hybrid analog of PTH and PTH-related peptide designed to strongly bind to the R0 conformation while having a short circulating half-life similar to that of PTH. When tested in cHP patients, Enebo eliminated the need for calcium (Ca) (88% patients) and Vitamin D (92% patients) supplementation, while maintaining serum Ca levels over a full 24 hours. Urinary calcium (uCa) was rapidly decreased and was normalized in 92% of patients who were hypercalciuric prior to treatment. To explore the mechanism of the potent effect of Enebo on uCa, a binding assay specific for the R0 conformation of the PTH1R confirmed the high affinity of Enebo versus natural PTH1-34, with IC50 = 1.5nM and 30.9nM, respectively. Postulating that the high-affinity binding of Enebo to R0 should result in longer tissue retention in vivo, we examined the tissue levels of PTH1-34 and Enebo in male, 200-300g rats using quantitative whole-body autoradiography (QWBA) following subcutaneous injection of 3H-labeled Enebo and PTH1-34. Tissue levels of the compounds were normalized based on the concentrations observed in cardiac blood. The highest level observed for both compounds was in the renal cortex, the major site of active PTH-induced Ca reabsorption, with peak concentrations observed 30 minutes post-injection. Six hours after injection, 96.3% of PTH1-34 had disappeared from the renal cortex, as compared with only 61.4% of Enebo, despite both compounds having similar circulating half-lives. With respect to activating the PTH1R, both compounds showed a similar increase in cAMP in human embryonic kidney (HEK293) cells transfected with the PTH1R and the Green Downward cADDis cAMP biosensor, with EC50 of 0.32 and 0.48nM for Enebo and PTH1-34, respectively. To mimic the effect of rapid elimination in vivo, the ligands were added to the cells, washed-off after 15 minutes, and cAMP production followed over the next 16 hours. After washout, the cAMP signal induced by PTH1-34 rapidly returned to baseline in less than 60 minutes, whereas the cAMP signal induced by Enebo gradually decreased and was still evident after 16 hours. Collectively, these data confirm high affinity of Enebo for the R0 conformation of the PTH1R, as well as prolonged receptor signaling and retention by the renal cortex, clearly differentiating Enebo from PTH1-34 and providing strong rationale for the efficacy of Enebo in reducing uCa in cHP patients. Presentation: 6/3/2024

6564 A Case of Severe Hypercalcemia in Carney's Complex

Abstract Disclosure: J. Chippior: None. M. Rayan: None. L. Wright: None. L.S. Kirschner: Grant Recipient; Self; Medpace, Sparrow, Corcept Therapeutics, Corcept, Spruce, CinCor, Crinetics, Adrenas. S.W. Ing: Grant Recipient; Self; Alexion Pharmaceuticals, Inc., Amgen Inc, Radius Health, Inc, Takeda, Ultragenyx, Amolyt, Bridge Bio. Carney complex is a rare syndrome with fewer than 750 reported cases, characterized by lentigniosis, myxomas, and the occurrence of both endocrine and non-endocrine tumors. An inactivating mutation PRKAR1A, which encodes for the type 1A regulatory subunit of Protein kinase A, is responsible for over 70% of familial cases and 37% of sporadic cases. A 31-year-old female with a history of Carney complex, Cushing syndrome due to primary pigmented nodular adrenocortical disease (PPNAD), facial lentigines, spinal Schwannomas, and recent ischemic stroke secondary to embolization of a cardiac atrial myxoma, was transferred to our medical center for hyperemesis and severe hypercalcemia. Albumin-corrected serum calcium measured 15.4 mg/dL (8.6-10.5 mg/dL), ionized calcium 7.8 mg/dL (4.6-5.3 mg/dL) associated with suppressed PTH, normal PTH-related protein, and elevated bone turnover markers, C-telopeptide 4,273 pg/ml (150-635) and Procollagen Type 1 N-terminal Propeptide 222 mcg/L (19-83). Evaluation for etiology of hypercalcemia revealed elevated 1,25 dihydroxy vitamin D at 107.0 pg/ml (20-79) and elevated interleukin-6 at 11 pg/ml (&amp;lt;6.4). IL-6 is expressed by cardiac myxomas. Serum angiotensin-converting enzyme level was 50 U/L (16-85) and infectious workup was negative for potential granulomatous causes of elevated calcitriol (including testing for tuberculosis, histoplasma, blastomyces, bartonella, and coccidioides). CT angiogram of the abdomen and pelvis (completed in preparation of atrial myxoma resection) revealed ground-glass opacities in bilateral posterior lung fields and multiple pulmonary nodules, with the largest measuring 8 mm. After treatment with aggressive intravenous fluid resuscitation, intramuscular calcitonin, and intravenous zoledronic acid, corrected calcium normalized to 9.4 mg/dL at discharge. Urinary cortisol was 6.2 mcg per 24-hr (3.5-45), for which she started hydrocortisone 20 mg daily to treat suspected adrenal insufficiency as excess cortisol secretion in PPNAD may be episodic. Thus remission of hypercortisolism may have unmasked an underlying hypercalcemia, and adrenal insufficiency itself may have contributed to this finding. She had excision of left atrial myxoma and has continued to exhibit normal calcium levels on steroid replacement therapy. Repeat chest CT to further characterize these pulmonary findings is planned. This case illustrates a unique constellation of endocrine features associated with severe hypercalcemia in Carney complex. Presentation: 6/1/2024

6564 A Case Of Severe Hypercalcemia In Carney's Complex

Abstract Disclosure: J. Chippior: None. M. Rayan: None. L. Wright: None. L.S. Kirschner: Grant Recipient; Self; Medpace, Sparrow, Corcept Therapeutics, Corcept, Spruce, CinCor, Crinetics, Adrenas. S.W. Ing: Grant Recipient; Self; Alexion Pharmaceuticals, Inc., Amgen Inc, Radius Health, Inc, Takeda, Ultragenyx, Amolyt, Bridge Bio. Carney complex is a rare syndrome with fewer than 750 reported cases, characterized by lentigniosis, myxomas, and the occurrence of both endocrine and non-endocrine tumors. An inactivating mutation PRKAR1A, which encodes for the type 1A regulatory subunit of Protein kinase A, is responsible for over 70% of familial cases and 37% of sporadic cases. A 31-year-old female with a history of Carney complex, Cushing syndrome due to primary pigmented nodular adrenocortical disease (PPNAD), facial lentigines, spinal Schwannomas, and recent ischemic stroke secondary to embolization of a cardiac atrial myxoma, was transferred to our medical center for hyperemesis and severe hypercalcemia. Albumin-corrected serum calcium measured 15.4 mg/dL (8.6-10.5 mg/dL), ionized calcium 7.8 mg/dL (4.6-5.3 mg/dL) associated with suppressed PTH, normal PTH-related protein, and elevated bone turnover markers, C-telopeptide 4,273 pg/ml (150-635) and Procollagen Type 1 N-terminal Propeptide 222 mcg/L (19-83). Evaluation for etiology of hypercalcemia revealed elevated 1,25 dihydroxy vitamin D at 107.0 pg/ml (20-79) and elevated interleukin-6 at 11 pg/ml (&amp;lt;6.4). IL-6 is expressed by cardiac myxomas. Serum angiotensin-converting enzyme level was 50 U/L (16-85) and infectious workup was negative for potential granulomatous causes of elevated calcitriol (including testing for tuberculosis, histoplasma, blastomyces, bartonella, and coccidioides). CT angiogram of the abdomen and pelvis (completed in preparation of atrial myxoma resection) revealed ground-glass opacities in bilateral posterior lung fields and multiple pulmonary nodules, with the largest measuring 8 mm. After treatment with aggressive intravenous fluid resuscitation, intramuscular calcitonin, and intravenous zoledronic acid, corrected calcium normalized to 9.4 mg/dL at discharge. Urinary cortisol was 6.2 mcg per 24-hr (3.5-45), for which she started hydrocortisone 20 mg daily to treat suspected adrenal insufficiency as excess cortisol secretion in PPNAD may be episodic. Thus remission of hypercortisolism may have unmasked an underlying hypercalcemia, and adrenal insufficiency itself may have contributed to this finding. She had excision of left atrial myxoma and has continued to exhibit normal calcium levels on steroid replacement therapy. Repeat chest CT to further characterize these pulmonary findings is planned. This case illustrates a unique constellation of endocrine features associated with severe hypercalcemia in Carney complex. Presentation: 6/1/2024

Personal Genetic-Hypertension Odyssey From Phenotypes to Genotypes and Targets.

Hypertension requires increased systemic vascular resistance. Thus far, Mendelian hypertension-related genes are related to salt retention, an indirect regulatory effect. With the identification of mutated, overactive, PDE3A (phosphodiesterase 3A), we have uncovered a more direct vasoconstrictive mechanism. The autosomal-dominant syndrome features another specific phenotype, brachydactyly type E. Hypertension and the bony phenotype invariably occur together. We distinguished between these phenotypes by examining individual pedigrees. We implicated the gene encoding the parathyroid hormone-related peptide in the brachydactyly. We identified the hypertensive mechanisms as involving regulatory-region, gain-of-function, exon 4 rare pathogenic variants, in the cAMP-cGMP-catabolizing enzyme, PDE3A. We generated rodent models that recapitulate all human phenotypes. Comparisons not only allowed pathogenic insights into the human condition but also provided intervention models. Moreover, we identified rare pathogenic variants in exon 13 encoding the enzymatic pocket. These patients had identical phenotypes, also corroborated in a rodent model, which produced the same human phenotypes. These data could allow the differentiation between a target organ and blood pressure phenotype. The research allows visualization of enzymatic processes at the intracellular nanodomain level. The scope of this project has elucidated genetic mechanisms important to cartilage development, possibly cancer metastases, and findings relevant to cardiovascular regulation via systemic vascular resistance. For our team, the project was an educational/scientific adventure over a professional lifetime.

Recurrent Hypercalcemia in a Patient with Hodgkin Lymphoma with Concurrent Elevated Parathyroid Hormone-Related Peptide (PTHrP) and Vitamin D

Recurrent Hypercalcemia in a Patient with Hodgkin Lymphoma with Concurrent Elevated Parathyroid Hormone-Related Peptide (PTHrP) and Vitamin D

Utility of N-terminal Parathyroid Hormone-Related Protein in Evaluating Hypercalcemia in Patients with CKD

Utility of N-terminal Parathyroid Hormone-Related Protein in Evaluating Hypercalcemia in Patients with CKD

Receptor activity-modifying protein modulation of parathyroid hormone-1 receptor function and signaling.

Receptor activity-modifying proteins (RAMPs) are known to modulate the pharmacology and function of several G-protein-coupled receptors (GPCRs), including the parathyroid hormone 1 receptor (PTH1R). However, the precise effects of different RAMPs on PTH1R signalling and trafficking remain poorly understood. This study investigated the impact of RAMP2 and RAMP3 on PTH1R function using a range of PTH and PTH-related protein (PTHrP)-derived ligands. We employed FRET imaging to assess PTH1R interactions with RAMPs. Cell surface expression of PTH1R was evaluated in the presence of RAMPs. PTH1R-mediated cAMP accumulation, β-arrestin recruitment, and calcium signalling were measured in response to various ligands. Antibody-capture scintillation proximity assays were used to examine G-protein activation patterns. PTH1R preferentially interacted with RAMP2 and, to a lesser extent, RAMP3, but not with RAMP1. RAMP3 co-expression reduced cell surface expression of PTH1R. RAMP2 significantly enhanced PTH1R-mediated signalling responses to PTH (1-34), PTHrP (1-34), PTH (1-84), and PTH (1-17) analogue ZP2307, while RAMP3 co-expression attenuated or abolished these responses. Full-length PTHrP analogues exhibited lower potency and efficacy than PTHrP (1-34) in activating PTH1R. RAMP2 increased the potency and/or efficacy of these analogues, whereas RAMP3 reduced these responses. RAMP2 differentially modulated G-protein activation by PTH1R in a ligand-dependent manner, with PTH (1-34) and PTHrP (1-34) inducing distinct patterns of G-protein subtype activation. These findings highlight the complex role of RAMPs in regulating PTH1R signalling and trafficking, revealing differential effects of RAMP2 and RAMP3 on receptor function. The data suggest that targeting the PTH1R/RAMP2 complex may be a promising strategy for developing novel bone anabolic therapies by leveraging biased agonism and functional selectivity. Further research using physiologically relevant models is needed to elucidate the therapeutic potential of this approach.

Regulation of Skeletal Development and Maintenance by Runx2 and Sp7.

Runx2 (runt related transcription factor 2) and Sp7 (Sp7 transcription factor 7) are crucial transcription factors for bone development. The cotranscription factor Cbfb (core binding factor beta), which enhances the DNA-binding capacity of Runx2 and stabilizes the Runx2 protein, is necessary for bone development. Runx2 is essential for chondrocyte maturation, and Sp7 is partly involved. Runx2 induces the commitment of multipotent mesenchymal cells to osteoblast lineage cells and enhances the proliferation of osteoprogenitors. Reciprocal regulation between Runx2 and the Hedgehog, fibroblast growth factor (Fgf), Wnt, and parathyroid hormone-like hormone (Pthlh) signaling pathways and Dlx5 (distal-less homeobox 5) plays an important role in these processes. The induction of Fgfr2 (Fgf receptor 2) and Fgfr3 expression by Runx2 is important for the proliferation of osteoblast lineage cells. Runx2 induces Sp7 expression, and Runx2+ osteoprogenitors become Runx2+Sp7+ preosteoblasts. Sp7 induces the differentiation of preosteoblasts into osteoblasts without enhancing their proliferation. In osteoblasts, Runx2 is required for bone formation by inducing the expression of major bone matrix protein genes, including Col1a1 (collagen type I alpha 1), Col1a2, Spp1 (secreted phosphoprotein 1), Ibsp (integrin binding sialoprotein), and Bglap (bone gamma carboxyglutamate protein)/Bglap2. Bglap/Bglap2 (osteocalcin) regulates the alignment of apatite crystals parallel to collagen fibrils but does not function as a hormone that regulates glucose metabolism, testosterone synthesis, and muscle mass. Sp7 is also involved in Co1a1 expression and regulates osteoblast/osteocyte process formation, which is necessary for the survival of osteocytes and the prevention of cortical porosity. SP7 mutations cause osteogenesis imperfecta in rare cases. Runx2 is an important pathogenic factor, while Runx1, Runx3, and Cbfb are protective factors in osteoarthritis development.

Role of hedgehog signaling in the pathogenesis and therapy of heterotopic ossification.

Heterotopic ossification (HO) is a pathological process that generates ectopic bone in soft tissues. Hedgehog signaling (Hh signaling) is a signaling pathway that plays an important role in embryonic development and involves three ligands: sonic hedgehog (Shh), Indian hedgehog (Ihh) and desert hedgehog (Dhh). Hh signaling also has an important role in skeletal development. This paper discusses the effects of Hh signaling on the process of HO formation and describes several signaling molecules that are involved in Hh-mediated processes: parathyroid Hormone-Related Protein (PTHrP) and Fkbp10 mediate the expression of Hh during chondrogenesic differentiation. Extracellular signal-regulated kinase (ERK), GNAs and Yes-Associated Protein (YAP) interact with Hh signaling to play a role in osteogenic differentiation. Runt-Related Transcription Factor 2 (Runx2), Mohawk gene (Mkx) and bone morphogenetic protein (BMP) mediate Hh signaling during both chondrogenic and osteogenic differentiation. This paper also discusses possible therapeutic options for HO, lists several Hh inhibitors and explores whether they could serve as emerging targets for the treatment of HO.

Eiken syndrome with parathyroid hormone resistance due to a novel parathyroid hormone receptor type 1 mutation: clinical features and functional analysis.

We report two patients of east African ancestry with the same novel homozygous variant in the parathyroid hormone receptor type 1 (PTH1R). Both patients shared skeletal features including brachydactyly, extensive metacarpal pseudoepiphyses, elongated cone-shaped epiphyses, ischiopubic hypoplasia, deficient sacral ossification, suggestive of Eiken syndrome. Strikingly, both patients exhibited clinically manifest parathyroid hormone (PTH) resistance with hypocalcaemia and elevated serum phosphate levels. These laboratory and clinical abnormalities initially suggested pseudohypoparathyroidism, which is typically associated with GNAS abnormalities. In both patients, however, a homozygous novel PTH1R variant was identified (c.710T > A; p.IIe237Asn, p.I237N) that is located in the second transmembrane helical domain. Previously, others have reported a patient with a nearby PTH1R mutation (D241E) who presented with similar clinical features, e.g. delayed bone mineralization as well as clinical PTH resistance. Functional analysis of the effects of both novel PTH1R variants (I237N- and D241E-PTH1R) in HEK293 reporter cells transfected with plasmid DNA encoding the wild-type or mutant PTH1Rs demonstrated increased basal cAMP signalling for both variants, with relative blunting of responses to both PTH and PTH-related peptide (PTHrP) ligands. The clinical presentation of PTH resistance and delayed bone mineralization combined with the functional properties of the mutant PTH1Rs suggest that this form of Eiken syndrome results from alterations in PTH1R-mediated signalling in response to both canonical ligands, PTH and PTHrP.

The G protein-coupled receptor ADGRG6 maintains mouse growth plate homeostasis through IHH signaling.

The cartilage growth plate is essential for maintaining skeletal growth; however, the mechanisms governing postnatal growth plate homeostasis are still poorly understood. Using approaches of molecular mouse genetics and spatial transcriptomics applied to formalin-fixed, paraffin-embedded tissues, we show that ADGRG6/GPR126, a cartilage-enriched adhesion G protein-coupled receptor (GPCR), is essential for maintaining slow-cycling resting zone cells, appropriate chondrocyte proliferation and differentiation, and growth plate homeostasis in mice. Constitutive ablation of Adgrg6 in osteochondral progenitor cells with Col2a1Cre leads to a shortened resting zone, formation of cell clusters within the proliferative zone, and an elongated hypertrophic growth plate, marked by limited expression of parathyroid hormone-related protein (PTHrP) but increased Indian Hedgehog (IHH) signaling throughout the growth plate. Attenuation of smoothened-dependent hedgehog signaling restored the Adgrg6 deficiency-induced expansion of hypertrophic chondrocytes, confirming that IHH signaling can promote chondrocyte hypertrophy in a PTHrP-independent manner. In contrast, postnatal ablation of Adgrg6 in mature chondrocytes with AcanCreERT2, induced after the formation of the resting zone, does not affect PTHrP expression but causes an overall reduction of growth plate thickness marked by increased cell death specifically in the resting zone cells and a general reduction of chondrocyte proliferation and differentiation. Spatial transcriptomics reveals that ADGRG6 is essential for maintaining chondrocyte homeostasis by regulating osteogenic and catabolic genes in all the zones of the postnatal growth plates, potentially through positive regulation of SOX9 expression. Our findings elucidate the essential role of a cartilage-enriched adhesion GPCR in regulating cell proliferation and hypertrophic differentiation by regulation of PTHrP/IHH signaling, maintenance of slow-cycle resting zone chondrocytes, and safeguarding chondrocyte homeostasis in postnatal mouse growth plates.