Elevated Phosphate Research Articles

ODP542 Challenging Management of Fasting Hypoglycemia in a Patient With Pancreatic Neuroendocrine Tumor and Lupus Nephritis

Abstract Background Derangements of calcium metabolism are common in patients with cancer.1 Unlike hypercalcemia which is well-recognized, little is known about hypocalcemia of malignancy.2 In fact, there were only 2 studies published to date that investigated its incidence among cancer patients. 2,3 Clinical Case: A 40-year-old male consulted an ENT specialist for a 2×2 cm left parotid mass of 3 years duration. There was no fever, cough, night sweats or significant weight loss. Histological analysis of the mass revealed parotid gland adenocarcinoma. Tumor metastases to lung and spine were seen on PET CT scan. Consequently, he received 18 cycles of chemotherapy and 10 sessions of radiotherapy without surgery because of the advanced stage. He initially exhibited good response with the treatment. A year after, he developed shortness of breath, numbness of hands with seizure episode. Chest x-ray showed pleural effusion and osteoblastic changes while cranial CT scan revealed brain metastasis. Bone scintigraphy was evident for multiple osseous metastases. He underwent stereotactic brain surgery, and thoracentesis with pleural fluid analysis consistent with malignancy. Biochemical tests were remarkable for hypocalcemia (0.99, n>1.12 mmol/L, normomagnesemia (0.89, n>0.74 mmol/L), low vitamin D (11.6, n>30 ng/mL), elevated phosphate (1.73; n: <1.49 mg/dL), and elevated intact PTH which eventually dropped (157.7 to 9, n>15 pg/mL). Ultrasound of the neck revealed diffuse thyroid calcifications. Despite oral calcium supplementation (14.4 grams elemental calcium), calcium infusion (1.35 mg/kg/hour elemental calcium) and vitamin D supplementation (6000 IU cholecalciferol and 0.5 mg calcitriol daily), he remained hypocalcemic. His condition eventually worsened, and he succumbed to death in due course despite aggressive measures provided. Conclusion To our knowledge, this is the first case illustrating the therapeutic challenge in addressing an intractable hypocalcemia in the setting of an advanced parotid carcinoma in our country.Reference: (1)Carl Blomqvist. A Hospital Survey of Hypocalcemia in Patients with Malignant Disease. Acta Med Scand. 1986;220. (2) Schattner et al. Hypocalcaemia of Malignancy. The Netherlands Journal of Medicine. July 2016, Vol. 74, No. 6. (3) Goncalves. Hypocalcemia in cancer patients: An exploratory Study. Porto Biomed. J. (2019) 4: 4 Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

ODP110 Parathyroid Hormone Independent Hypercalcaemia Secondary to Granulomatous Inflammation: Could This Be Melioidosis?

Abstract Background Hypercalcemia has been described in patients with most granulomatous disorders, most commonly in sarcoidosis and tuberculosis, but never in melioidosis. Clinical Case A 45-year-old woman presented with two-day history of abdominal pain, nausea, lethargy and intermittent fevers during Darwin's wet season. Medications included daily methadone, diazepam and mirtazapine. On examination she was febrile with stable hemodynamics and left upper quadrant tenderness. Investigations revealed pancytopenia, deranged liver function tests with a cholestatic pattern, raised CRP of 53 (<5mg/L) and normal serum calcium. Blood cultures were negative but melioidosis serology was positive (indirect hemagglutination antibody (IHA) titer 1: 1280). Abdominal computed tomography demonstrated hepatosplenomegaly with peripheral splenic infarcts and a 7mm intrasplenic aneurysm, but no abscesses. PET showed no hypermetabolic lesions. After extensive investigation, she was treated for presumptive culture-negative melioidosis with intravenous meropenem. Clinically she improved despite gradual elevation in serum calcium which peaked at 3.31 (2.1-2.6mmol/L) five weeks after presentation. Initial biochemical investigations revealed suppressed PTH at 0.5 (1.4-9.1pmol/L), low 25-hydroxy-Vitamin D of 26 (50-150nmol/L), elevated phosphate at 2.43 (0.75-1.5mmol/L) and moderate renal impairment (eGFR 34mL/min/1.73m 2). Hypercalciuria was present with urine calcium to creatinine ratio of 812 (100-580mmol/mol). Her 1,25-dihydroxy-Vitamin D (calcitriol) was elevated at 219 (48-190pmol/L) suggestive of PTH-independent hypercalcemia. The patient remained asymptomatic of hypercalcemia and aggressive intravenous hydration produced minimal biochemical improvement. Renal impairment precluded bisphosphonate use. She was commenced on prednisolone 50mg while continuing intravenous meropenem. Serum calcium normalized in 10 days and prednisolone dose was gradually tapered. A liver biopsy was performed due to persistent liver derangement and histology showed granulomatous inflammation. She completed six weeks of antibiotics for melioidosis and was discharged on day 50, with a planned six months of eradication therapy. Outpatient follow-up demonstrated normal serum calcium after cessation of prednisolone with decreasing IHA titer (1: 320). We hypothesize that this case reflects PTH-independent hypercalcemia secondary to granulomatous inflammation, presumptively caused by melioidosis. The rise in calcium after commencement of antibiotics and normalization following initiation of glucocorticoid therapy, with no rebound post cessation, is consistent with successful therapy of an infection with granulomatous inflammation. Activated macrophages within granulomas have unregulated 1-alpha-hydroxylase expression which is thought to increase conversion of 25-hydroxy-Vitamin D to calcitriol and therefore increase intestinal calcium absorption. Treatment with glucocorticoids inhibits calcitriol synthesis leading to quick resolution of hypercalcemia. While use of glucocorticoids are commonplace in the management of granulomatous disease due to sarcoidosis, they have been increasingly recognized as an important risk factor for melioidosis, considered to result from suppression of innate immunity. However, use of glucocorticoids if required is not contraindicated once antibiotic therapy for melioidosis has commenced. Conclusion In areas where melioidosis is endemic, clinicians should be aware of hypercalcemia and the potential risks associated with its treatment. Presentation: No date and time listed

ODP652 Challenges in the Management of Hypocalcemia in a Patient with Advanced Stage of Parotid Gland Adenocarcinoma

Abstract Background Derangements of calcium metabolism are common in patients with cancer. 1 Unlike hypercalcemia which is well-recognized, little is known about hypocalcemia of malignancy. 2 In fact, there were only 2 studies published to date that investigated its incidence among cancer patients. 2,3 Clinical Case: A 40-year-old male consulted an ENT specialist for a 2×2 cm left parotid mass of 3 years duration. There was no fever, cough, night sweats or significant weight loss. Histological analysis of the mass revealed parotid gland adenocarcinoma. Tumor metastases to lung and spine were seen on PET CT scan. Consequently, he received 18 cycles of chemotherapy and 10 sessions of radiotherapy without surgery because of the advanced stage. He initially exhibited good response with the treatment. A year after,he developed shortness of breath, numbness of hands with seizure episode. Chest x-ray showed pleural effusion and osteoblastic changes while cranial CT scan revealed brain metastasis. Bone scintigraphywas evident for multiple osseous metastases. He underwent stereotactic brain surgery, and thoracentesis with pleural fluid analysis consistent with malignancy. Biochemical tests were remarkable for hypocalcemia (0.99, n>1.12 mmol/L, normomagnesemia (0.89, n>0.74 mmol/L), low vitamin D (11.6, n>30 ng/mL), elevated phosphate (1.73; n: <1.49 mg/dL), and elevated intact PTH which eventually dropped (157.7 to 9, n>15 pg/mL). Ultrasound of the neck revealed diffuse thyroid calcifications. Despite oral calcium supplementation (14.4 grams elemental calcium),calcium infusion (1.35 mg/kg/hour elemental calcium) and vitamin D supplementation (6000 IU cholecalciferol and 0.5 mg calcitriol daily), he remained hypocalcemic. His condition eventually worsened, and he succumbed to death in due course despite aggressive measures provided. Conclusion To our knowledge, this is the first case illustrating the therapeutic challenge in addressing an intractable hypocalcemia in the setting of an advanced parotid carcinoma inour country. Reference: (1)Carl Blomqvist. A Hospital Survey of Hypocalcemia in Patients with Malignant Disease. Acta Med Scand. 1986;220. (2) Schattner et al. Hypocalcaemia of Malignancy. The Netherlands Journal of Medicine. July 2016, Vol. 74, No. 6. (3) Goncalves. Hypocalcemia in cancer patients: An exploratory Study. Porto Biomed. J. (2019) 4: 4 Presentation: No date and time listed

Differences in Phosphate and Parathyroid Hormone Concentrations over the Day among Patients on Hemodialysis.

Elevated serum phosphate and parathyroid hormone (PTH) concentrations are associated with cardiovascular events, bone disease, and mortality in patients on maintenance hemodialysis. Although circadian changes are known in people with CKD, it is unknown whether differences occur in these parameters over the course of a day in people receiving hemodialysis. We used clinical data from Fresenius Medical Care US dialysis clinics to determine how the time of day when measurements were collected (hemodialysis treatment start time) may be associated with serum phosphate and PTH concentrations. We used harmonic regression to assess these associations while accounting for demographic data and treatment parameters. A total of 96,319 patients receiving maintenance hemodialysis were included in this analysis. Patients had a mean age of 64±14 years, 43% were women, and dialysis start times ranged from 3:00 am to 7:59 pm. The mean serum phosphate concentration was 5.2±1.5 mg/dl, and the median PTH was 351 pg/ml (interquartile range [IQR], 214-547). In fully adjusted models, serum phosphate had a nadir at 11:00 am of 4.97 (IQR, 4.94-5.01) mg/dl and a peak at 7:00 pm of 5.56 (IQR, 5.50-5.62) mg/dl. Serum PTH had a nadir at 9:00 am of 385 (IQR, 375-395) pg/ml and a peak at 7:00 pm of 530 (IQR, 516-547) pg/ml. Among patients receiving maintenance hemodialysis, concentrations of PTH and phosphate before a dialysis session vary with the time of day that these values are measured. Consideration of whether these values were obtained at peak or nadir times of the day may be important in treatment decisions.

Association between admission serum phosphate and risk of acute kidney injury in critically ill patients with rhabdomyolysis: A retrospective study based on MIMIC-Ⅲ

BackgroundThe incidence of acute kidney injury (AKI) is high in critically ill patients with rhabdomyolysis. Limited evidence was proved of the association between serum phosphate levels at intensive care unit (ICU) admission and the subsequent risk of AKI. Our study aims to assess if serum phosphate levels at admission were independently associated with AKI risk in these patients. MethodsThis study extracted and analyzed data from Medical Information Mart for Intensive Care-Ⅲ (MIMIC-Ⅲ, version1.4). Rhabdomyolysis was defined as a peak creatine kinase (CK) level higher than 1000 U/L. Serum phosphate was measured within the first day into the ICU and was categorized to 4 groups (<2.6, 2.6-3.4, 3.5-4.5, >4.5mg/dl). AKI was defined according to the Kidney Disease Improving Global Outcome (KDIGO) guidelines. Adjusted smoothing spline plots and multivariable logistic regressions were carried out to explode the association between serum phosphate and risk of AKI. Subgroup analyse was applied to verify the consistency of the association. ResultsThree hundred and twenty-one patients (68% male) diagnosed as rhabdomyolysis were eligible for this analysis. AKI occurred in 204 (64%) patients of total. Incidence of AKI with admission serum phosphate groups<2.6, 2.6-3.4, 3.5-4.5 and>4.5mg/dl were 53%, 57%, 68% and 76%, respectively. Smoothing spline curve showed that there was a positive curve between the elevated phosphate values and increasing risk of AKI, and there was no threshold saturation effect. In multivariable logistic regression, OR was 1.2 (95%CI 1.0-1.5, P=0.035, P trend=0.041) after adjusting confounders. Subgroup analyses proved the consistency of the relationship in these patients, possibly, except in the strata of potassium. ConclusionIn rhabdomyolysis patients admitted to ICU, serum phosphate levels at admission were independently associated with an increased risk of AKI. As phosphate levels rise, the risk of AKI increased.

Sex Differences in Phosphate Homeostasis: Females Excrete More Phosphate and Calcium After an Oral Phosphate Challenge.

Dietary consumption of phosphate is increasing, and elevated serum phosphate is associated with increased cardiovascular disease (CVD) risk. Sex differences in phosphate homeostasis and response to changes in dietary phosphate intake, which are not captured by clinically measured analytes, may contribute to differences in CVD presentation and bone disease. To assess sex differences in acute phosphate homeostasis in response to a single oral phosphate challenge. Cross-sectional. General community. 78 participants (40-76 years) with measured glomerular filtration rate >60 mL/min/1.73 m2 and no clinically diagnosed CVD and 14 young healthy adults. To elucidate subtle alterations in phosphate homeostasis, we employ an acute challenge whereby the hormonal response, circulating mineral levels, and urinary excretion are assessed following an oral challenge of phosphate. Although both males and females had similar changes in circulating phosphate, calcium, and parathyroid hormone in response to the challenge, females excreted ∼1.9x more phosphate and ∼2.7x more calcium than males, despite not consuming calcium. These sex differences were recapitulated in healthy young adults. This excretion response did not correlate to age, serum phosphate, or estradiol levels. The females with greater excretion of phosphate had higher levels of bone resorption markers compared to formation markers. Taken together, these data identify sex differences in acute phosphate homeostasis, specifically that females may mobilize and excrete endogenous sources of calcium and phosphate in response to oral phosphate compared to males. While high levels of dietary phosphate negatively impact bone, our results suggest that females may incur more risk from these diets.

Central gene transcriptional regulatory networks shaping monocyte development in bone marrow.

The development of monocytes in bone marrow is a complex process with multiple steps. We used RNA-seq data to analyze the transcriptome profiles in developing stages of monocytes, including hematopoietic stem cells (HSCs), common myeloid progenitors (CMPs), granulocyte–monocyte progenitors (GMPs), and monocytes. We found that genes related to potassium and other cation transmembrane activities and ion binding were upregulated during the differentiation of HSCs into CMPs. Protein transport and membrane surface functional molecules were significantly upregulated in the GMP stage. The CD42RAC and proteasome pathways are significantly upregulated during the development of HSCs into monocytes. Transcription factors Ank1, Runx2, Hmga2, Klf1, Nfia, and Bmyc were upregulated during the differentiation of HSCs into CMPs; Gfi1 and Hmgn2 were highly expressed during the differentiation of CMPs into GMPs; Seventeen transcription factors including Foxo1, Cdkn2d, Foxo3, Ep300, Pias1, Nfkb1, Creb1, Bcl6, Ppp3cb, Stat5b, Nfatc4, Mef2a, Stat6, Ifnar2, Irf7, Irf5, and Cebpb were identified as potentially involved in the development of GMPs into monocytes in mice and humans. In metabolism pathway regulation, HSCs have high glucose, lipid, and nucleic acid metabolism activities; CMPs mainly up regulate the TCA cycle related genes; and GMPs have extremely active metabolisms, with significantly elevated pentose phosphate pathway, TCA cycle, histidine metabolism, and purine metabolism. In the monocyte phase, the tricarboxylic acid (TCA) cycle is reduced, and the anaerobic glycolysis process becomes dominated. Overall, our studies offer the kinetics and maps of gene transcriptional expressions and cell metabolisms during monocyte development in bone marrow.

Long-Term Excessive Dietary Phosphate Intake Increases Arterial Blood Pressure, Activates the Renin-Angiotensin-Aldosterone System, and Stimulates Sympathetic Tone in Mice.

Increased dietary phosphate intake has been associated with severity of coronary artery disease, increased carotid intima–media thickness, left ventricular hypertrophy (LVH), and increased cardiovascular mortality and morbidity in individuals with normal renal function as well as in patients suffering from chronic kidney disease. However, the underlying mechanisms are still unclear. To further elucidate the cardiovascular sequelae of long-term elevated phosphate intake, we maintained male C57BL/6 mice on a calcium, phosphate, and lactose-enriched diet (CPD, 2% Ca, 1.25% P, 20% lactose) after weaning them for 14 months and compared them with age-matched male mice fed a normal mouse diet (ND, 1.0% Ca, 0.7% P). Notably, the CPD has a balanced calcium/phosphate ratio, allowing the effects of elevated dietary phosphate intake largely independent of changes in parathyroid hormone (PTH) to be investigated. In agreement with the rationale of this experiment, mice maintained on CPD for 14 months were characterized by unchanged serum PTH but showed elevated concentrations of circulating intact fibroblast growth factor-23 (FGF23) compared with mice on ND. Cardiovascular phenotyping did not provide evidence for LVH, as evidenced by unchanged LV chamber size, normal cardiomyocyte area, lack of fibrosis, and unchanged molecular markers of hypertrophy (Bnp) between the two groups. However, intra-arterial catheterization revealed increases in systolic pressure, mean arterial pressure, and pulse pressure in mice fed the CPD. Interestingly, chronically elevated dietary phosphate intake stimulated the renin–angiotensin–aldosterone system (RAAS) as evidenced by increased urinary aldosterone in animals fed the CPD, relative to the ND controls. Furthermore, the catecholamines epinephrine, norepinephrine, and dopamine as well as the catecholamine metabolites metanephrine. normetanephrine and methoxytyramine as measured by mass spectrometry were elevated in the urine of mice on CPD, relative to mice on the ND. These changes were partially reversed by switching 14-month-old mice on CPD back to ND for 2 weeks. In conclusion, our data suggest that excess dietary phosphate induces a rise in blood pressure independent of secondary hyperparathyroidism, and that this effect may be mediated through activation of the RAAS and stimulation of the sympathetic tone.

Elevated pentose phosphate pathway flux supports appendage regeneration

A fundamental step in regeneration is rapid growth to replace lost tissue. Cells must generate sufficient lipids, nucleotides, and proteins to fuel rapid cell division. To define metabolic pathways underlying regenerative growth, we undertake a multimodal investigation of metabolic reprogramming in Xenopus tropicalis appendage regeneration. Regenerating tissues have increased glucose uptake; however, inhibition of glycolysis does not decrease regeneration. Instead, glucose is funneled to the pentose phosphate pathway (PPP), which is essential for full tail regeneration. Liquid chromatography-mass spectrometry (LC-MS) metabolite profiling reveals increased nucleotide and nicotinamide intermediates required for cell division. Using single-cell RNA sequencing (scRNA-seq), we find that highly proliferative cells have increased transcription of PPP enzymes and not glycolytic enzymes. Further, PPP inhibition results in decreased cell division specifically in regenerating tissue. Our results inform a model wherein regenerating tissues direct glucose toward the PPP, yielding nucleotide precursors to drive regenerative cell proliferation.

S3039 Hepatic Undifferentiated Pleomorphic Sarcoma Mimicking Liver Abscess

Introduction: Undifferentiated pleomorphic sarcoma (UPS) has been recognized as one of the most common malignant soft tissue tumors. It usually affects the elderly population and involves the extremities. However, hepatic UPS is rare. Fewer than 200 cases have been reported. Primary hepatic UPS occurs in late adulthood. It typically presents with a solid hepatic mass with regional lymph node involvement. Occasionally, UPS mimics hepatic infection clinically. Here we report a case of hepatic UPS, mimicking the presentations of liver abscess. Case Description/Methods: A 44-year-old female with a past medical history of hypertension presented at the emergency department (ED) for right upper abdominal pain for one week. The pain was described as dull, 5/10 on a pain scale, non-radiating, and associated with nausea, fever, and chills. The pain was worsened by sitting up and alleviated by lying down. At ED, the patient’s vital signs were within normal range. Physical examination showed right upper quadrant tenderness, without rebound, guarding, or rigidity. She was found to have slightly elevated liver enzymes and alkaline phosphate. CT of the abdomen revealed an irregular hypodense mass at the right hepatic lobe measuring 14.3 x 11.6 cm and a 2.6cm enlarged porta hepatis lymph node. The patient was admitted for further evaluation. Concerning possible liver abscess, the blood culture was collected, and the patient was treated with empiric antibiotics. Tumor markers including AFP, and CEA were within normal range. The blood culture was negative for infection. To rule out malignancy, PET scan was performed and showed a hypermetabolic mass with necrotic components in the liver. The patient underwent a liver biopsy. The pathology revealed extensively necrotic pleomorphic round cells with eosinophilic cytoplasm and variably sized, oval nuclei, indicating hepatic undifferentiated pleomorphic sarcoma. Oncology was consulted and recommended doxorubicin and ifosfamide for neoadjuvant chemotherapy. The patient was discharged to home to follow up with oncology as outpatient for chemotherapy and surgery for tumor resection. (Figure) Discussion: Hepatic UPS is a rare malignant mesenchymal tumor. Nonspecific clinical symptoms, unmarkable laboratory findings, and rapid tumor growth lead to poor prognosis. Physicians should be aware of this rare disease as a differential diagnosis for liver mass. Surgical resection is the preferred treatment. Chemotherapy could be used as neoadjuvant or adjuvant treatment to improve the survival rate.Figure 1.: a large liver mass noticed on CT of abdomen with contrast.

S3074 Lecithin-Cholesterol Acyltransferase Deficiency From Statin-Induced Autoimmune Hepatitis

Introduction: A 65yr female presented to the hospital with complaints of pruritis and fatigue that had been ongoing for two months. Upon admission she was found to have transaminitis with AST and ALT at 392 and 400 respectively. (reference lab values AST 0-37 ; ALT 0-35) along with a total bilirubin of 25.1 mg/dL. (Ref 0-1.2). She reported that 1 month ago she went to a primary care doctor and was started on insulin, a calcium channel blocker and atorvastatin. A liver biopsy was done which revealed subacute liver injury- autoimmune pattern of injury (de novo versus secondary) +/- medication injury. This was confirmed by further positive autoimmune markers suggestive of autoimmune hepatitis. Case Description/Methods: The patient presented to the ER complaining of intense pruritis, dark urine, pale stools, and jaundice. She had recently started atorvastatin, amlodipine and insulin for newly diagnosed diabetes mellitus and essential hypertension. All home medications were stopped at admission except insulin. She had no prior history of liver disease and no family history of liver disease. She did not take any supplements and had never consumed alcohol. Initial labs revealed a transaminitis with AST and ALT at 392 and 400 IU/L respectively. (reference lab values AST 0-37 ; ALT 0-35 )along with alkaline phosphate at 2147 IU/L. (Ref 44-121). Initial imaging with a right upper quadrant ultrasound was normal. Patient continued to have a transaminitis and elevated alkaline phosphate that were not improving. A MRCP was done which showed hepatomegaly, mild periportal edema, slightly heterogenous enhancement-conglomerate of findings suggests nonspecific hepatitis. An EUS liver biopsy was performed which revealed subacute liver injury- autoimmune pattern of injury (de novo versus secondary) +/- medication injury. Additional laboratory testing revealed positive autoimmune markers; pointing to a drug induced autoimmune hepatitis as the likely diagnosis. Discussion: Patient was started on prednisone and discharged on a steroid taper once her liver function tests began to improve. She followed up in liver clinic and was noted to have developed hypercholesteremia with total cholesterol of 1,375 MG/DL (Ref 170-199 MG/DL), LDL of 1220 Mg/DL and HDL of 162 MG/DL. A cholesterol esters assay was ordered and referral for genetic testing made for likely acquired lecithin cholesterol acyltransferase deficiency in the setting of statin induced autoimmune hepatitis. Previous cholesterol tests prior to starting atorvastatin were normal.

Microbial Composition of Freshwater Marsh Sediment Responds more Strongly to Microcosm Seawater Addition than Simulated Nitrate or Phosphate Eutrophication.

As sea level rise impacts coastal wetlands, saltmarsh will overtake coastal freshwater marsh in many areas, but changes in the sediment microbiome in response to saltwater intrusion are difficult to predict. Coastal freshwater marsh sediment was exposed to ambient, brackish, and saline conditions as well as to elevated nitrate and phosphate to model the combined stresses of saltwater intrusion and coastal eutrophication. Initially, sediment prokaryotic composition was similar to prior studies of freshwater marsh but diverged over time, reflecting the magnitude of increase in saltwater. There was no observed effect of nutrient amendment, potentially ranking seawater intrusion as a higher-importance compositional driver. Although the previously described loss of methanogenic populations and promotion of sulfate reducers in response to saltwater exposure was observed, taxonomic distribution was not similar to typical meso-polyhaline wetlands. Without colonization by marine taxa, such a community may be short-lived naturally, ultimately equilibrating with more common saltmarsh species. However, the recapitulation of salinity concentration by freshwater sediment microbial composition demonstrates the overwhelming nature of saltwater intrusion relative to other drivers like eutrophication.

Photosynthetic and ultrastructural responses of the chlorophyte Lobosphaera to the stress caused by a high exogenic phosphate concentration

Biotechnology of microalgae holds promise for sustainable using of phosphorus, a finite non-renewable resource. Responses of the green microalga Lobosphaera sp. IPPAS C-2047 to elevated inorganic phosphate (Pi) concentrations were studied. Polyphosphate (PolyP) accumulation and ultrastructural rearrangements were followed in Lobosphaera using light and electron microscopy and linked to the responses of the photosynthetic apparatus probed with chlorophyll fluorescence. High tolerance of Lobosphaera to ≤ 50g L-1 Pi was accompanied by a retention of photosynthetic activity and specific induction of non-photochemical quenching (NPQ up to 4; Fv/Fm around 0.7). Acclimation of the Lobosphaera to the high Pi was accompanied by expansion of the thylakoid lumen and accumulation of the carbon-rich compounds. The toxic effect of the extremely high (100g L-1) Pi inhibited the growth by ca. 60%, induced a decline in photosynthetic activity and NPQ along with contraction of the lumen, destruction of the thylakoids, and depletion of starch reserves. The Lobosphaera retained viability at the Pi in the range of 25-100g L-1 showing moderate an increase of intracellular P content (to 4.6% cell dry weight). During the initial high Pi exposure, the vacuolar PolyP biosynthesis in Lobosphaera was impaired but recovered upon acclimation. Synthesis of abundant non-vacuolar PolyP inclusions was likely a manifestation of the emergency acclimation of the cells converting the Pi excess to less metabolically active PolyP. We conclude that the remarkable Pi tolerance of Lobosphaera IPPAS C-2047 is determined by several mechanisms including rapid conversion of the exogenic Pi into metabolically safe PolyP, the acclamatory changes in the cell population structure. Possible involvement of NPQ in the high Pi resilience of the Lobosphaera is discussed.

Binding and inhibitory activities: A novel oral therapeutic agent for the treatment of hyperphosphataemia rats

Novel oral therapeutic agents based on inhibition or binding activity without adverse events in CKD patients are urgently needed. Here, 5/6 nephrectomy (NX) rats were used to construct a CKD model. Aminated cellulose (AC711), which is metal-free, non-absorbable, and low-volume expansive, was used as a novel oral therapeutic agent for hyperphosphataemia treatment in rats. The efficacy of AC711 on serum and urinary phosphate levels, the expression of type II sodium-dependent phosphate cotransporter (NPT2b), and type III Na-dependent phosphate cotransporter (PiT-1/2) was examined. Serum fibroblast growth factor-23 (FGF-23) levels, parathyroid hormone (PTH) levels, and the phenotypic transformation of vascular smooth muscle cell markers (smooth muscle 22 (SM22) and Runx2) are considered an adaptive response to elevated serum phosphate levels. A similar efficacy of AC711 was observed on serum and urinary phosphate levels when the same dose of AC711 and sevelamer was administered to 5/6 NX rats. The decreasing expression of NPT2b, PiT-1, and PiT-2 was examined in the AC711 groups in a dose-dependent manner. The sevelamer and AC711-MD groups for FGF-23 and PTH indicated no significant difference. The down-regulation of Runx2 expression and up-regulation of SM22 expression were seen in the AC711 groups in a dose-dependent manner. Two suppression mechanisms (binding and inhibiting activities) were observed in the gastrointestinal (GI) tract in the AC711 groups. A novel oral phosphate binder, AC711, showed both binding and inhibition characteristics. The low-volume expansion of AC711 following exposure to simulated intestinal fluid provides the potential therapeutic benefits with the advantage of moderate GI side effects.

Identification of six novel variants from nine Chinese families with hypophosphatemic rickets

BackgroundHypophosphatemic rickets (HR) is a rare genetic disorder associated with renal phosphate wasting and characterized by bone defects. Inactivating mutations in the phosphate regulating endopeptidase homolog X‑linked gene (PHEX) account for most cases of HR. The aim of this study was to identify causative variants in nine unrelated Chinese families associated with HR, and to determine potential pathogenicity of the identified variants.MethodsGenomic DNA was isolated from the peripheral blood of HR patients and their healthy relatives, followed by next-generation sequencing and/or Sanger sequencing. In silico prediction combined with conservation analysis was performed to assess the effects of the variants, and 3D protein modeling was conducted to predict the functional effects on the encoded protein.ResultsAll HR patients recruited in this study displayed bone deformities and tooth agenesis, as well as reduced serum phosphate levels and elevated urine phosphate levels. Nine PHEX variants were identified in eight families, including four novel variants (c.1661_1726del, c.980A > G, c.1078A > T, and c.1017_1051dup). Of the nine identified PHEX variants, five caused a truncated protein, two caused an altered amino acid, and the other two were the canonical splicing variants. Novel variants c.1336G > A and c.1364 T > C in SLC34A3 were also found in one family. Conservation analysis showed that all the amino acids corresponding to the missense variants were highly conserved. In silico analysis and 3D protein structure modeling confirmed the pathogenicity of these variants.ConclusionsThis study identified four novel variants in PHEX and two novel variants in SLC34A3 in a Chinese cohort with HR. Our findings highlight the dominant role of PHEX in HR, and expand the genotypic and phenotypic spectra of this disorder.

UNDERSTANDING TROPONINS: HOW IMPORTANT IT IS

Troponins (TnT &amp; CTnI) as cardiac biomarkers found to be superior to creatinine Kinase – MB (CK – MB) as indicators of myocardial damage.1 They have proven to be useful in diagnosis and risk satisfaction of patients presenting with acute chest pain in emergency room.2 Troponins are released as a result of myocardial damage regardless of etiology. Other than ischemia there are myriad conditions that may cause elevation of troponin levels. Normal findings mentioned in the literature are cardiac troponins T:&lt;0.1ng/ml, cardiac troponin I:&lt;0.03ng/ml &amp; high sensitivity troponin T&lt;14ng/l for women &amp; &lt;22ng/l for men.3 There is a difference in the rise and fall pattern with old generation and modern assays. With the older generation assays elevated troponin levels can be detected 6-12 hours after onset of myocardial injury peaking at 24 hours with a decline up to 2 weeks. With modern assays elevation is detected in 3-4 hours after onset of myocardial injury. Its recommended to recheck troponins at 6-12 hours and up to 24 hours after symptoms onset2 &amp; Non-ST segment myocardial infection can be detected earlier i.e. at 3-4 hours after symptoms onset.4 The important point clinician should be aware is to know abnormal reference range for the assays used in practice, causes of troponin elevation other than myocardial infarction.5- 9 Next important step is collection of blood sample for troponins. It is collected as whole blood through venipuncture. No fasting and no special preparation are needed and can be collected at any time of the day. Troponin T requires heparinized test tubes while EDTA or heparinized tubes for troponin.10,11 The turnaround time for troponin measurement in setting of chest pain should be 60 minutes and all laboratories to achieve this time.12 In patients with borderline elevation of troponins need for seminal testing is important. The initial recommendations were to check the markers every 6 hours until expected peak was reached. Now more sensitive assays are available and very low concentration of elevated cardiac markers can be detected and evidence has demonstrated that checking troponins 3-4 hours after initial draw can help in early diagnosis.13 Last but not the least analytical factors that may lead to falsely elevated troponins results include incompletely clotted specimen,14 Heterophile antibodies15 Rheumatoid factor16 elevated bilirubin levels17 immune complex formation18 markedly elevated alkaline Phosphates levels19 and analyzer malfunction.20 In conclusion, levels of troponins should be interpreted in relation to history and ancillary tests. False-positive results should be kept in mind in decision making, if elevated, troponins will provide a visa to take these patients to catheterization laboratory unnecessary.

Innate Immunity in Calcinosis Cutis

Calcinosis cutis is the deposition of calcium salts in the skin and subcutaneous tissue, manifesting as variably shaped papules, nodules, and plaques that can substantially impair quality of life. The pathophysiology of calcinosis cutis involves dysregulation of proinflammatory cytokines, leukocytes, and other components of the innate immune system. In some conditions associated with calcinosis cutis, elevated serum calcium, phosphate, and vitamin D may also perturb innate immunity. The mechanisms by which these lead to cutaneous and subcutaneous calcification likely parallel those seen in vascular calcification. The role of aberrant innate immunity is further supported by the association between various autoantibodies with calcinosis cutis, such as anti-MDA5, anti-NXP2, anti-centromere, and anti-topoisomerase I. Treatments for calcinosis cutis remain limited and largely experimental, although mechanistically many therapies appear to focus on dampening innate immune responses. Further research is needed to better understand the innate immune pathophysiology and establish treatment options based on randomized-controlled trials.

Abstract 2408: Elevated phosphate promotes a pro-migration phenotype in lung cancer

Abstract Despite advances in treatment, metastasis remains a major cause of cancer-associated death in the US. Thus, identification of intrinsic and extrinsic factors that promote or inhibit tumor migration and invasion are critical. Increasingly, the dysregulation of cellular metabolism is being linked not only to tumor growth but tumor cell migration. Phosphorus (Pi) is an essential micronutrient necessary for energy metabolism, cell signaling via ATP, formation of nucleic acids and the cell membrane, and mineralization of the skeleton. Unsurprisingly, high levels of Pi are observed in tumor tissue compared to normal tissue, consistent with a demand for this metabolite in rapidly growing cells. Further, our lab has reported that high extracellular Pi increases cell growth, alters gene expression and protein translation, and promotes angiogenesis. High dietary Pi levels has also been shown to increase tumorigenesis in both lung and skin cancer models. However, it remains unknown how phosphate metabolism affects tumor migration and invasion. Here we report that high levels of Pi increases gene expression of EMT-related genes such as Twist, Vimentin, CD44, and OPN in the lung cancer cell line, A549. Further, increased Pi promotes tumor cell migration in a transwell assay and decreased Pi levels or inhibition of phosphate transport by phosphonoformic acid (PFA) impaired migration. Suggesting that high Pi can promote a pro-migration phenotype by upregulating EMT in cancer cells and by increasing directed cell migration. Ongoing research will focus on how intracellular Pi flux changes during cell invasion using a novel FRET-based phosphate sensor in vitro and the effect of Pi inhibition on metastasis in vivo. These findings provide further evidence for the role dysregulated Pi metabolism in promoting cancer progression and the potential for therapeutic intervention with broad impacts on patient outcomes. Citation Format: Jamie Arnst, George Beck. Elevated phosphate promotes a pro-migration phenotype in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2408.

MO448: Progressive Tubular Injury Caused by High Phosphate Intake is Associated With Activation of STAT3/KIM-1 Signalling and Macrophage Recruitment in Mice

Abstract BACKGROUND AND AIMS Dietary phosphate intake greatly exceeds the recommended daily allowance in the Western population. Elevated phosphate levels are linked to increased cardiovascular and all-cause mortality, impaired bone health and premature ageing. The renal effects of chronic high dietary phosphate intake for healthy individuals are still not clear. METHOD Male C57BL/6 mice received a 2% high phosphate diet (HPD) or a 0.8% normal phosphate diet (NPD) for 1 to 6 months. We collected blood, urine and kidneys to investigate phosphate metabolism and progression of kidney injury. In vitro analysis for the toxic effects of phosphate or the phosphaturic hormones parathyroid hormone (PTH) and fibroblast growth factor (FGF) 23 was done in HK-2 humane proximal tubule (PT) cells. RESULTS HPD in mice caused hyperphosphataemia despite increased phosphaturia. Both intact FGF23 and PTH plasma levels were significantly increased in mice on HPD at 1 month onward. Mice on HPD showed significantly increased creatinine levels and albuminuria. Histopathological analysis of the kidneys revealed increased PT injury in mice receiving HPD compared to NPD controls from 2 months onward followed by a rapid progression of organ damage from month 5 to 6. Picrosirius red staining of kidney sections showed a progressively increased accumulation of collagen fibers in renal cortex of mice on HPD, which was significant from 4 months onward and accompanied by enhanced expression of transforming growth factor-β1, collagen 1 and fibronectin. The significant induction of the tubular injury marker Havcr1, encodes for Kim-1, started already after 1 month of feeding HPD, progressed over time and was strongest after 6 months. Immunofluorescence staining revealed Kim-1 localization to damaged PT, which was most prominent after 3 and 6 months of HPD. Renal Kim-1 protein levels were positively associated with tubular injury score and tubulointerstitial fibrosis. Histological staining and quantification revealed significantly increased number of pStat3+ cells in PTs of HPD-fed mice at months 1, 2, 5 and 6, indicating activated Stat3 signaling. The number of pStat3+ cells was positively associated with Kim-1 synthesis. Inflammation cytokine array with kidney tissue lysates showed, among others, enhanced expression of monocyte chemoattractant protein (MCP)-1 and macrophage colony-stimulating factor (MCSF) in HPD-fed mice. The mRNA expression of Csf1 (encodes for MCSF) was upregulated in HPD-fed mice and most pronounced after 6 months. Ccl2 mRNA expression (encodes for MCP-1) was significantly enhanced after 3, 5 and 6 months of HPD compared to NPD. Immunofluorescence staining showed a strong and specific induction of MCP-1 in PT epithelial cells due to HPD. The HPD-induced Ccl2 expression was positively associated with Kim-1 expression indicating an interaction between pStat3/Kim-1 signalling pathway and MCP-1. Immunohistochemical staining and flow cytometry analysis revealed a significantly increased recruitment of F4/80+ macrophages in renal tissue of HPD-fed mice after 3 and 6 months. The enhanced renal macrophage recruitment in the entire HPD cohort was associated with increased MCP-1 protein synthesis and increased tubular injury score. In vitro experiments using HK-2 cells showed that stimulation with FGF23 or high phosphate, but not PTH, induced the phosphorylation of STAT3. Interestingly, only treatment with high phosphate significantly upregulated HAVCR1 and CCL2 expression. CONCLUSION Chronic oral phosphate load in mice induced progressive inflammatory kidney injury dominated by macrophages. The direct toxic effects of phosphate are mediated by Stat3/Kim-1 signalling activation in PT cells. Our data support the hypothesis that kidney injury induced by widespread high dietary phosphate intake may cause a global health problem and underline the urgent need for clinical studies on this issue.

Hyperphosphatemia Contributes to Skeletal Muscle Atrophy in the Absence and Presence of Chronic Kidney Disease

BackgroundChronic Kidney Disease (CKD) is a public health epidemic that is associated with elevated serum levels of phosphate (hyperphosphatemia) as well as skeletal muscle atrophy, but this interconnection is poorly understood. Elevated phosphate (Pi) has direct effects on smooth muscle cells and induces vascular calcification. Pi also induces the production of the hormone fibroblast growth factor (FGF) 23 in bone. We wanted to test if Pi induces atrophy and/or FGF23 expression in skeletal muscle cells. Furthermore, we analyzed skeletal muscle on a functional, histological and molecular level in three models of hyperphosphatemia – two CKD models, i.e. mice with global deletion of collagen 4a3 (Col4a3‐/‐) and wildtype mice receiving an adenine‐rich diet, as well as wildtype mice on a high Pi diet with normal kidney function. Finally, we determined the effect of a low Pi diet on skeletal muscle in Col4a3‐/‐ mice.MethodsC2C12 myotubes were treated with 1‐5 mM Pi for 24 hours, followed by qPCR expression analysis of atrophy genes (atrogenes), including MT1, Trim63, and Fbox32, as well as FGF23. Furthermore, we studied Col4a3‐/‐ mice receiving normal chow or a 0.2% Pi diet at 10 weeks of age. We also analyzed C57Bl/6 mice receiving an adenine‐rich (0.2%) diet for 14 weeks or a 3% Pi diet for 3 months. We analyzed grip strength, hindlimb area by MRI, muscle wet weight, cross‐sectional area of individual muscle fibers immuno‐labeled with anti‐laminin by fluorescence microscopy, and expression levels of atrogenes by qPCR and of FGF23 by qPCR and ELISA.ResultsPi treatments increased the expression levels of atrogenes as well as FGF23 in C2C12 myotubes. In the three mouse models, grip strength and cross‐sectional area of myofibers were significantly reduced, and the expression levels of atrogenes were significantly elevated when compared to respective controls. Furthermore, we detected elevations in the mRNA and protein levels of FGF23 in the hindlimb muscles of all models. Additionally, the two CKD models showed significant reductions in muscle weight and hindlimb area. Administration of a 0.2% Pi diet protected Col4a3‐/‐ mice from developing skeletal muscle atrophy.ConclusionElevated Pi induces myotube atrophy and FGF23 expression in vitro. Mouse models with hyperphosphatemia not only develop skeletal muscle atrophy, but also produce FGF23 in skeletal muscle tissue in the presence and absence of CKD, and a low Pi diet protects the skeletal muscle in CKD mice. Pharmacological approaches targeting Pi uptake or excretion, or inhibition of Pi’s direct actions on tissues might alleviate various CKD‐associated pathologies. Future studies need to determine whether skeletal muscle‐derived FGF23 contributes to tissue injury or is protective against Pi‐induced damage.