NH4Cl Treatment Prevents Tissue Calcification in Klotho Deficiency.

Klotho, a cofactor in suppressing 1,25(OH)2D3 formation, is a powerful regulator of mineral metabolism. Klotho-hypomorphic mice (kl/kl) exhibit excessive plasma 1,25(OH)2D3, Ca(2+), and phosphate concentrations, severe tissue calcification, volume depletion with hyperaldosteronism, and early death. Calcification is paralleled by overexpression of osteoinductive transcription factor Runx2/Cbfa1, Alpl, and senescence-associated molecules Tgfb1, Pai-1, p21, and Glb1. Here, we show that NH4Cl treatment in drinking water (0.28 M) prevented soft tissue and vascular calcification and increased the life span of kl/kl mice >12-fold in males and >4-fold in females without significantly affecting extracellular pH or plasma concentrations of 1,25(OH)2D3, Ca(2+), and phosphate. NH4Cl treatment significantly decreased plasma aldosterone and antidiuretic hormone concentrations and reversed the increase of Runx2/Cbfa1, Alpl, Tgfb1, Pai-1, p21, and Glb1 expression in aorta of kl/kl mice. Similarly, in primary human aortic smooth muscle cells (HAoSMCs), NH4Cl treatment reduced phosphate-induced mRNA expression of RUNX2/CBFA1, ALPL, and senescence-associated molecules. In both kl/kl mice and phosphate-treated HAoSMCs, levels of osmosensitive transcription factor NFAT5 and NFAT5-downstream mediator SOX9 were higher than in controls and decreased after NH4Cl treatment. Overexpression of NFAT5 in HAoSMCs mimicked the effect of phosphate and abrogated the effect of NH4Cl on SOX9, RUNX2/CBFA1, and ALPL mRNA expression. TGFB1 treatment of HAoSMCs upregulated NFAT5 expression and prevented the decrease of phosphate-induced NFAT5 expression after NH4Cl treatment. In conclusion, NH4Cl treatment prevents tissue calcification, reduces vascular senescence, and extends survival of klotho-hypomorphic mice. The effects of NH4Cl on vascular osteoinduction involve decrease of TGFB1 and inhibition of NFAT5-dependent osteochondrogenic signaling.

Effect of carbon monoxide donor CORM-2 on vitamin D3 metabolism.

BACKGROUND/AIMS: Carbon monoxide (CO) interferes with cytochrome-dependent cellular functions and acts as gaseous transmitter. CO is released from CO-releasing molecules (CORM) including tricarbonyl-dichlororuthenium (II) dimer (CORM-2), molecules considered for the treatment of several disorders including vascular dysfunction, inflammation, tissue ischemia and organ rejection. Cytochrome P450-sensitive function include formation of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) by renal 25-hydroxyvitamin D3 1-alpha-hydroxylase (Cyp27b1). The enzyme is regulated by PTH, FGF23 and klotho. 1,25(OH)2D3 regulates Ca(2+) and phosphate transport as well as klotho expression. The present study explored, whether CORM-2 influences 1,25(OH)2D3 formation and klotho expression. METHODS: Mice were treated with intravenous CORM-2 (20 mg/kg body weight). Plasma 1,25(OH)2D3 and FGF23 concentrations were determined by ELISA, phosphate, calcium and creatinine concentrations by colorimetric methods, transcript levels by quantitative RT-PCR and protein expression by western blotting. Fgf23 mRNA transcript levels were further determined in rat osteosarcoma UMR106 cells without or with prior treatment for 24 hours with 20 µM CORM-2. RESULTS: CORM-2 injection within 24 hours significantly increased FGF23 plasma levels and decreased 1,25(OH)2D3 plasma levels, renal Cyp27b1 gene expression as well as renal klotho protein abundance and transcript levels. Moreover, treatment of UMR106 cells with CORM-2 significantly increased Fgf23 transcript levels. CONCLUSION: CO-releasing molecule CORM-2 enhances FGF23 expression and release and decreases klotho expression and 1,25(OH)2D3 synthesis. © 2013 S. Karger AG, Basel.

OSR1-sensitive small intestinal Na+ transport.

The oxidative stress responsive kinase 1 (OSR1) contributes to WNK (with no K)-dependent regulation of renal tubular salt transport, renal salt excretion, and blood pressure. Little is known, however, about a role of OSR1 in the regulation of intestinal salt transport. The present study thus explored whether OSR1 is expressed in intestinal tissue and whether small intestinal Na(+)/H(+) exchanger (NHE), small intestinal Na(+)-glucose cotransport (SGLT1), and/or colonic epithelium Na(+) channel (ENaC) differ between knockin mice carrying one allele of WNK-resistant OSR1 (osr1(+/KI)) and wild-type mice (osr1(+/+)). OSR1 protein abundance was determined by Western blotting, cytosolic pH from BCECF fluorescence, NHE activity from Na(+)-dependent realkalinization following an ammonium pulse, SGLT1 activity from glucose-induced current, and colonic ENaC activity from amiloride-sensitive transepithelial current in Ussing chamber experiments. As a result, OSR1 protein was expressed in small intestine of both osr1(+/KI) mice and osr1(+/+) mice. Daily fecal Na(+), K(+), and H(2)O excretion and jejunal SGLT1 activity were lower, whereas small intestinal NHE activity and colonic ENaC activity were higher in osr1(+/KI) mice than in osr1(+/+) mice. NHE3 inhibitor S-3226 significantly reduced NHE activity in both genotypes but did not abrogate the difference between the genotypes. Plasma osmolarity, serum antidiuretic hormone, plasma aldosterone, and plasma corticosterone concentrations were similar in both genotypes. Small intestinal NHE3 and colonic α-ENaC protein abundance were not significantly different between genotypes, but colonic phospho-ß-ENaC (ser633) was significantly higher in osr1(+/KI) mice. In conclusion, OSR1 is expressed in intestine and partial WNK insensitivity of OSR1 increases intestinal NHE activity and colonic ENaC activity.

DOCA sensitive pendrin expression in kidney, heart, lung and thyroid tissues.

BACKGROUND/AIMS: Pendrin (SLC26A4), a transporter accomplishing anion exchange, is expressed in inner ear, thyroid gland, kidneys, lung, liver and heart. Loss or reduction of function mutations of SLC26A4 underlie Pendred syndrome, a disorder invariably leading to hearing loss with enlarged vestibular aqueducts and in some patients to hypothyroidism and goiter. Renal pendrin expression is up-regulated by mineralocorticoids such as aldosterone or deoxycorticosterone (DOCA). Little is known about the impact of mineralocorticoids on pendrin expression in extrarenal tissues. METHODS: The present study utilized RT-qPCR and Western blotting to quantify the transcript levels and protein abundance of Slc26a4 in murine kidney, thyroid, heart and lung prior to and following subcutaneous administration of 100 mg/kg DOCA. RESULTS: Slc26a4 transcript levels as compared to Gapdh transcript levels were significantly increased by DOCA treatment in kidney, heart, lung and thyroid. Accordingly pendrin protein expression was again significantly increased by DOCA treatment in kidney, heart, lung and thyroid. CONCLUSION: The observations reveal mineralocorticoid sensitivity of pendrin expression in kidney, heart, thyroid and lung.

Enhanced FGF23 serum concentrations and phosphaturia in gene targeted mice expressing WNK-resistant SPAK.

BACKGROUND: The WNK-dependent STE20/SPS1-related proline/alanine-rich kinase (SPAK) regulates the renal thiazide sensitive NaCl cotransporter (NCC) and the renal furosemide sensitive Na+, K+, 2Cl- cotransporter (NKCC2) and thus participates in the regulation of renal salt excretion, extracellular fluid volume and blood pressure. Inhibition of NCC leads to anticalciuria. Moreover, NCC is also expressed in osteoblasts where it is implicated in the regulation of bone mineralization. Osteoblasts further influence mineral metabolism by releasing the phosphaturic hormone FGF23. The present study explored, whether SPAK participates in the regulation of calcium-phosphate homeostasis. METHODS: FGF23 serum levels and phosphate homeostasis were analyzed in gene targeted mice expressing SPAK resistant to WNK-dependent activation (spak(tg/tg)) and in mice expressing wild type SPAK (spak(wt/wt)). RESULTS: Serum FGF23 level was significantly higher, urinary phosphate excretion significantly larger and serum phosphate concentration significantly lower in spak(tg/tg) mice than in spak(wt/wt) mice. Urinary calcium excretion was significantly decreased in spaktg/tg mice. Serum levels of calcitriol and PTH were not significantly different between the genotypes. Bone density was significantly increased in spak(tg/tg) mice compared to spak(wt/wt) mice. Treatment of spak(wt/wt) mice with HCT increased FGF23 serum levels, and led to phosphaturia and hypophosphatemia. CONCLUSIONS: SPAK is a strong regulator of FGF23 formation, bone mineralization and renal Ca2+ and phosphate excretion.

OSR1-sensitive renal tubular phosphate reabsorption.

BACKGROUND: The oxidative stress-responsive kinase 1 (OSR1) participates in the WNK-(with no K) kinase dependent regulation of renal salt excretion and blood pressure. Little is known, however, about the role of OSR1 in the regulation of further renal transport systems. The present study analyzed the effect of OSR1 on NaPiIIa, the major renal tubular phosphate transporter. METHODS: Immunohistochemistry and confocal microscopy were employed to determine renal localization of OSR1 and NaPiIIa. To elucidate the effect of OSR on NaPiIIa activity, cRNA encoding NaPiIIa was injected into Xenopus oocytes with or without additional injection of cRNA encoding OSR1, and phosphate transport was estimated from phosphateinduced currents determined with dual electrode voltage clamp. To elucidate the in vivo significance of OSR1 serum phosphate and hormone concentrations as well as urinary phosphate output of mice carrying one allele of WNK-resistant OSR1 (osr1tg/(+)) were compared to the respective values of wild type mice (osr1(+/+)). RESULTS: NaPiIIa and OSR1 were both expressed in proximal renal tubule cells. Coexpression of OSR1 significantly up-regulated phosphate-induced currents in NaPiIIa-expressing Xenopus oocytes. Despite decreased serum phosphate concentration urinary phosphate excretion was significantly increased and NaPiIIa protein abundance in the brush border membrane significantly reduced in osr1tg/(+) mice as compared to osr1(+/+) mice. Serum PTH and calcitriol levels were similar in osr1tg/(+) mice and in osr1(+/+) mice, serum FGF23 concentration was, however, significantly higher in osr1tg/(+) mice than in osr1(+/+) mice. CONCLUSIONS: OSR1 is expressed in proximal renal tubules and participates in the regulation of FGF23 release and renal tubular phosphate transport.

Tumor suppressor gene adenomatous polyposis coli downregulates intestinal transport.

Loss of function mutations of the tumor suppressor gene adenomatous polyposis coli (APC) underly the familial adenomatous polyposis. Mice carrying an inactivating mutation in the apc gene (apc (Min/+)) similarly develop intestinal polyposis. APC is effective at least in part by degrading ß-catenin and lack of APC leads to markedly enhanced cellular ß-catenin levels. ß-Catenin has most recently been shown to upregulate the Na+/K+ ATPase. The present study, thus, explored the possibility that APC could influence intestinal transport. The abundance and localization of ß-catenin were determined utilizing Western blotting and confocal microscopy, the activity of the electrogenic glucose carrier (SGLT1) was estimated from the glucose-induced current in jejunal segments utilizing Ussing chamber experiments and the Na+/H+ exchanger (NHE3) activity from Na+ -dependent re-alkalinization of cytosolic pH (ΔpH(i)) following an ammonium pulse employing BCECF fluorescence. As a result, ß-catenin abundance in intestinal tissue was significantly higher in apc (Min/+) mice than in wild-type mice (apc (+/+)). The ß-catenin protein was localized in the basolateral membrane. Both, the glucose-induced current and ΔpH(i) were significantly higher in apc (Min/+) mice than in apc (+/+) mice. In conclusion, intestinal electrogenic transport of glucose and intestinal Na+/H+ exchanger activity are both significantly enhanced in apc (Min/+) mice, pointing to a role of APC in the regulation of epithelial transport.

Downregulation of Klotho expression by dehydration.

Klotho, a transmembrane protein, protease, and hormone mainly expressed in renal tissue counteracts aging. Overexpression of Klotho substantially prolongs the life span. Klotho deficiency leads to excessive formation of 1,25(OH)(2)D(3), growth deficit, accelerated aging, and early death. Aging is frequently paralleled by dehydration, which is considered to accelerate the development of age-related disorders. The present study explored the possibility that dehydration influences Klotho expression. Klotho transcript levels were determined by RT-PCR, and Klotho protein abundance was detected by Western blotting in renal tissue from hydrated and 36-h-dehydrated mice as well as in human embryonic kidney (HEK293) cells. Dehydration was followed by a significant decline of renal Klotho transcript levels and protein abundance, accompanied by an increase in plasma osmolarity as well as plasma ADH, aldosterone, and 1,25(OH)(2)D(3) levels. Antidiuretic hormone (ADH; 50 nM) and aldosterone (1 µM) significantly decreased Klotho transcription and protein expression in HEK293 cells. In conclusion, the present observations disclose a powerful effect of dehydration on Klotho expression, an effect at least partially mediated by enhanced release of ADH and aldosterone.

p38 MAPK activation and function following osmotic shock of erythrocytes.

p38 protein kinase is activated by hyperosmotic shock, participates in the regulation of cell volume sensitive transport and metabolism and is involved in the regulation of various physiological functions including cell proliferation and apoptosis. Similar to apoptosis of nucleated cells, erythrocytes may undergo suicidal death or eryptosis, which is paralleled by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Triggers of eryptosis include hyperosmotic shock, which increases cytosolic Ca(2+) activity and ceramide formation. The present study explored whether p38 kinase is expressed in human erythrocytes, is activated by hyperosmotic shock and participates in the regulation of eryptosis. Western blotting was utilized to determine phosphorylation of p38 kinase, forward scatter to estimate cell volume, annexin V binding to depict phosphatidylserine exposure and Fluo3 fluorescence to estimate cytosolic Ca(2+) activity. As a result, erythrocytes express p38 kinase, which is phosphorylated upon osmotic shock (+550 mM sucrose). Osmotic shock decreased forward scatter, increased annexin V binding and increased Fluo3 fluorescence, all effects significantly blunted by the p38 kinase inhibitors SB203580 (2 µM) and p38 Inh III (1 µM). In conclusion, p38 kinase is expressed in erythrocytes and participates in the machinery triggering eryptosis following hyperosmotic shock.

Inhibition of Kir2.1 (KCNJ2) by the AMP-activated protein kinase.

The inward rectifier K(+) channel Kir2.1 participates in the maintenance of the cell membrane potential in a variety of cells including neurons and cardiac myocytes. Mutations of KCNJ2 encoding Kir2.1 underlie the Andersen-Tawil syndrome, a rare disorder clinically characterized by periodic paralysis, cardiac arrhythmia and skeletal abnormalities. The maintenance of the cardiac cell membrane potential is decreased in ischaemia, which is known to stimulate the AMP-activated serine/threonine protein kinase (AMPK). This energy-sensing kinase stimulates energy production and limits energy utilization. The present study explored whether AMPK regulates Kir2.1. To this end, cRNA encoding Kir2.1 was injected into Xenopus oocytes with and without additional injection of wild type AMPK (AMPKα1+AMPKß1+AMPKγ1), of the constitutively active (γR70Q)AMPK (α1ß1γ1(R70Q)), of the kinase dead mutant (αK45R)AMPK (α1(K45R)ß1γ1), or of the ubiquitin ligase Nedd4-2. Kir2.1 activity was determined in two-electrode voltage-clamp experiments. Moreover, Kir2.1 protein abundance in the cell membrane was determined by immunostaining and subsequent confocal imaging. As a result, wild type and constitutively active AMPK significantly reduced Kir2.1-mediated currents and Kir2.1 protein abundance in the cell membrane. Expression of wild type Nedd4-2 or of Nedd4-2(S795A) lacking an AMPK phosphorylation consensus sequence downregulated Kir2.1 currents. The effect of wild type Nedd4-2 but not of Nedd4-2(S795A) was significantly augmented by additional coexpression of AMPK. In conclusion, AMPK is a potent regulator of Kir2.1. AMPK is at least partially effective through phosphorylation of the ubiquitin ligase Nedd4-2.

Predicting Response to Chemotherapy in Patients With Newly Diagnosed High-Risk Neuroblastoma: A Report From the International Neuroblastoma Risk Group.

PURPOSE: Metaiodobenzylguanidine (MIBG) scans are a radionucleotide imaging modality that undergo Curie scoring to semiquantitatively assess neuroblastoma burden, which can be used as a marker of therapy response. We hypothesized that a convolutional neural network (CNN) could be developed that uses diagnostic MIBG scans to predict response to induction chemotherapy. METHODS: We analyzed MIBG scans housed in the International Neuroblastoma Risk Group Data Commons from patients enrolled in the Children's Oncology Group high-risk neuroblastoma study ANBL12P1. The primary outcome was response to upfront chemotherapy, defined as a Curie score ≤ 2 after four cycles of induction chemotherapy. We derived and validated a CNN using two-dimensional whole-body MIBG scans from diagnosis and evaluated model performance using area under the receiver operating characteristic curve (AUC). We also developed a clinical classification model to predict response on the basis of age, stage, and MYCN amplification. RESULTS: Among 103 patients with high-risk neuroblastoma included in the final cohort, 67 (65%) were responders. Performance in predicting response to upfront chemotherapy was equivalent using the CNN and the clinical model. Class-activation heatmaps verified that the CNN used areas of disease within the MIBG scans to make predictions. Furthermore, integrating predictions using a geometric mean approach improved detection of responders to upfront chemotherapy (geometric mean AUC 0.73 v CNN AUC 0.63, P < .05; v clinical model AUC 0.65, P < .05). CONCLUSION: We demonstrate feasibility in using machine learning of diagnostic MIBG scans to predict response to induction chemotherapy for patients with high-risk neuroblastoma. We highlight improvements when clinical risk factors are also integrated, laying the foundation for using a multimodal approach to guiding treatment decisions for patients with high-risk neuroblastoma.

Acetazolamide sensitive tissue calcification and aging of klotho-hypomorphic mice.

UNLABELLED: Klotho, a protein expressed mainly in the kidney, is required for the inhibitory effect of FGF23 on renal 1,25(OH)2D3 formation. Klotho counteracts vascular calcification and diverse age-related disorders. Klotho-hypomorphic mice (kl/kl) suffer from severe vascular calcification and rapid aging. The calcification is at least in part caused by excessive 1,25(OH)2D3, Ca(2+), and phosphate concentrations in blood, which trigger osteogenic signaling including upregulation of alkaline phosphatase (Alpl). As precipitation of calcium and phosphate is fostered by alkaline pH, extracellular acidosis could counteract tissue calcification. In order to induce acidosis, acetazolamide was added to drinking water (0.8 g/l) of kl/kl and wild-type mice. As a result, acetazolamide treatment of kl/kl mice partially reversed the growth deficit, tripled the life span, almost completely reversed the calcifications in trachea, lung, kidney, stomach, intestine, and vascular tissues, the excessive aortic alkaline phosphatase mRNA levels and the plasma concentrations of osteoprotegerin, osteopontin as well as fetuin-A, without significantly decreasing FGF23, 1,25(OH)2D3, Ca(2+), and phosphate plasma concentrations. In primary human aortic smooth muscle cells, acidotic environment prevented phosphate-induced alkaline phosphatase mRNA expression. The present study reveals a completely novel effect of acetazolamide, i.e., interference with osteoinductive signaling and tissue calcification in kl/kl mice. KEY MESSAGES: Klotho deficient (kl/kl) mice suffer from hyperphosphatemia with dramatic tissue calcification. Acetazolamide (ACM) treatment partially reversed the growth deficit of kl/kl mice. In kl/kl mice, ACM reversed tissue calcification despite continued hyperphosphatemia. ACM tripled the life span of kl/kl mice. In human aortic smooth muscle cells, low extracellular pH prevented osteogenic signaling.

Apigenin-induced suicidal erythrocyte death.

Apigenin, a flavone in fruits and vegetables, stimulates apoptosis and thus counteracts cancerogenesis. Erythrocytes may similarly undergo suicidal cell death or eryptosis, characterized by cell shrinkage and phosphatidylserine exposure at the cell surface. Triggers of eryptosis include increase of cytosolic Ca(2+) activity ([Ca(2+)](i)), ceramide formation and ATP depletion. The present study explored the effect of apigenin on eryptosis. [Ca(2+)](i) was estimated from Fluo3-fluorescence, cell volume from forward scatter, phosphatidylserine exposure from annexin V binding, hemolysis from hemoglobin release, ceramide utilizing antibodies, and cytosolic ATP with luciferin-luciferase. A 48 h exposure to apigenin significantly increased [Ca(2+)](i) (≥ 1 µM), increased ceramide formation (15 µM), decreased ATP concentration (15 µM), decreased forward scatter (≥ 1 µM), and increased annexin V binding (≥ 5 µM) but did not significantly modify hemolysis. The effect of 15 µM apigenin on annexin V binding was blunted by Ca(2+) removal. The present observations reveal novel effects of apigenin, i.e. stimulation of Ca(2+) entry, ceramide formation and ATP depletion in erythrocytes with subsequent triggering of suicidal erythrocyte death, paralleled by cell shrinkage and phosphatidylserine exposure.

Overexpression of kinase-negative protein kinase Cdelta in pancreatic beta-cells protects mice from diet-induced glucose intolerance and beta-cell dysfunction.

OBJECTIVE: In vitro models suggest that free fatty acid-induced apoptotic beta-cell death is mediated through protein kinase C (PKC)delta. To examine the role of PKCdelta signaling in vivo, transgenic mice overexpressing a kinase-negative PKCdelta (PKCdeltaKN) selectively in beta-cells were generated and analyzed for glucose homeostasis and beta-cell survival. RESEARCH DESIGN AND METHODS: Mice were fed a standard or high-fat diet (HFD). Blood glucose and insulin levels were determined after glucose loads. Islet size, cleaved caspase-3, and PKCdelta expression were estimated by immunohistochemistry. In isolated islet cells apoptosis was assessed with TUNEL/TO-PRO3 DNA staining and the mitochondrial potential by rhodamine-123 staining. Changes in phosphorylation and subcellular distribution of forkhead box class O1 (FOXO1) were analyzed by Western blotting and immunohistochemistry. RESULTS: PKCdeltaKN mice were protected from HFD-induced glucose intolerance. This was accompanied by increased insulin levels in vivo, by an increased islet size, and by a reduced staining of beta-cells for cleaved caspase-3 compared with wild-type littermates. In accordance, long-term treatment with palmitate increased apoptotic cell death of isolated islet cells from wild-type but not from PKCdeltaKN mice. PKCdeltaKN overexpression protected islet cells from palmitate-induced mitochondrial dysfunction and inhibited nuclear accumulation of FOXO1 in mouse islet and INS-1E cells. The inhibition of nuclear accumulation of FOXO1 by PKCdeltaKN was accompanied by an increased phosphorylation of FOXO1 at Ser256 and a significant reduction of FOXO1 protein. CONCLUSIONS: Overexpression of PKCdeltaKN in beta-cells protects from HFD-induced beta-cell failure in vivo by a mechanism that involves inhibition of fatty acid-mediated apoptosis, inhibition of mitochondrial dysfunction, and inhibition of FOXO1 activation.