Masashi Masuda, Yuji Shiozaki and Makoto Miyazaki : Chapter 10 - Lipotoxicity in the pathogenesis of chronic kidney disease complications, Academic Press, Aug. 2023.
Minori Uga, Ichiro Kaneko, Yuji Shiozaki, Megumi Koike, Naoko Tsugawa, W. Peter Jurutka, Ken-ichi Miyamoto and Hiroko Segawa : The Role of Intestinal Cytochrome P450s in Vitamin D Metabolism, Biomolecules, 14, 6, 2024.
(Summary)
Vitamin D hydroxylation in the liver/kidney results in conversion to its physiologically active form of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. 1,25(OH)2D3 controls gene expression through the nuclear vitamin D receptor (VDR) mainly expressed in intestinal epithelial cells. Cytochrome P450 (CYP) 24A1 is a catabolic enzyme expressed in the kidneys. Interestingly, a recently identified mutation in another CYP enzyme, CYP3A4 (gain-of-function), caused type III vitamin D-dependent rickets. CYP3A are also expressed in the intestine, but their hydroxylation activities towards vitamin D substrates are unknown. We evaluated CYP3A or CYP24A1 activities on vitamin D action in cultured cells. In addition, we examined the expression level and regulation of CYP enzymes in intestines from mice. The expression of CYP3A or CYP24A1 significantly reduced 1,25(OH)2D3-VDRE activity. Moreover, in mice, Cyp24a1 mRNA was significantly induced by 1,25(OH)2D3 in the intestine, but a mature form (approximately 55 kDa protein) was also expressed in mitochondria and induced by 1,25(OH)2D3, and this mitochondrial enzyme appears to hydroxylate 25OHD3 to 24,25(OH)2D3. Thus, CYP3A or CYP24A1 could locally attenuate 25OHD3 or 1,25(OH)2D3 action, and we suggest the small intestine is both a vitamin D target tissue, as well as a newly recognized vitamin D-metabolizing tissue.
(Keyword)
1,25(OH) D 2 3 / 25OHD 3 / CYP24A1 / CYP3A / vitamin D
Shinobu Miyazaki-Anzai, Masashi Masuda, Audrey L Keenan, Yuji Shiozaki, Jose G Miranda and Makoto Miyazaki : Activation of the IKK2/NF-κB pathway in VSMCs inhibits calcified vascular stiffness in CKD, JCI Insight, 9, 7, 12:e174977, 2024.
(Summary)
IKK2/NF-κB pathway-mediated inflammation in vascular smooth muscle cells (VSMCs) has been proposed to be an etiologic factor in medial calcification and stiffness. However, the role of the IKK2/NF-κB pathway in medial calcification remains to be elucidated. In this study, we found that chronic kidney disease (CKD) induces inflammatory pathways through the local activation of the IKK2/NF-κB pathway in VMSCs associated with calcified vascular stiffness. Despite reducing the expression of inflammatory mediators, complete inhibition of the IKK2/NF-κB pathway in vitro and in vivo unexpectedly exacerbated vascular mineralization and stiffness. In contrast, activation of NF-κB by SMC-specific IκBα deficiency attenuated calcified vascular stiffness in CKD. Inhibition of the IKK2/NF-κB pathway induced cell death of VSMCs by reducing anti-cell death gene expression, whereas activation of NF-κB reduced CKD-dependent vascular cell death. In addition, increased calcification of extracellular vesicles through the inhibition of the IKK2/NF-κB pathway induced mineralization of VSMCs, which was significantly reduced by blocking cell death in vitro and in vivo. This study reveals that activation of the IKK2/NF-κB pathway in VSMCs plays a protective role in CKD-dependent calcified vascular stiffness by reducing the release of apoptotic calcifying extracellular vesicles.
(Keyword)
Humans / NF-kappa B / Signal Transduction / Vascular Stiffness / Muscle, Smooth, Vascular / Renal Insufficiency, Chronic
Kazuya Tanifuji, Yuji Shiozaki, Megumi Koike, Minori Uga, Aoi Komiya, Mizuki Miura, Ayami Higashi, Takaaki Shimohata, Akira Takahashi, Noriko Ishizuka, Hisayoshi Hayashi, Yasuhiro Ichida, Shuichi Ohtomo, Naoshi Horiba, Ken-ichi Miyamoto and Hiroko Segawa : Effects of EOS789, a novel pan-phosphate transporter inhibitor, on phosphate metabolism : Comparison with a conventional phosphate binder, The Journal of Medical Investigation : JMI, 70, 1,2, 260-270, 2023.
(Summary)
Inorganic phosphate (Pi) binders are the only pharmacologic treatment approved for hyperphosphatemia. However, Pi binders induce the expression of intestinal Pi transporters and have limited effects on the inhibition of Pi transport. EOS789, a novel pan-Pi transporter inhibitor, reportedly has potent efficacy in treating hyperphosphatemia. We investigated the properties of EOS789 with comparison to a conventional Pi binder. Protein and mRNA expression levels of Pi transporters were measured in intestinal and kidney tissues from male Wistar rats fed diets supplemented with EOS789 or lanthanum carbonate (LC). 32Pi permeability was measured in intestinal tissues from normal rats using a chamber. Increased protein levels of NaPi-2b, an intestinal Pi transporter, and luminal Pi removal were observed in rats treated with LC but not in rats treated with EOS789. EOS789 but not LC suppressed intestinal protein levels of the Pi transporter Pit-1 and sodium/hydrogen exchanger isoform 3. 32Pi flux experiments using small intestine tissues from rats demonstrated that EOS789 may affect transcellular Pi transport in addition to paracellular Pi transport. EOS789 has differing regulatory effects on Pi metabolism compared to LC. The properties of EOS789 may compensate for the limitations of LC therapy. The combined or selective use of EOS789 and conventional Pi binders may allow tighter control of hyperphosphatemia. J. Med. Invest. 70 : 260-270, February, 2023.
(Keyword)
Rats / Male / Animals / Phosphate Transport Proteins / Rats, Wistar / Hyperphosphatemia / Intestinal Absorption / Phosphates
Sumire Sasaki, Yuji Shiozaki, Ai Hanazaki, Megumi Koike, Kazuya Tanifuji, Minori Uga, Kota Kawahara, Ichiro Kaneko, Yasuharu Kawamoto, Pattama Wiriyasermkul, Tomoka Hasegawa, Norio Amizuka, Ken-ichi Miyamoto, Shushi Nagamori, Yoshikatsu Kanai and Hiroko Segawa : Tmem174, a regulator of phosphate transporter prevents hyperphosphatemia., Scientific Reports, 12, 1, 6353, 2022.
(Summary)
Renal type II sodium-dependent inorganic phosphate (Pi) transporters NaPi2a and NaPi2c cooperate with other organs to strictly regulate the plasma Pi concentration. A high Pi load induces expression and secretion of the phosphaturic hormones parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23) that enhance urinary Pi excretion and prevent the onset of hyperphosphatemia. How FGF23 secretion from bone is increased by a high Pi load and the setpoint of the plasma Pi concentration, however, are unclear. Here, we investigated the role of Transmembrane protein 174 (Tmem174) and observed evidence for gene co-expression networks in NaPi2a and NaPi2c function. Tmem174 is localized in the renal proximal tubules and interacts with NaPi2a, but not NaPi2c. In Tmem174-knockout (KO) mice, the serum FGF23 concentration was markedly increased but increased Pi excretion and hypophosphatemia were not observed. In addition, Tmem174-KO mice exhibit reduced NaPi2a responsiveness to FGF23 and PTH administration. Furthermore, a dietary Pi load causes marked hyperphosphatemia and abnormal NaPi2a regulation in Tmem174-KO mice. Thus, Tmem174 is thought to be associated with FGF23 induction in bones and the regulation of NaPi2a to prevent an increase in the plasma Pi concentration due to a high Pi load and kidney injury.
Sumire Sasaki, Megumi Koike, Kazuya Tanifuji, Minori Uga, Kota Kawahara, Aoi Komiya, Mizuki Miura, Yamato Harada, Yuki Hamaguchi, Shohei Sasaki, Yuji Shiozaki, Ichiro Kaneko, Ken-ichi Miyamoto and Hiroko Segawa : Dietary polyphosphate has a greater effect on renal damage and FGF23 secretion than dietary monophosphate, The Journal of Medical Investigation : JMI, 69, 3, 173-179, 2022.
(Summary)
Phosphate (Pi)-containing food additives are used in several forms. Polyphosphate (PPi) salt has more harmful effects than monophosphate (MPi) salt on bone physiology and renal function. This study aimed to analyze the levels of parathyroid hormone PTH and fibroblast growth factor 23 (FGF23) and the expression of renal / intestinal Pi transport-related molecules in mice fed with an MPi or PPi diet. There were no significant differences in plasma Pi concentration and fecal Pi excretion levels between mice fed with the high-MPi and PPi diet. However, more severe tubular dilatation, interstitial fibrosis, and calcification were observed in the kidneys of mice fed with the high PPi diet versus the MPi diet. Furthermore, there was a significant increase in serum FGF23 levels and a decrease in renal phosphate transporter protein expression in mice fed with the PPi diet versus the MPi diet. Furthermore, the high MPi diet was associated with significantly suppressed expression and activity of intestinal alkaline phosphatase protein. In summary, PPi has a more severe effect on renal damage than MPi, as well as induces more FGF23 secretion. Excess FGF23 may be more involved in inflammation, fibrosis, and calcification in the kidney. J. Med. Invest. 69 : 173-179, August, 2022.
Yuji Shiozaki, Shinobu Miyazaki-Anzai, Kayo Okamura, L. Audrey Keenan, Masashi Masuda and Makoto Miyazaki : GPAT4-Generated Saturated LPAs Induce Lipotoxicity through Inhibition of Autophagy by Abnormal Formation of Omegasomes, iScience, 23, 5, 101105, 2020.
(Summary)
Excessive levels of saturated fatty acids are toxic to vascular smooth muscle cells (VSMCs). We previously reported that mice lacking VSMC-stearoyl-CoA desaturase (SCD), a major enzyme catalyzing the detoxification of saturated fatty acids, develop severe vascular calcification from the massive accumulation of lipid metabolites containing saturated fatty acids. However, the mechanism by which SCD deficiency causes vascular calcification is not completely understood. Here, we demonstrate that saturated fatty acids significantly inhibit autophagic flux in VSMCs, contributing to vascular calcification and apoptosis. Mechanistically, saturated fatty acids are accumulated as saturated lysophosphatidic acids (LPAs) (i.e. 1-stearoyl-LPA) possibly synthesized through the reaction of GPAT4 at the contact site between omegasomes and the MAM. The accumulation of saturated LPAs at the contact site causes abnormal formation of omegasomes, resulting in accumulation of autophagosomal precursor isolation membranes, leading to inhibition of autophagic flux. Thus, saturated LPAs are major metabolites mediating autophagy inhibition and vascular calcification.
Ai Hanazaki, Kayo Ikuta, Shohei Sasaki, Sumire Sasaki, Megumi Koike, Kazuya Tanifuji, Yuki Arima, Ichiro Kaneko, Yuji Shiozaki, Sawako Tatsumi, Tomoka Hasegawa, Norio Amizuka, Ken-ichi Miyamoto and Hiroko Segawa : Role of sodium-dependent Pi transporter/Npt2c on Pi homeostasis in klotho knockout mice different properties between juvenile and adult stages., Physiological Reports, 8, 3, e14324, 2020.
(Summary)
SLC34A3/NPT2c/NaPi-2c/Npt2c is a growth-related NaPi cotransporter that mediates the uptake of renal sodium-dependent phosphate (Pi). Mutation of human NPT2c causes hereditary hypophosphatemic rickets with hypercalciuria. Mice with Npt2c knockout, however, exhibit normal Pi metabolism. To investigate the role of Npt2c in Pi homeostasis, we generated -klotho /Npt2c (KL2cDKO) mice and analyzed Pi homeostasis. -Klotho (KLKO) mice exhibit hyperphosphatemia and markedly increased kidney Npt2c protein levels. Genetic disruption of Npt2c extended the lifespan of KLKO mice similar to that of -Klotho /Npt2a mice. Adult KL2cDKO mice had hyperphosphatemia, but analysis of Pi metabolism revealed significantly decreased intestinal and renal Pi (re)absorption compared with KLKO mice. The 1,25-dihydroxy vitamin D3 concentration was not reduced in KL2cDKO mice compared with that in KLKO mice. The KL2cDKO mice had less severe soft tissue and vascular calcification compared with KLKO mice. Juvenile KL2cDKO mice had significantly reduced plasma Pi levels, but Pi metabolism was not changed. In Npt2cKO mice, plasma Pi levels began to decrease around the age of 15 days and significant hypophosphatemia developed within 21 days. The findings of the present study suggest that Npt2c contributes to regulating plasma Pi levels in the juvenile stage and affects Pi retention in the soft and vascular tissues in KLKO mice.
Shohei Kohno, Yuji Shiozaki, L. Audrey Keenan, Shinobu Miyazaki-Anzai and Makoto Miyazaki : An N-terminal-truncated isoform of FAM134B (FAM134B-2) regulates starvation-induced hepatic selective ER-phagy, Life Science Alliance, 2, 3, 2019.
(Summary)
Autophagy is a conserved system that adapts to nutrient starvation, after which proteins and organelles are degraded to recycle amino acids in response to starvation. Recently, the ER was added to the list of targets of autophagic degradation. Autophagic degradation pathways of bulk ER and the specific proteins sorted through the ER are considered key mechanisms in maintaining ER homeostasis. Four ER-resident proteins (FAM134B, CCPG1, SEC62, and RTN3) have been identified as ER-resident cargo receptors, which contain LC3-interacting regions. In this study, we identified an N-terminal-truncated isoform of FAM134B (FAM134B-2) that contributes to starvation-induced ER-related autophagy. Hepatic FAM134B-2 but not full-length FAM134B (FAM134B-1) is expressed in a fed state. Starvation drastically induces FAM134B-2 but no other ER-resident cargo receptors through transcriptional activation by C/EBPβ. C/EBPβ overexpression increases FAM134B-2 recruitment into autophagosomes and lysosomal degradation. FAM134B-2 regulates lysosomal degradation of ER-retained secretory proteins such as ApoCIII. This study demonstrates that the C/EBPβ-FAM134B-2 axis regulates starvation-induced selective ER-phagy.
Toru Fujii, Hiroko Segawa, Ai Hanazaki, Shiori Nishiguchi, Sakura Minoshima, Akiko Ohi, Rieko Tominaga, Sumire Sasaki, Kazuya Tanifuji, Megumi Koike, Yuki Arima, Yuji Shiozaki, Ichiro Kaneko, Mikiko Ito, Sawako Tatsumi and Ken-ichi Miyamoto : Role of the putative PKC phosphorylation sites of the type IIc sodium-dependent phosphate transporter in parathyroid hormone regulation., Clinical and Experimental Nephrology, 23, 7, 898-907, 2019.
(Summary)
Injection of parathyroid hormone (PTH) rapidly stimulates renal Pi excretion, in part by downregulating NaPi-IIa (Npt2a/SLC34A1) and NaPi-IIc (Npt2c/SLC34A3) transporters. The mechanisms underlying the effects of PTH on NaPi-IIc are not fully elucidated. We analyzed the effect of PTH on inorganic phosphate (Pi) reabsorption in Npt2a-/- mice to eliminate the influence of Npt2a on renal Pi reabsorption. In opossum kidney (OK) cells and Xenopus oocytes, we investigated the effect of NaPi-IIc transporter phosphorylation. Studies of mice with mutations of NaPi-IIc protein in which serine and threonine were replaced with either alanine (A), which prevents phosphorylation, or aspartic acid (D), which mimics the charged state of phosphorylated NaPi-IIc, were also performed to evaluate the involvement of phosphorylation in the regulation of transport function. The Npt2a-/- experiments showed that PTH administration rapidly inactivated NaPi-IIc function in the apical membrane of proximal tubular cells. Analysis of mutant proteins (S71, S138, T151, S174, T583) at putative protein kinase C sites, revealed that S138 markedly suppressed the function and cellular expression of mouse NaPi-IIc in Xenopus oocytes and OK cells. In addition, 138D had a short half-life compared with wild-type protein. The present study suggests that acute regulation of NaPi-IIc protein by PTH is involved in the inactivation of Na+-dependent Pi cotransporter activity and that phosphorylation of the transporter is involved in the rapid modification.
(Keyword)
Animals / Cell Line / Female / Kidney Tubules, Proximal / Male / Mice, Knockout / Opossums / Parathyroid Hormone / Peptide Fragments / Phosphates / Phosphorylation / Protein Kinase C / Protein Processing, Post-Translational / Protein Stability / Renal Reabsorption / Sodium-Phosphate Cotransporter Proteins, Type IIa / Sodium-Phosphate Cotransporter Proteins, Type IIc / Time Factors / Xenopus
Kayo Ikuta, Hiroko Segawa, Ai Hanazaki, Toru Fujii, Ichiro Kaneko, Yuji Shiozaki, Sawako Tatsumi, Yasuko Ishikawa and Ken-ichi Miyamoto : Systemic network for dietary inorganic phosphate adaptation among three organs., Pflügers Archiv : European Journal of Physiology, 471, 1, 123-136, 2018.
(Summary)
Inorganic phosphate (Pi) secretion from the salivary glands and dietary Pi are key Pi sources. The regulatory mechanisms of Pi homeostasis in the salivary glands are unknown. We investigated how salivary Pi concentrations are regulated by dietary Pi in mouse models. Dietary manipulation significantly changed the levels of Npt2b protein in the salivary gland ductal cells. In addition, rapid feeding on a high-Pi diet increased the saliva Pi concentrations and led to rapid endocytosis of Npt2b in the apical membranes of the duct cells. Global Npt2b± mice exhibited increased salivary Pi concentrations and intestine-specific deletion of Npt2b after high Pi loading increased the salivary Pi concentrations. These findings indicate that Npt2b levels in the salivary glands affect the salivary Pi concentration and are regulated by dietary Pi. Intestinal Npt2b levels might also affect salivary Pi concentrations as well as renal Pi excretion. These findings suggest Pi is endogenously recycled by salivary Pi secretion, intestinal Pi absorption, and renal Pi excretion.
Toru Fujii, Yuji Shiozaki, Hiroko Segawa, Shiori Nishiguchi, Ai Hanazaki, Miwa Noguchi, Ruri Kirino, Sumire Sasaki, Kazuya Tanifuji, Megumi Koike, Mizuki Yokoyama, Yuki Arima, Ichiro Kaneko, Sawako Tatsumi, Mikiko Ito and Ken-ichi Miyamoto : Analysis of opossum kidney NaPi-IIc sodium-dependent phosphate transporter to understand Pi handling in human kidney., Clinical and Experimental Nephrology, 23, 3, 313-324, 2018.
(Summary)
The role of Na+-dependent inorganic phosphate (Pi) transporters in the human kidney is not fully clarified. Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is caused by loss-of-function mutations in the IIc Na+-dependent Pi transporter (NPT2c/Npt2c/NaPi-IIc) gene. Another Na+-dependent type II transporter, (NPT2A/Npt2a/NaPi-IIa), is also important for renal Pi reabsorption in humans. In mice, Npt2c deletion does not lead to hypophosphatemia and rickets because Npt2a compensates for the impaired Pi reabsorption. To clarify the differences between mouse and human, we investigated the relation between NaPi-IIa and NaPi-IIc functions in opossum kidney (OK) cells. We cloned NaPi-IIc from OK cells and created opossum NaPi-IIc (oNaPi-IIc) antibodies. We used oNaPi-IIc small interference (si)RNA and investigated the role of NaPi-IIc in Pi transport in OK cells. We cloned opossum kidney NaPi-IIc cDNAs encoding 622 amino acid proteins (variant1) and examined their pH- and sodium-dependency. The antibodies reacted specifically with 75-kDa and 150-kDa protein bands, and the siRNA of NaPi-IIc markedly suppressed endogenous oNaPi-IIc in OK cells. Treatment with siRNA significantly suppressed the expression of NaPi-4 (NaPi-IIa) protein and mRNA. oNaPi-IIc siRNA also suppressed Na+/H+ exchanger regulatory factor 1 expression in OK cells. These findings suggest that NaPi-IIc is important for the expression of NaPi-IIa (NaPi-4) protein in OK cells. Suppression of Npt2c may downregulate Npt2a function in HHRH patients.
Shohei Sasaki, Hiroko Segawa, Ai Hanazaki, Ruri Kirino, Toru Fujii, Kayo Ikuta, Miwa Noguchi, Sumire Sasaki, Megumi Koike, Kazuya Tanifuji, Yuji Shiozaki, Ichiro Kaneko, Sawako Tatsumi, Takaaki Shimohata, Yoshichika Kawai, Sonoko Narisawa, Luis José Millán and Ken-ichi Miyamoto : A Role of Intestinal Alkaline Phosphatase 3 (Akp3) in Inorganic Phosphate Homeostasis., Kidney & Blood Pressure Research, 43, 5, 1409-1424, 2018.
(Summary)
Hyperphosphatemia is a serious complication of late-stage chronic kidney disease (CKD). Intestinal inorganic phosphate (Pi) handling plays an important role in Pi homeostasis in CKD. We investigated whether intestinal alkaline phosphatase 3 (Akp3), the enzyme that hydrolyzes dietary Pi compounds, is a target for the treatment of hyperphosphatemia in CKD. We investigated Pi homeostasis in Akp3 knockout mice (Akp3-/-). We also studied the progression of renal failure in an Akp3-/- mouse adenine treated renal failure model. Plasma, fecal, and urinary Pi and Ca concentration were measured with commercially available kit, and plasma fibroblast growth factor 23, parathyroid hormone, and 1,25(OH)2D3 concentration were measured with ELISA. Brush border membrane vesicles were prepared from mouse intestine using the Ca2+ precipitation method and used for Pi transport activity and alkaline phosphatase activity. In vivo intestinal Pi absorption was measured with oral 32P administration. Akp3-/- mice exhibited reduced intestinal type II sodium-dependent Pi transporter (Npt2b) protein levels and Na-dependent Pi co-transport activity. In addition, plasma active vitamin D levels were significantly increased in Akp3-/- mice compared with wild-type animals. In the adenine-induced renal failure model, Akp3 gene deletion suppressed hyperphosphatemia. The present findings indicate that intestinal Akp3 deletion affects Na+-dependent Pi transport in the small intestine. In the adenine-induced renal failure model, Akp3 is predicted to be a factor contributing to suppression of the plasma Pi concentration.
Yuji Shiozaki, Kayo Okamura, Shohei Kohno, L. Audrey Keenan, Kristina Williams, Xiaoyun Zhao, S. Wallace Chick, Shinobu Miyazaki-Anzai and Makoto Miyazaki : The CDK9 - cyclin T1 complex mediates saturated fatty acid-induced vascular calcification by inducing expression of the transcription factor CHOP, The Journal of Biological Chemistry, 293, 44, 17008-17020, 2018.
(Summary)
Vascular calcification (or mineralization) is a common complication of chronic kidney disease (CKD) and is closely associated with increased mortality and morbidity rates. We recently reported that activation of the activating transcription factor 4 (ATF4) pathway through the saturated fatty acid (SFA)-induced endoplasmic reticulum (ER) stress response plays a causative role in CKD-associated vascular calcification. Here, using mouse models of CKD, we 1) studied the contribution of the proapoptotic transcription factor CCAAT enhancer-binding protein homologous protein (CHOP) to CKD-dependent medial calcification, and 2) we identified an additional regulator of ER stress-mediated CHOP expression. Transgenic mice having smooth muscle cell (SMC)-specific CHOP expression developed severe vascular apoptosis and medial calcification under CKD. Screening of a protein kinase inhibitor library identified 16 compounds, including seven cyclin-dependent kinase (CDK) inhibitors, that significantly suppressed CHOP induction during ER stress. Moreover, selective CDK9 inhibitors and CRISPR/Cas9-mediated CDK9 reduction blocked SFA-mediated induction of CHOP expression, whereas inhibitors of other CDK isoforms did not. Cyclin T1 knockout inhibited SFA-mediated induction of CHOP and mineralization, whereas deletion of cyclin T2 and cyclin K promoted CHOP expression levels and mineralization. Of note, the CDK9-cyclin T1 complex directly phosphorylated and activated ATF4. These results demonstrate that the CDK9-cyclin T1 and CDK9-cyclin T2/K complexes have opposing roles in CHOP expression and CKD-induced vascular calcification. They further reveal that the CDK9-cyclin T1 complex mediates vascular calcification through CHOP induction and phosphorylation-mediated ATF4 activation.
Shinobu Miyazaki-Anzai, Masashi Masuda, Shohei Kohno, Moshe Levi, Yuji Shiozaki, L. Audrey Keenan and Makoto Miyazaki : Simultaneous inhibition of FXR and TGR5 exacerbates atherosclerotic formation, Journal of Lipid Research, 59, 9, 1709-1713, 2018.
(Summary)
Simultaneous activation of bile acid receptors farnesoid X receptor (FXR) and G protein-coupled bile acid receptor 1 (TGR5) by INT-767 significantly reduces atherosclerotic formation. In this study, we investigated the effect of simultaneous inactivation of these bile acid receptors in atherosclerosis and which bile acid receptor mediates the anti-atherogenic effect of INT-767. To investigate the role of simultaneous inactivation of FXR and TGR5 in vivo, we generated LDL receptor knockout (LDLR) KO mice with FXR and TGR5 dual deficiency, which exhibited severe atherosclerosis and aortic inflammation through nuclear factor κΒ activation. The lipid-lowering effects of INT-767 were completely blocked by FXR single deficiency but not TGR5 single deficiency. INT-767 was able to block atherosclerotic formation and decrease levels of aortic cytokines and chemokines in LDLR KO mice under either FXR or TGR5 single deficiency. Dual deficiency of FXR and TGR5 completely blocked the anti-atherogenic and anti-inflammatory effects of INT-767 in LDLR KO mice. We demonstrated that 1) FXR and TGR5 dual deficiency exacerbated the development of atherosclerosis and 2) the anti-atherogenic effect of INT-767 requires the anti-inflammatory effect but not the lipid-lowering effect through the simultaneous activation of FXR and TGR5. Our results indicate that dual activation of FXR and TGR5 is a promising strategy for treating atherosclerosis.
(Keyword)
Animals / atherosclerosis / Bile Acids and Salts / Gene Knockout Techniques / male / Mice / Mice, Inbred C57BL / Receptors, Cytoplasmic and Nuclear / Receptors, G-Protein-Coupled / Receptors, LDL
Masashi Masuda, Shinobu Miyazaki-Anzai, Audrey L. Keenan, Yuji Shiozaki, Kayo Okamura, Wallace S. Chick, Kristina Williams, Xiaoyun Zhao, Shaikh Mizanoor Rahman, Yin Tintut, Christopher M. Adams and Makoto Miyazaki : Activating transcription factor-4 promotes mineralization in vascular smooth muscle cells., JCI Insight, 1, 18, e88646, 2016.
(Summary)
Emerging evidence indicates that upregulation of the ER stress-induced pro-osteogenic transcription factor ATF4 plays an important role in vascular calcification, a common complication in patients with aging, diabetes, and chronic kidney disease (CKD). In this study, we demonstrated the pathophysiological role of ATF4 in vascular calcification using global Atf4 KO, smooth muscle cell-specific (SMC-specific) Atf4 KO, and transgenic (TG) mouse models. Reduced expression of ATF4 in global ATF4-haplodeficient and SMC-specific Atf4 KO mice reduced medial and atherosclerotic calcification under normal kidney and CKD conditions. In contrast, increased expression of ATF4 in SMC-specific Atf4 TG mice caused severe medial and atherosclerotic calcification. We further demonstrated that ATF4 transcriptionally upregulates the expression of type III sodium-dependent phosphate cotransporters (PiT1 and PiT2) by interacting with C/EBP . These results demonstrate that the ER stress effector ATF4 plays a critical role in the pathogenesis of vascular calcification through increased phosphate uptake in vascular SMCs.
Sawako Tatsumi, A Miyagawa, Ichiro Kaneko, Yuji Shiozaki, Hiroko Segawa and Ken-ichi Miyamoto : Regulation of renal phosphate handling: inter-organ communication in health and disease., Journal of Bone and Mineral Metabolism, 34, 1, 1-10, 2016.
(Summary)
In this review, we focus on the interconnection of inorganic phosphate (Pi) homeostasis in the network of the bone-kidney, parathyroid-kidney, intestine-kidney, and liver-kidney axes. Such a network of organ communication is important for body Pi homeostasis. Normalization of serum Pi levels is a clinical target in patients with chronic kidney disease (CKD). Particularly, disorders of the fibroblast growth factor 23/klotho system are observed in early CKD. Identification of phosphaturic factors from the intestine and liver may enhance our understanding of body Pi homeostasis and Pi metabolism disturbances in CKD patients.
Yuji Shiozaki, Hiroko Segawa, Saori Ohnishi, Akiko Ohi, Mikiko Ito, Ichiro Kaneko, Shinsuke Kido, Sawako Tatsumi and Ken-ichi Miyamoto : Relationship between sodium-dependent phosphate transporter (NaPi-IIc) function and cellular vacuole formation in opossum kidney cells., The Journal of Medical Investigation : JMI, 62, 3-4, 209-218, 2015.
(Summary)
NaPi-IIc/SLC34A3 is a sodium-dependent inorganic phosphate (Pi) transporter in the renal proximal tubules and its mutations cause hereditary hypophosphatemic rickets with hypercalciuria (HHRH). In the present study, we created a specific antibody for opossum SLC34A3, NaPi-IIc (oNaPi-IIc), and analyzed its localization and regulation in opossum kidney cells (a tissue culture model of proximal tubular cells). Immunoreactive oNaPi-IIc protein levels increased during the proliferative phase and decreased during differentiation. Moreover, stimulating cell growth upregulated oNaPi-IIc protein levels, whereas suppressing cell proliferation downregulated oNaPi-IIc protein levels. Immunocytochemistry revealed that endogenous and exogenous oNaPi-IIc proteins localized at the protrusion of the plasma membrane, which is a phosphatidylinositol 4,5-bisphosphate (PIP2) rich-membrane, and at the intracellular vacuolar membrane. Exogenous NaPi-IIc also induced cellular vacuoles and localized in the plasma membrane. The ability to form vacuoles is specific to electroneutral NaPi-IIc, and not electrogenic NaPi-IIa or NaPi-IIb. In addition, mutations of NaPi-IIc (S138F and R468W) in HHRH did not cause cellular PIP2-rich vacuoles. In conclusion, our data anticipate that NaPi-IIc may regulate PIP2 production at the plasma membrane and cellular vesicle formation.
Kengo Nomura, Sawako Tatsumi, Atsumi Miyagawa, Yuji Shiozaki, Shohei Sasaki, Ichiro Kaneko, Mikiko Ito, Shinsuke Kido, Hiroko Segawa, Mitsue Sano, Tsutomu Fukuwatari, Katsumi Shibata and Ken-ichi Miyamoto : Hepatectomy-related hypophosphatemia: a novel phosphaturic factor in the liver-kidney axis., Journal of the American Society of Nephrology, 25, 4, 761-772, 2014.
(Summary)
Marked hypophosphatemia is common after major hepatic resection, but the pathophysiologic mechanism remains unknown. We used a partial hepatectomy (PH) rat model to investigate the molecular basis of hypophosphatemia. PH rats exhibited hypophosphatemia and hyperphosphaturia. In renal and intestinal brush-border membrane vesicles isolated from PH rats, Na(+)-dependent phosphate (Pi) uptake decreased by 50%-60%. PH rats also exhibited significantly decreased levels of renal and intestinal Na(+)-dependent Pi transporter proteins (NaPi-IIa [NaPi-4], NaPi-IIb, and NaPi-IIc). Parathyroid hormone was elevated at 6 hours after PH. Hyperphosphaturia persisted, however, even after thyroparathyroidectomy in PH rats. Moreover, DNA microarray data revealed elevated levels of nicotinamide phosphoribosyltransferase (Nampt) mRNA in the kidney after PH, and Nampt protein levels and total NAD concentration increased significantly in the proximal tubules. PH rats also exhibited markedly increased levels of the Nampt substrate, urinary nicotinamide (NAM), and NAM catabolites. In vitro analyses using opossum kidney cells revealed that NAM alone did not affect endogenous NaPi-4 levels. However, in cells overexpressing Nampt, the addition of NAM led to a marked decrease in cell surface expression of NaPi-4 that was blocked by treatment with FK866, a specific Nampt inhibitor. Furthermore, FK866-treated mice showed elevated renal Pi reabsorption and hypophosphaturia. These findings indicate that hepatectomy-induced hypophosphatemia is due to abnormal NAM metabolism, including Nampt activation in renal proximal tubular cells.
Sakiko Haito-Sugino, Mikiko Ito, Akiko Ohi, Yuji Shiozaki, Natsumi Kangawa, Takashi Nishiyama, Fumito Aranami, Shohei Sasaki, Ayaka Mori, Shinsuke Kido, Sawako Tatsumi, Hiroko Segawa and Ken-ichi Miyamoto : Processing and stability of type IIc sodium-dependent phosphate cotransporter mutations in patients with hereditary hypophosphatemic rickets with hypercalciuria., American Journal of Physiology, Cell Physiology, 302, 9, C1316-30, 2011.
(Summary)
Mutations in the apically located Na(+)-dependent phosphate (NaPi) cotransporter, SLC34A3 (NaPi-IIc), are a cause of hereditary hypophosphatemic rickets with hypercalciuria (HHRH). We have characterized the impact of several HHRH mutations on the processing and stability of human NaPi-IIc. Mutations S138F, G196R, R468W, R564C, and c.228delC in human NaPi-IIc significantly decreased the levels of NaPi cotransport activities in Xenopus oocytes. In S138F and R564C mutant proteins, this reduction is a result of a decrease in the V(max) for P(i), but not the K(m). G196R, R468W, and c.228delC mutants were not localized to oocyte membranes. In opossum kidney (OK) cells, cell surface labeling, microscopic confocal imaging, and pulse-chase experiments showed that G196R and R468W mutations resulted in an absence of cell surface expression owing to endoplasmic reticulum (ER) retention. G196R and R468W mutants could be partially stabilized by low temperature. In blue native-polyacrylamide gel electrophoresis analysis, G196R and R468W mutants were either denatured or present in an aggregation complex. In contrast, S138F and R564C mutants were trafficked to the cell surface, but more rapidly degraded than WT protein. The c.228delC mutant did not affect endogenous NaPi uptake in OK cells. Thus, G196R and R468W mutations cause ER retention, while S138F and R564C mutations stimulate degradation of human NaPi-IIc in renal epithelial cells. Together, these data suggest that the NaPi-IIc mutants in HHRH show defective processing and stability.
金子 一郎, 宇賀 穂, Yuji Shiozaki, Ken-ichi Miyamoto and Hiroko Segawa : The physiological function of vitamin D which maintains phosphate homeostasis, Vitamins, 95, 56, 280-285, 2021.
(Summary)
Inorganic phosphate (Pi) plays essential roles in many biological processes. Blood Pi level should be maintained at a constant range because hypophosphatemia causes rickets/osteomalacia, whereas hyperphosphatemia causes cardiovascular events in chronic kidney disease (CKD) or hemodialysis patients. As a phosphaturic hormone, parathyroid hormone (PTH) or fibroblast growth factor (FGF) 23 strongly suppresses Pi reabsorption. 1,25-Dihydroxyvitamin D[1,25(OH) <sub>2</sub>D] up-regulates Pi absorption in intestines, and regulates urinary Pi excretion through PTH or FGF23. <br> Activated FGF23 signal leads urinary Pi excretion, and hypophosphatemic rickets (FGF23-related hypophosphatemic rickets). Rickets characterized as vitamin D deficiency also associates with impaired renal Pi reabsorption in juvenile stage in addition to low Pi absorption in intestines. Now, burosumab (FGF23 neutralizing antibody) has a beneficent effect on FGF23-related hypophosphatemic rickets/osteomalacia. The antibody increases renal Pi reabsorption and 1,25(OH) <sub>2</sub>D production. On the other hand, treatment with vitamin D alone in an animal model of X-linked hypophosphatemic rickets can potentially increase renal Pi reabsorption, despite of excess FGF23, and improve hypophosphatemia and bone phenotypes. <br> CKD patients often accompany with mineral bone disorder (CKD-MBD) such as hyperparathyroidism, cardiovascular calcification, and osteodystrophy. Prevention and treatment for hyperphosphatemia or hypovitaminosis D would improve their QOL or mortality. <br> Thus, renal Pi reabsorption is important for bone and mineral homeostasis. We focus on the metabolism of Pi and vitamin D to understand the molecular mechanism for the onset and prevention of diseases associated with Pi and vitamin D.
Aoi Komiya, Ayami Higashi, Megumi Koike, Yuji Shiozaki and Hiroko Segawa : リン代謝調節機構―腸管リン酸輸送の理解ー, Journal of Japanese Society of Nutrition and Food Science, 77, 4, 247-253, Apr. 2024.
Hiroko Segawa, Yuji Shiozaki, Ichiro Kaneko and Ken-ichi Miyamoto : The Role of Sodium-Dependent Phosphate Transporter in Phosphate Homeostasis., Journal of Nutritional Science and Vitaminology, 61 Suppl, S119-121, 2015.
(Summary)
Inorganic phosphate (Pi) is an essential compound for several biologic functions. Pi levels outside the normal range, however, contribute to several pathological processes. Hypophosphatemia leads to bone abnormalities, such as rickets/osteomalacia. Hyperphosphatemia contributes to vascular calcification in patients with chronic kidney disease and hemodialysis patients and is independently associated with cardiac mortality.Pi homeostasis is regulated by the coordinated function of renal and intestinal sodium-dependent phosphate (NaPi) transporters with dietary Pi, parathyroid hormone, 1,25-dihydroxyvitamin D3, and fibroblast growth factor 23. The type II NaPi transporter/SLC34 family, with three members identified to date, is mainly responsible for Pi homeostasis in the body. SLC34A1 and SCL34A3 are predominantly expressed in the kidney, whereas SLC34A2 is expressed in the small intestine. The role of each SLC34 in the body was recently established by studies of gene-targeted mice. Mutation of SLC34A1 causes Fanconi syndrome and mutation of SLC34A3 causes autosomal recessive hereditary hypophosphatemic rickets with hypercalciuria. SLC34A2 is thought to be a major intestinal NaPi transporter and mutation of SLC34A2 causes pulmonary alveolar microlithiasis. A detailed understanding of Pi regulation in the body is important toward maintaining health.
Hiroko Segawa, Yuji Shiozaki, Sakura Minoshima and Ken-ichi Miyamoto : [Updates on rickets and osteomalacia: the role of NaPi-2c/SLC34A3 and hypophosphataemic rickets]., Clinical Calcium, 23, 10, 1445-1450, Oct. 2013.
(Summary)
Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) , an autosomal recessive disorder first identified in a large Bedouin tribe, is characterized by hypophosphatemia secondary to renal inorganic phosphate (Pi) wasting, resulting in increased serum1,25-dihydroxyvitamin D3 concentrations with associated intestinal calcium hyperabsorption, hypercalciuria, rickets, and osteomalacia. Recent studies identified several mutations in the NaPi-2c/NPT2c transporter gene (SLC34A3) as the cause of HHRH. The fact that HHRH is caused by NaPi-2c loss-of-function mutations is compatible with the HHRH phenotype and the prevailing view of renal Pi regulation. The NaPi-2c mutants in HHRH show defective processing and stability.
Shinsuke Kido, Kengo Nomura, Shohei Sasaki, Yuji Shiozaki, Hiroko Segawa and Sawako Tatsumi : [Information about phosphorus additives and nutritional counseling]., Clinical Calcium, 22, 10, 1583-1591, Oct. 2012.
(Summary)
Hyperphosphatemia is a common disorder in patients with chronic kidney disease (CKD) , and may result in hyperparathyroidism and renal osteodystrophy. Hyperphosphatemia also may contribute to deterioration vascular calcification and increase mortality. Hence, correction and prevention of hyperphosphatemia is a main component of the management of CKD. This goal is usually approached both by administering phosphorus binders and by restricting dietary phosphorus (P) intake. Dietary intake of phosphorus (P) is derived largely from foods with high protein content or food additives and is an important determinant of P balance in patient with CKD. Food additives (PO4) can dramatically increase the amount of P consumed in the daily diet, especially because P is more readily absorbed in its inorganic form. In addition, information about the P content and type in prepared foods is often unavailable or misleading. Therefore, during dietary counseling of patients with CKD, we recommended that they consider both the absolute dietary P content and the P-to-protein ratio of foods and meals including food additives.
Phosphaturia has been documented following cadmium (Cd) exposure in both humans and experimental animals. Fibroblast growth factor 23 (FGF23) serves as an essential phosphate homeostasis pathway in the bone-kidney axis. In the present study, we investigated the effects of Cd on phosphate (Pi) homeostasis in mice. Following Cd injection into C57BL/6J mice, plasma FGF23 concentration significantly increased. The urinary Pi excretion level was significantly higher in the Cd-injected C57BL/6J mice than in the control group. Plasma Pi concentration decreased only slightly in the Cd-injected mice compared with the control group. No changes were observed in the concentration of the plasma parathyroid hormone and 1,25-dihydroxy vitamin D(3) in both groups of mice. We observed a decrease in phosphate transport activity and also a decrease in the expression level of renal phosphate transporter Npt2c, but not that of Npt2a. Furthermore, we examined the effect of Cd on Npt2c in Npt2a-knockout (KO) mice, which expresses Npt2c as a major NaPi cotransporter. Injecting Cd to Npt2aKO mice induced a significant increase in plasma FGF23 concentration and urinary Pi excretion level. Furthermore, we observed decreases in phosphate transport activity and renal Npt2c expression level in the Cd-injected Npt2a KO mice. The present study suggests that hypophosphatemia induced by Cd may be closely associated with FGF23.
Yuji Shiozaki, UGA Minori, Megumi Koike, KOMIYA Aoi, SHIBAHARA Shion, Ayami Higashi, Ken-ichi Miyamoto and Hiroko Segawa : Identification of functional domains of TMEM174 on PTH-induced endocytosis of NaPi2a, Physiology, Biology and Pathology of Phosphate Gordon Research Conference, Renaissance Tuscany Il Ciocco, Via Giovanni Pascoli, Lucca, Italy, Feb. 2025.
2.
Yuji Shiozaki, Minori Uga, Mizuki Miura, Aoi Komiya, Kazuya Tanifuji, Megumi Koike, Ken-ichi Miyamoto and Hiroko Segawa : Analysis of regulation of Tmem174 expression by Pi concentration and PTH signaling in opossum kidney cells., Physiology, Biology and Pathology of Phosphate Gordon Research Conference, Grand Galvez in Galveston, Texas, United States, Feb. 2023.
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Minori Uga, Ichiro Kaneko, Sumire Sasaki, Megumi Koike, Kazuya Tanifuji, Yuji Shiozaki, Peter W. Jurutka and Hiroko Segawa : The role of intestinal Cytochrome P450 in vitamin D metabolism, 22nd International Congress of Nutrition in Tokyo, Dec. 2022.