Ichiro Kaneko

Six pyridine analogs of (E)-3-(3-(1,2,3,4-tetrahydro-1,1,4,4,6-pentamethylnaphthalen-7-yl)-4-hydroxyphenyl)acrylic acid-or CD3254 (11)-in addition to two novel analogs of 1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)-1H-benzo[d][1,2,3]triazole-5-carboxylic acid (CBt-PMN or 23) were prepared and evaluated for selective retinoid-X-receptor (RXR) agonism alongside bexarotene (1), an FDA-approved drug for cutaneous T-cell lymphoma (CTCL). Treatment with 1 often elicits side-effects by disrupting or provoking other RXR-dependent nuclear receptors and cellular pathways. All analogs were assessed through modeling for their ability to bind RXR and then evaluated in human colon and kidney cells employing an RXR-RXR mammalian-2-hybrid (M2H) system and in an RXRE-controlled transcriptional assay. The EC50 values for these analogs, and their corresponding effectiveness in activating both LXR/LXRE and the Sterol Regulatory Element Binding Protein (SREBP) promoter in comparison to 1, suggests that these compounds likely display a range of therapeutic potential and differential side effect profiles. Several analogs also exhibited reduced retinoic-acid-receptor (RAR) cross-signaling implying that they possess enhanced selectivity towards activation of cellular RXR versus RAR pathways. These results show that modifying potent rexinoids such as CD3254 or partial agonists such as CBt-PMN can result in improved target receptor selectivity and enhanced potency, such as compounds 26, 27 and 28 in this study, compared with approved therapeutics such as compound 1, where these three compounds exhibited similar potency as 1, but 26 and 27 lower RAR and SREBP activation than 1.

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.

The SARS-CoV-2 main protease is an essential molecule for viral replication and is often targeted by medications to treat the infection. In this study, we investigated the possible inhibitory action of endogenous quinones on the enzyme. Recombinant SARS-CoV-2 main protease was exposed to tryptamine-4,5-dione (TD) or quinone from 5-hydroxyindoleacetic acid (Q5HIAA). As a result, the protease activity was considerably decreased in a dose-dependent manner. The IC50 values of the quinones toward the enzyme were approximately 0.28 μM (TD) and 0.49 μM (Q5HIAA). Blot analyses using specific antibodies to quinone-modified proteins revealed that quinones were adducted to the enzyme at concentrations as low as 0.12 μM. Intact mass analyses showed that one or two quinone molecules were covalently adducted onto the main protease. Chymotrypsin-digested main protease analyses revealed that the quinones bind to thiol residues at the enzyme's active site. When TD or Q5HIAA were exposed to cultured cells expressing the viral enzyme, quinone-modified enzyme was identified in the cell lysate, suggesting that even extracellularly generated quinones could react with the viral enzyme expressed in an infected cell. Thus, these endogenous quinones could act as inhibitors of the viral enzyme.

Abstract 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.

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.

Irritable bowel syndrome (IBS) is one of the most common gastrointestinal disorders and affects approximately 4% of the global population. The diagnosis of IBS can be made based on symptoms using the validated Rome criteria and ruling out commonly occurring organic diseases. Although biomarkers exist for "IBS mimickers" such as celiac disease and inflammatory bowel disease (IBD), no such test exists for IBS. DNA microarrays of colonic tissue have been used to identify disease-associated variants in other gastrointestinal (GI) disorders. In this study, our objective was to identify biomarkers and unique gene expression patterns that may define the pathological state of IBS. Mucosal tissue samples were collected from the sigmoid colon of 29 participants (11 IBS and 18 healthy controls). DNA microarray analysis was used to assess gene expression profiling. Extraction and purification of RNA were then performed and used to synthesize cDNA. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) was employed to identify differentially expressed genes in patients diagnosed with IBS compared to healthy, non-IBS patient-derived cDNA. Additional testing probed vitamin D-mediated regulation of select genes associated with serotonergic metabolism. DNA microarray analyses led to the identification of 858 differentially expressed genes that may characterize the IBS pathological state. After screening a series of genes using a combination of gene ontological analysis and RT-qPCR, this spectrum of potential IBS biomarkers was narrowed to 23 genes, some of which are regulated by vitamin D. Seven putative IBS biomarkers, including genes involved in serotonin metabolism, were identified. This work further supports the hypothesis that IBS pathophysiology is evident within the human transcriptome and that vitamin D modulates differential expression of genes in IBS patients. This suggests that IBS pathophysiology may also involve vitamin D deficiency and/or an irregularity in serotonin metabolism.

There is considerable interest in identifying effective and safe drugs for neurodegenerative disorders. Cell culture and animal model work have demonstrated that modulating gene expression through RXR-mediated pathways may mitigate or reverse cognitive decline. However, because RXR is a dimeric partner for several transcription factors, activating off-target transcription is a concern with RXR ligands (rexinoids). This off-target gene modulation leads to unwanted side effects that can include low thyroid function and significant hyperlipidemia. There is a need to develop rexinoids that have binding specificity for subsets of RXR heterodimers, to drive desired gene modulation, but that do not induce spurious effects. Herein, we describe experiments in which we analyze a series of novel and previously reported rexinoids for their ability to modulate specific gene pathways implicated in neurodegenerative disorders employing a U87 cell culture model. We demonstrate that, compared to the FDA-approved rexinoid bexarotene (1), several of these compounds are equally or more effective at stimulating gene expression via LXREs or Nurr1/NBREs and are superior at inducing ApoE and/or tyrosine hydroxylase (TH) gene and protein expression, including analogs 8, 9, 13, 14, 20, 23, and 24, suggesting a possible therapeutic role for these compounds in Alzheimer's or Parkinson's disease (PD). A subset of these potent RXR agonists can synergize with a presumed Nurr1 ligand and antimalarial drug (amodiaquine) to further enhance Nurr1/NBREs-directed transcription. This novel discovery has potential clinical implications for treatment of PD since it suggests that the combination of an RXR agonist and a Nurr1 ligand can significantly enhance RXR-Nurr1 heterodimer activity and drive enhanced therapeutic expression of the TH gene to increase endogenous synthesis of dopamine. These data indicate that is it possible and prudent to develop novel rexinoids for testing of gene expression and side effect profiles for use in potential treatment of neurodegenerative disorders, as individual rexinoids can have markedly different gene expression profiles but similar structures.

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 α-klotho-/- /Npt2c-/- (KL2cDKO) mice and analyzed Pi homeostasis. α-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 α-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.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

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.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

X-linked hypophosphatemia (XLH), the most common form of inheritable rickets, is caused by inactivation of phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) and leads to fibroblast growth factor (FGF) 23-dependent renal inorganic phosphate (Pi) wasting. In the present study, we investigated whether maintaining Pi homeostasis with a potent vitamin D3 analog, eldecalcitol [1α,25-dihydroxy-2β-(3-hydroxypropyloxy) vitamin D3; ED71], could improve hypophosphatemic rickets in a murine model of XLH, the Hyp mouse. Vehicle, ED71, or 1,25-dihydroxyvitamin D was subcutaneously injected five times weekly in wild-type (WT) and Hyp mice for 4 weeks, from 4 to 8 weeks of age. Injection of ED71 into WT mice suppressed the synthesis of renal 1,25-dihydroxyvitamin D and promoted phosphaturic activity. In contrast, administration of ED71 to Hyp mice completely restored renal Pi transport and NaPi-2a protein levels, although the plasma-intact FGF23 levels were further increased. In addition, ED71 markedly increased the levels of the scaffold proteins, renal sodium-hydrogen exchanger regulatory factor 1, and ezrin in the Hyp mouse kidney. Treatment with ED71 increased the body weight and improved hypophosphatemia, the bone volume/total volume, bone mineral content, and growth plate structure in Hyp mice. Thus, ED71 causes FGF23 resistance for phosphate reabsorption and improves rachitic bone phenotypes in Hyp mice. In conclusion, ED71 has opposite effects on phosphate homeostasis in WT and Hyp mice. Analysis of Hyp mice treated with ED71 could result in an additional model for elucidating PHEX abnormalities.

BACKGROUND/AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Members of the fibroblast growth factor (FGF) 19 subfamily, including FGF23, FGF15/19, and FGF21, have a role as endocrine factors which influence the metabolism of inorganic phosphate (Pi) and vitamin D, bile acid, and energy. It has been reported that dietary Pi regulates circulating FGF23. In this study, the short-term effects of dietary Pi restriction on the expression of FGF19 subfamily members in mice were analyzed. An initial analysis confirmed plasma FGF23 levels positively correlated with the amount of dietary Pi. On the other hand, ileal Fgf15 gene expression, but not hepatic Fgf21 gene expression, was up-regulated by dietary Pi restriction. In addition, we observed the increase of plasma 1,25-dihydroxyvitamin D [1,25(OH)2D] levels by dietary Pi restriction, and the up-regulation of ileal Fgf15 mRNA expression by 1,25(OH)2D3 and vitamin D receptor (VDR). Importantly, dietary Pi restriction-induced Fgf15 gene expression was prevented in VDR-knockout mice. Furthermore, diurnal variations of plasma triglyceride concentrations and hepatic mRNA expression of the bile acid synthesis enzyme Cyp7a1 as one of Fgf15 negative target genes was influenced by dietary Pi restriction. These results suggest that dietary Pi restriction up-regulates ileal Fgf15 gene expression through 1,25(OH)2D3 and VDR, and may affect hepatic bile acid homeostasis.