Hiroshi Watanabe

Cancer cachexia is frequently associated with altered pharmacokinetics and increased chemotherapy toxicity due to the downregulation of cytochrome P450 (CYP) enzymes. However, the molecular mechanisms driving this broad metabolic suppression remain poorly understood. This study investigated whether tumor-derived parathyroid hormone-related protein (PTHrP) is associated with, and may contribute to, suppression of multiple CYP families. In a rat cachexia model, protein expression of CYP3A, CYP1A, CYP2C, CYP2D, and CYP2E1 was significantly downregulated in both the liver and small intestine. Consistent with these changes, pharmacokinetic analyses using a CYP substrate cocktail demonstrated markedly increased AUC and reduced clearance for probe drugs. In vitro experiments showed that PTHrP treatment reduced these CYP isoforms in primary rat hepatocytes. In human data sets, analysis of The Cancer Genome Atlas (TCGA) hepatocellular carcinoma (HCC) data set revealed a significant negative correlation between PTHrP and CYP gene expression, together with enrichment of NF-κB-related transcriptional programs. Furthermore, multimodal analysis using single-cell RNA sequencing and spatial transcriptomics demonstrated that PTHrP-high tumor regions exhibit suppressed xenobiotic metabolism. Additionally, in breast cancer liver metastases, high tumor PTHrP expression correlated with reduced CYP expression in surrounding nontumor hepatocytes, consistent with a possible paracrine relationship. Collectively, these results support an association between tumor-derived PTHrP and suppression of drug-metabolizing programs. They further suggest that PTHrP may be one contributing factor, but not definitive proof of a principal suppressor, and should therefore be considered a candidate biomarker requiring further mechanistic and clinical validation.

Human serum albumin (HSA) is the most abundant circulating protein and a central mediator of extracellular redox homeostasis. Its antioxidant function has been attributed primarily to the free thiol at cysteine residue 34 (Cys34), whose progressive oxidation—yielding nonmercaptoalbumin isoforms HNA1 and HNA2—serves as a sensitive biomarker of systemic oxidative stress. Advances in electrospray ionisation time-of-flight mass spectrometry have enabled precise quantification of Cys34 modifications, establishing cysteinylated albumin as a clinically applicable marker in chronic kidney disease, chronic liver disease, diabetes, and ageing-related functional decline. Beyond its role as a biomarker, oxidized albumin is now recognized as an active pathogenic mediator in preclinical models, with emerging evidence implicating ferroptosis as a potential mechanism of renal tubular cell injury, and contributing to cardiovascular risk and sarcopenia. A previously unrecognized dimension of albumin redox biology has also emerged: the presence of endogenous oxidized polysulfide bonds (Ox-PSS; RSS_n-R', n > 1) across multiple intramolecular cysteine bridges. Quantified using the newly developed Elimination Method for Sulfide from Polysulfide, albumin polysulfides are dynamically regulated by oxidative stress and are significantly depleted in chronic kidney disease and early hepatitis, even when conventional Cys34-based markers remain unchanged. Polysulfide depletion impairs both Site II drug-binding capacity and reactive oxygen species scavenging activity, with direct implications for pharmacokinetics and therapeutic efficacy. These findings collectively reframe HSA as a dynamic polysulfide reservoir and extracellular redox regulator, highlighting albumin polysulfide modulation as a compelling emerging therapeutic strategy.

Dysregulation of hepatokines has been observed in patients with metabolic dysfunction-associated steatotic liver disease (MASLD), suggesting their involvement in disease progression. In this study, we aimed to investigate the role of α1-acid glycoprotein (AGP), a hepatokine, in the progression of MASLD and to evaluate its potential therapeutic utility. Reanalysis of hepatic RNA sequencing datasets from patients with fatty liver disease showed that expression of the hepatic AGP gene decreased with disease progression. A high-fat diet (HFD)-induced mouse model of MASLD also showed decreased plasma and hepatic AGP levels. Whole-body AGP-knockout (AGP-KO) mice were fed a HFD for 12 weeks. Compared to wild-type mice, AGP-KO mice fed a HFD displayed further exacerbated hepatic steatosis, obesity, adipose tissue inflammation, and impaired glucose tolerance, suggesting a protective role for endogenous AGP against MASLD. Morphological abnormalities of hepatic mitochondria were observed in AGP-KO mice. Human-derived AGP (hAGP) was isolated and purified from plasma fraction V supernatant derived from human blood donations using ion-exchange column chromatography. hAGP exerted a protective effect against palmitate-induced lipotoxicity in hepatocytes, adipocytes, and macrophages. Exogenous administration of hAGP to MASLD mice attenuated disease progression by reducing hepatic mitochondrial dysfunction and adipose tissue inflammation. In conclusion, decreased endogenous AGP levels may contribute to the disease progression of MASLD. AGP has the potential to serve as a novel therapeutic agent against MASLD by targeting both liver and adipose tissue.

Key Points: Indoxyl sulfate (IS) is involved in the liver-kidney axis by inducing renal ferroptosis by selenium deficiency due to suppression of selenoprotein P (SEPP1) expression in the liver. IS concentrations were negatively associated with selenium concentrations and SEPP1 concentrations in serum from patients on dialysis. We propose the potential therapeutic strategy targeting the IS/SEPP1/glutathione peroxidase 4 pathway for CKD. Background: The relationship between the progression of CKD and trace element deficiencies has attracted considerable attention. However, many aspects of trace element deficiency and the molecular mechanisms of CKD pathology remain unclear. In this study, we hypothesized that uremic toxins are involved in trace element deficiencies, which contribute to the progression of CKD. Methods: Adenine-induced CKD mice were used for in vivo study. Cultured hepatocytes were used for in vitro study. Results: Seventeen trace elements in plasma of CKD mice were measured using inductively coupled plasma mass spectrometry. Among these, selenium (Se) was identified as the trace element most significantly affected by the administration of oral spherical activated carbon (AST-120), an oral spherical activated carbon. CKD mice displayed reduced levels of Se in plasma, which was restored after the administration of AST-120. In vivo and in vitro experiments showed the uremic toxin indoxyl sulfate (IS) decreased expression of the Se transport protein selenoprotein P (SEPP1) in the liver. IS suppressed SEPP1 expression through increased production of reactive oxygen species by the organic anion transporting polypeptide/aryl hydrocarbon receptor/NADPH oxidase pathway. Increased reactive oxygen species led to the downregulation of transcription factors for SEPP1, such as AMP-activated protein kinase/PGC-1α and microRNA-34a/HNF4α. Analysis of serum from patients on hemodialysis also suggested that IS is involved in reducing serum SEPP1 levels and exacerbating Se deficiency. Combination therapy with AST-120 and sodium selenite restored the supply of Se to the kidneys and increased glutathione peroxidase 4 expression, thereby exerting renoprotective effects by suppression of ferroptosis. Conclusions: This study highlights the key role IS plays in Se deficiency and renal ferroptosis by suppressing hepatic SEPP1 expression. The findings suggest potential therapeutic strategies that target IS and Se deficiency for the management of CKD.

Background: CD169⁺ macrophages in lymph nodes (LNs) activate cytotoxic T lymphocytes (CTLs), which play a crucial role in anticancer immunity, through antigen presentation and co-stimulation by CD169. Interferon alpha (IFNα) is capable of inducing the CD169⁺ phenotype of macrophages; however, its clinical applications have been hindered by pharmacokinetic limitations—low LN distribution and an inability to target macrophages. To overcome these issues, this study genetically fused mouse IFNα (mIFNα) with mannosylated mouse serum albumin (Man-MSA), and investigated the antitumor effects of the hybrid protein (Man-MSA-mIFNα) or its add-on effects with programmed death-ligand 1 (PD-L1) blockade. Methods: To confirm the possibility of CD169⁺ macrophage-mediated T cell priming, positional information about individual immune cells in LNs of cancer patients was obtained. Traits of Man-MSA-mIFNα, which was prepared using Pichia pastoris to form the high-mannose structure, were characterized by several physicochemical methods. To evaluate the lymphatic drainage of Man-MSA-mIFNα, radioiodine or Cy5-labeled Man-MSA-mIFNα was subcutaneously administered in mice, and then the radioactivity or fluorescence in LNs was analyzed. CD169-diphtheria toxin (DT) receptor (CD169-DTR) mice in which LN CD169⁺ macrophages can be depleted by DT injection were used to verify whether the antitumor effect of Man-MSA-mIFNα is dependent on LN CD169⁺ macrophages. Results: Multiplex tissue imaging predicted close proximity of CD169⁺ macrophages and T cells and positive correlation between the number of CD169⁺ macrophages and T cells in neighborhoods in LNs of cancer patients. Physicochemical analyses showed that Man-MSA-mIFNα was formed from the fusion of the intact Man-MSA and mIFNα. Man-MSA-mIFNα efficiently induced the CD169⁺ phenotype of macrophages by its high LN distribution and macrophage-targeting capability, and significantly exerted antitumor activity through CD8⁺ T cell activation in the LNs, whereas its antitumor effects were canceled in CD169-DTR mice. Finally, combination therapy with PD-L1 blockade markedly suppressed tumor growth in MB49-bearing mice, which exhibit resistance to PD-L1 blockade monotherapy. Conclusions: The present study successfully designed and developed Man-MSA-mIFNα, which efficiently induces the CD169⁺ phenotype in LN macrophages, contributing to the antitumor immunity. The findings suggest that our novel strategy targeting CD169⁺ macrophages could be a promising immunotherapy for cancer patients who are unresponsive to immune checkpoint inhibitors. Graphical

Key Points: Omega-3 polyunsaturated fatty acids prevent AKI to CKD transition and renal fibrosis. Eicosapentaenoic acid metabolites 18-hydroxyeicosapentaenoic acid, 17,18-epoxyeicosatetraenoic acid, and 17,18-dihydroxyeicosatetraenoic acid have antifibrotic effects. Background: AKI is an established risk factor for developing CKD. Recently, the renoprotective effect of omega-3 polyunsaturated fatty acids (ω3PUFAs) has attracted attention. The aims of this study were to evaluate the effect of ω3PUFAs on the transition of AKI to CKD and to identify fatty acid active metabolites in renal tissue. Methods: Two mice models of the AKI to CKD transition (7-week, male) and unilateral ureteral obstruction–induced renal fibrosis (11-week, male) were fed linseed oil, rich in ω3PUFAs (Lin group), or with soybean oil, low in ω3PUFAs (Soy group). Renal fatty acids and metabolites composition in mice were measured by liquid chromatography-mass spectrometry. Rat renal fibroblast cells were used for in vitro study. Results: At day 14 after 35 minutes of bilateral renal ischemia reperfusion, significant increase in survival was observed in the Lin group compared with the Soy group. Using the 30-minute bilateral renal ischemia–reperfusion model (AKI to CKD model), the Lin group showed attenuated renal tissue damage and fibrosis. In addition, the antifibrotic effect of the Lin group was also observed in the unilateral ureteral obstruction renal fibrosis model. In the two mice models, levels of eicosapentaenoic acid (EPA) and its metabolites were significantly elevated in renal tissue of mice fed with Lin. Cultured NRK-49F incubated with EPA and its metabolites 18-hydroxyeicosapentaenoic acid, 17,18-epoxyeicosatetraenoic acid, and 17,18-dihydroxyeicosatetraenoic acid displayed suppressed TGF-β1–stimulated α-smooth muscle actin protein expression. These effects were suppressed in the presence of an inhibitor of a cytochrome P450 involved in EPA metabolism. This observation suggests that the EPA metabolites have antifibrotic effects. Conclusions: ω3PUFAs prevent the AKI to CKD transition and renal fibrosis. Moreover, the EPA metabolites 18-hydroxyeicosapentaenoic acid, 17,18-epoxyeicosatetraenoic acid, and 17,18-dihydroxyeicosatetraenoic acid were found to have antifibrotic effects.

The plasma protein alpha(1)-acid glycoprotein (AGP) primarily affects the pharmacokinetics of basic drugs. There are two AGP variants in humans, A and F1*S, exhibiting distinct drug-binding selectivity. Elucidation of the drug-binding selectivity of human AGP variants is essential for drug development and personalized drug therapy. Herein, we aimed to establish the contribution of amino acids 112 and 114 of human AGP to drug-binding selectively. Both amino acids are located in the drug-binding region and differ between the variants. Phe112/Ser114 of the A variant and its equivalent residues in the F1*S variant (Leu112/Phe114) were swapped with each other. Binding experiments were then conducted using the antiarrhythmic drug disopyramide, which selectively binds to the A variant. A significant decrease in the bound fraction was observed in each singly mutated A protein (Phe112Leu or Ser114Phe). Moreover, the bound fraction of the double A mutant (Phe112Leu/Ser114Phe) was decreased to that of wild-type F1*S. Intriguingly, the double F1*S mutant (Leu112Phe/Phe114Ser), in which residues were swapped with those of the A variant, showed only partial restoration in binding. The triple F1*S mutant (Leu112Phe/Phe114Ser/Asp115Tyr), where position 115 is thought to contribute to the difference in pocket size between variants, showed a further recovery in binding to 70% of that of wild-type A. These results were supported by thermodynamic analysis and acridine orange binding, which selectively binds the A variant. Together, these data indicate that, in addition to direct interaction with Phe112 and Ser114, the binding pocket size contributed by Tyr115 is important for the drug-binding selectivity of the A variant.

Alpha(1)-Acid glycoprotein (AGP) is a primary binding protein for many basic drugs in plasma. The number of drugs that bind to AGP, such as molecular target anticancer drugs, has been continuously increasing. Since the plasma level of AGP fluctuates under various pathological conditions such as inflammation, it is important to evaluate the contribution of AGP to drug pharmacokinetics. Here, we generated conventional AGP-knockout (AGP-KO) mice and used them to evaluate the contribution of AGP. The pharmacokinetics of drugs that bind to two AGP variants (F1*S or A variants) or albumin were evaluated. Imatinib (a F1*S-binding drug) and disopyramide (an A-binding drug) or ibuprofen (an albumin-binding drug) were administered to wild-type (WT) and AGP-KO. The plasma level of imatinib and disopyramide decreased rapidly in AGP-KO as compared to WT. In AGP-KO, AUC and t(1/2) were decreased, then CLtot was increased. Compared with disopyramide, imatinib pharmacokinetics showed more marked changes in AGP-KO as compared to WT. The results seemed to be due to the difference in plasma level of each AGP variant (F1*S:A = 2-3:1). No differences were observed in ibuprofen pharmacokinetics between the WT and AGP-KO mice. In vitro experiments using plasma from WT and AGP-KO showed that unbound fractions of imatinib and disopyramide were higher in AGP-KO. These results suggest that the rapid elimination of imatinib and disopyramide in AGP-KO could be due to decreased protein binding to AGP. Taken together, the AGP-KO mouse could be a potential animal model for evaluating the contribution of AGP to the pharmacokinetics of various drugs.