IgM to IgG Class Switching Is a Necessary Step for Pemphigus Phenotype Induction in Desmoglein 3-Specific B Cell Receptor Knock-in Mouse.

Pemphigus vulgaris is an autoimmune blistering disease caused by IgG targeting desmoglein 3 (Dsg3), an adhesion molecule of keratinocytes. Anti-Dsg3 IgG production is prevented in healthy individuals, but it is unclear how Dsg3-specific B cells are regulated. To clarify the immunological condition regulating Dsg3-specific B cells, a pathogenic anti-Dsg3 Ig (AK23) knock-in mouse was generated. AK23 knock-in B cells developed normally without undergoing deletion or acquiring an anergic phenotype in vivo. The knock-in B cells showed Ca2+ influx upon IgM cross-linking and differentiated into AK23-IgG+ B cells after LPS and IL-4 stimulation in vitro that induced a pemphigus phenotype after adoptive transfer into Rag2 -/- mice. However, the knock-in mouse itself produced AK23-IgM but little IgG without blisters in vivo. Dsg3 immunization and skin inflammation caused AK23-IgG production and a pemphigus phenotype in vivo. Furthermore, Fcgr2b deficiency or haploinsufficiency spontaneously induced AK23-IgG production and a pemphigus phenotype with poor survival rates in AK23 knock-in mice. To assess Fcgr2b involvement in Ig class-switch efficiency, postswitch transcripts of B cells were quantified and significantly higher in Fcgr2b -/- and Fcgr2b +/- mice than wild-type mice in a gene dose-dependent manner. Finally, RNA sequencing revealed reduced expression of FCGR2B and FcγRIIB-related genes in patient B cells. These results indicated that Dsg3-specific B cells do not spontaneously perform pathogenic class switching in vivo, and pemphigus phenotype induction was prevented under normal conditions. Attenuated FcγRIIB signaling is also one of the drivers for pathogenic class switching and is consistent with immunological features identified from clinical samples. This study unveiled a characteristic immune state silencing autoreactive B cells in mice.

Peripheral tolerance by Treg via constraining OX40 signal in autoreactive T cells against desmoglein 3, a target antigen in pemphigus.

Antigen-specific peripheral tolerance is crucial to prevent the development of organ-specific autoimmunity. However, its function decoupled from thymic tolerance remains unclear. We used desmoglein 3 (Dsg3), a pemphigus antigen expressed in keratinocytes, to analyze peripheral tolerance under physiological antigen-expression conditions. Dsg3-deficient thymi were transplanted into athymic mice to create a unique condition in which Dsg3 was expressed only in peripheral tissue but not in the thymus. When bone marrow transfer was conducted from high-avidity Dsg3-specific T cell receptor-transgenic mice to thymus-transplanted mice, Dsg3-specific CD4+ T cells developed in the transplanted thymus but subsequently disappeared in the periphery. Additionally, when Dsg3-specific T cells developed in Dsg3-/- mice were adoptively transferred into Dsg3-sufficient recipients, the T cells disappeared in an antigen-specific manner without inducing autoimmune dermatitis. However, Dsg3-specific T cells overcame this disappearance and thus induced autoimmune dermatitis in Treg-ablated recipients but not in Foxp3-mutant recipients with dysfunctional Tregs. The molecules involved in disappearance were sought by screening the transcriptomes of wild-type and Foxp3-mutant Tregs. OX40 of Tregs was suggested to be responsible. Consistently, when OX40 expression of Tregs was constrained, Dsg3-specific T cells did not disappear. Furthermore, Tregs obtained OX40L from dendritic cells in an OX40-dependent manner in vitro and then suppressed OX40L expression in dendritic cells and Birc5 expression in Dsg3-specific T cells in vivo. Lastly, CRISPR/Cas9-mediated knockout of OX40 signaling in Dsg3-specific T cells restored their disappearance in Treg-ablated recipients. Thus, Treg-mediated peripheral deletion of autoreactive T cells operates as an OX40-dependent regulatory mechanism to avoid undesired autoimmunity besides thymic tolerance.

Problems, goals and solutions reported by cancer patients participating in group problem-solving therapy.

BACKGROUND: The purpose of the present study was to categorize and develop lists of contents of problems, goals, solution plans generated through the brainstorming work in therapy, and selected solutions for execution, that treated in problem-solving therapy (PST) for cancer patients in clinical settings, and to describe their characteristics. Additionally, examining the associations of problem domains with characteristics of participants, was also aimed. METHODS: We conducted content analysis using records of thirty-one cancer patients (M = 62.6 years old; SD = 10.5) who participated in PST program. RESULTS: Problems were categorized into four domains (e.g. psychological and existential problems; physical problems; social relations; social living environment). Participants under treatment at baseline reported psychological and existential problems most often (P < 0.05). Goals were categorized into four domains (e.g. improving mental health; improving physical functions; improving social relations and improving one's social living environment). Solution plans generated through the brainstorming work in therapy were categorized into four domains (e.g. emotion regulation/cognitive adjustment; health behaviors; adjustment of social relationships and adjusting one's social living environment). Selected solutions for execution were categorized into four domains (e.g. emotion regulation/cognitive adjustment; health behaviors; adjustment of social relationships and adjusting one's social living environment). CONCLUSIONS: We found that various problems, goals and solutions were treated in PST of realistic clinical setting. Creating lists based on our study and making use of it for the materials as aids while implementing the PST or being shared with patients and medical staff would be expected.

Activation of central/effector memory T cells and T-helper 1 polarization in malignant melanoma patients treated with anti-programmed death-1 antibody.

Human anti-programmed death-1 (PD-1) antibody possesses the capability to revitalize host T cells and has been an effective therapy for metastatic malignant melanoma (MM). The precise subsets of T cells predominantly activated by anti-PD-1, however, have not yet been clarified. In this study, peripheral blood mononuclear cells obtained from MM patients scheduled to receive anti-PD-1 (nivolumab) therapy, and healthy subjects (HS), were systematically examined on flow cytometry to identify changes in the proportion of immune cell subsets. Compared with HS, MM patients prior to therapy had an increased proportion of activated CD8+ T cells with effector memory phenotypes (Tem), and PD-1 positive subsets of CD4+ central memory T cells (Tcm) and T-helper (Th)17 cells. After a single course of anti-PD-1 therapy, MM patients had an increase in activated Tem and Tcm subsets of CD4+ and CD8+ T cells, and activated Th1 plus T-helper follicular 1 cells. There was no consistent change in the proportion of Tfh cells, B cells, natural killer cells, or dendritic cells. The observed activated phenotypes were attenuated during the course of therapy, but regulatory T cells belonging to the CD3+CD4+CD45RO+CD25high fraction increased at disease progression. Taken together, anti-PD-1 therapy modulates systemic immune reactions and exerts anti-tumor effects, not only by revitalizing Tem and Tcm of CD4+ and CD8+ T cells, but also via a shift to a Th1 phenotype.

Melanoma and Immune Checkpoint Inhibitors.

PURPOSE OF REVIEW: Prognosis of patients with advanced melanoma is dismal with a median overall survival of about 8 months and 5-year overall survival from a diagnosis of metastatic disease of roughly 10%. However, immune checkpoint inhibitors have brought indispensable benefits to melanoma patients. Here we will review the recent clinical efficacy and adverse events of immune checkpoint inhibitors for melanoma patients. RECENT FINDINGS: The immune checkpoint inhibitors increase confirmed objective response and prolong progression-free and overall survival of the afflicted patients in association with maintaining their quality of life. Although diverse immune-related adverse events occur, most of them are manageable by appropriate immunomodulating agents. Clinical efficacy of immune checkpoint inhibitors continues even after discontinuation of drugs. Compared with conventional therapeutic options, the immune checkpoint inhibitors appear to prolong the survival of patients with advanced melanoma. Further clinical trials are warranted to determine whether their combinatory use with other treatment options may augment benefits or not.

Nivolumab-induced thyroid dysfunction lacking antithyroid antibody is frequently evoked in Japanese patients with malignant melanoma.

BACKGROUND: Nivolumab, an anti-programmed cell death-1 monoclonal antibody, has improved the survival of patients with malignant melanoma. Despite its efficacy, nivolumab inconsistently induces thyroid dysfunction as an immune-related adverse event (irAE). This study aimed to evaluate nivolumab-induced thyroid dysfunction to determine the risks and mechanisms of thyroid irAEs. METHODS: After excluding 10 patients, data of 24 patients with malignant melanoma (aged 17-85 years; 54% female) were retrospectively analyzed. RESULTS: Thyroid irAEs were observed in seven patients (29%). Three patients had hypothyroidism after preceding transient thyrotoxicosis, and the other four patients had hypothyroidism without thyrotoxicosis. Levothyroxine-Na replacement was required in three patients. Antithyroid antibody (ATA) titer was elevated in one of four assessable patients. The average (±SD) time to onset of thyroid irAE was 33.6 (±21.9) weeks. The administration period of nivolumab was longer in patients with thyroid irAEs than in those without thyroid irAEs (P < 0.01). There were no significant differences between patients with and without thyroid irAEs regarding age, sex, tumor stage, response to nivolumab therapy, baseline thyroid function, antithyroid peroxidase antibody (anti-TPO Ab) and antithyroglobulin antibody (anti-Tg Ab). CONCLUSIONS: Thyroid dysfunction was a common irAE of nivolumab in malignant melanoma. Neither anti-TPO Ab nor anti-Tg Ab was associated with the risk for nivolumab-induced thyroid dysfunction. A conventional ATA-independent mechanism might be involved in thyroid irAEs. Further studies are required to clarify the mechanism and identify the predictive factors of thyroid irAEs.

Effect of Npt2b deletion on intestinal and renal inorganic phosphate (Pi) handling.

BACKGROUND: Hyperphosphatemia is common in chronic kidney disease and is associated with morbidity and mortality. The intestinal Na+-dependent phosphate transporter Npt2b is thought to be an important molecular target for the prevention of hyperphosphatemia. The role of Npt2b in the net absorption of inorganic phosphate (Pi), however, is controversial. METHODS: In the present study, we made tamoxifen-inducible Npt2b conditional knockout (CKO) mice to analyze systemic Pi metabolism, including intestinal Pi absorption. RESULTS: Although the Na+-dependent Pi transport in brush-border membrane vesicle uptake levels was significantly decreased in the distal intestine of Npt2b CKO mice compared with control mice, plasma Pi and fecal Pi excretion levels were not significantly different. Data obtained using the intestinal loop technique showed that Pi uptake in Npt2b CKO mice was not affected at a Pi concentration of 4 mM, which is considered the typical luminal Pi concentration after meals in mice. Claudin, which may be involved in paracellular pathways, as well as claudin-2, 12, and 15 protein levels were significantly decreased in the Npt2b CKO mice. Thus, Npt2b deficiency did not affect Pi absorption within the range of Pi concentrations that normally occurs after meals. CONCLUSION: These findings indicate that abnormal Pi metabolism may also be involved in tight junction molecules such as Cldns that are affected by Npt2b deficiency.

Inhibitory FcγRIIb-Mediated Soluble Antigen Clearance from Plasma by a pH-Dependent Antigen-Binding Antibody and Its Enhancement by Fc Engineering.

Fc engineering can modulate the Fc-FcγR interaction and thus enhance the potency of Abs that target membrane-bound Ags, but it has not been applied to Abs that target soluble Ags. In this study, we revealed a previously unknown function of inhibitory FcγRII in vivo and, using an Ab that binds to Ag pH dependently, demonstrated that the function can be exploited to target soluble Ag. Because pH-dependent Ab dissociates Ag in acidic endosome, its Ag clearance from circulation reflects the cellular uptake rate of Ag/Ab complexes. In vivo studies showed that FcγR but not neonatal FcR contributes to Ag clearance by the pH-dependent Ab, and when Fc binding to mouse FcγRII and III was increased, Ag clearance was markedly accelerated in wild-type mice and FcR γ-chain knockout mice, but the effect was diminished in FcγRII knockout mice. This demonstrates that mouse FcγRII efficiently promotes Ab uptake into the cell and its subsequent recycling back to the cell surface. Furthermore, when a human IgG1 Fc variant with selectively increased binding to human FcγRIIb was tested in human FcγRIIb transgenic mice, Ag clearance was accelerated without compromising the Ab half-life. Taken together, inhibitory FcγRIIb was found to play a prominent role in the cellular uptake of monomeric Ag/Ab immune complexes in vivo, and when the Fc of a pH-dependent Ab was engineered to selectively enhance human FcγRIIb binding, the Ab could accelerate soluble Ag clearance from circulation. We assume such a function would enhance the therapeutic potency of Abs that target soluble Ags.

Isolated ACTH deficiency probably induced by autoimmune-related mechanism evoked with nivolumab.

Nivolumab, an anti-programmed death-1 antibody, is a breakthrough treatment for several malignancies. Its specific adverse effects caused by autoimmunity are termed immune-related adverse events, which involve several endocrine dysfunctions. Herein, we report two cases of isolated adrenocorticotropic hormone (ACTH) deficiency induced by nivolumab for the treatment of metastatic malignant melanoma. Case 1 was a 39-year-old man and Case 2 was a 50-year-old woman, both of whom presented with progressive melanoma. After 13 courses of nivolumab administration, both cases were diagnosed with adrenal insufficiency. Despite their basal serum ACTH and cortisol levels being low with little response to corticotropin-releasing hormone loading, other anterior pituitary hormone levels were preserved. Based on these endocrinological data, isolated ACTH deficiency was diagnosed. Magnetic resonance imaging showed normal pituitary glands, excluding hypophysitis. Finally, hydrocortisone replacement enabled the patients to continue nivolumab treatment. Therefore, it is important to consider isolated ACTH syndrome as a possible and potentially severe immune-related adverse event of nivolumab, even when head magnetic resonance imaging of affected cases does not show enlargement. We should not misdiagnose hidden immune-related adverse events behind general complaints of malignancies such as general malaise and appetite loss, to allow successful treatment using this beneficial immune checkpoint inhibitor.

Immunomodulatory drugs act as inhibitors of DNA methyltransferases and induce PU.1 up-regulation in myeloma cells.

Immunomodulatory drugs (IMiDs) such as thalidomide, lenalidomide, and pomalidomide are efficacious in the treatment of multiple myeloma and significantly prolong their survival. However, the mechanisms of such effects of IMiDs have not been fully elucidated. Recently, cereblon has been identified as a target binding protein of thalidomide. Lenalidomide-resistant myeloma cell lines often lose the expression of cereblon, suggesting that IMiDs act as an anti-myeloma agent through interacting with cereblon. Cereblon binds to damaged DNA-binding protein and functions as a ubiquitin ligase, inducing degradation of IKZF1 and IKZF3 that are essential transcription factors for B and T cell development. Degradation of both IKZF1 and IKZF3 reportedly suppresses myeloma cell growth. Here, we found that IMiDs act as inhibitors of DNA methyltransferases (DMNTs). We previously reported that PU.1, which is an ETS family transcription factor and essential for myeloid and lymphoid development, functions as a tumor suppressor in myeloma cells. PU.1 induces growth arrest and apoptosis of myeloma cell lines. In this study, we found that low-dose lenalidomide and pomalidomide up-regulate PU.1 expression through inducing demethylation of the PU.1 promoter. In addition, IMiDs inhibited DNMT1, DNMT3a, and DNMT3b activities in vitro. Furthermore, lenalidomide and pomalidomide decreased the methylation status of the whole genome in myeloma cells. Collectively, IMiDs exert demethylation activity through inhibiting DNMT1, 3a, and 3b, and up-regulating PU.1 expression, which may be one of the mechanisms of the anti-myeloma activity of IMiDs.

Multimodal Imaging of Optic Nerve Head in Retinitis Pigmentosa.

BACKGROUND: A pallor optic nerve head (ONH) is one of the three features of retinitis pigmentosa (RP). This study aimed to assess the ONH prospectively by color tone, presence of hyper-reflective tissue, blood flow, retinal nerve fiber layer (RNFL) thickness, ganglion cell complex (GCC) and investigate the change in these parameters with and without ONH pallor. METHODS: The presence of ONH pallor was assessed by three independent examiners through careful examination using fundus photographs. The presence of a hyper-reflective structure on the ONH was carefully evaluated using a volume scan optical coherence tomography (OCT). RNFL thickness and ellipsoid zone (EZ) width around the macula were also evaluated by OCT. Laser speckle flowgraphy was used to measure the mean blur rate of the entire ONH area, which was subsequently divided into the vessel area (MV) and tissue area (MT). RESULTS: Twenty-eight eyes of 28 patients with RP (55.4 ± 16.23 years of age) were included. The pale ONH was observed in 10 (35%) eyes. Hyper-reflective structures were observed in seven (25%) eyes. No significant correlation was found between the pale ONH and the presence of a hyper-reflective structure (Pearson's chi-squared test, p = .364). The average of the ONH area, MV, and MT was 8.65 ± 3.08 AU, 17.81 ± 7.54 AU, and 6.4 ± 2.66 AU, respectively, which significantly decreased in patients with pallor ONH (all p < .05). The global RNFL thickness was 73.54 ± 18.82 µm. The nasal and superior quadrants and global RNFL thickness in patients with a pale ONH were significantly thinner than in patients without a pale ONH (all p < .05). The global and superior and inferior GCC thickness in patients with a pale ONH were significantly thinner than in patients without a pale ONH(all p < .05).There was no difference in the EZ width between patients with and without a pale ONH (p = .107). CONCLUSION: We conducted multiple assessments of the ONH in RP patients and investigated its clinical significance. Our findings suggest that ONH pallor may indicate a comprehensive change that emerges alongside the progression of retinal degeneration in RP. TRIAL REGISTRATION: This trial was retrospectively registered in the UMIN Clinical Trial Registry (UMIN ID: 000048168).

The influence of blood glucose on neutrophil function in individuals without diabetes.

We assessed the association of neutrophil function with glycated hemoglobin (HbA1c) levels in a Japanese general population. Participants were 809 males and females who were over 20 years old living in the Iwaki region in Aomori Prefecture located in northern Japan. Lifestyle parameters (smoking, alcohol consumption, and exercise habits), HbA1c and neutrophil function such as reactive oxygen species (ROS) production capability and phagocytic activity (PA) were measured. ROS production capability was measured before and after phagocytic stimulus to obtain basal ROS production and stimulated ROS production. Level of HbA1c had a positive correlation with basal ROS production (p=0.053), a negative correlation with stimulated ROS production (p=0.072) and PA (p=0.059) only in post-menopausal groups, and not in pre-menopausal groups. However, there were no correlations between levels of HbA1c and neutrophil functions in male. In conclusion, in the present study, despite the presence of diabetes, chronic hyperglycemia was found to cause an increase in daily basal ROS production of neutrophils, and increased susceptibility to infection caused by reduced neutrophilic reaction in females in their menopause. Therefore, from the oxidative point of view, strict glycemic control is necessary to prevent post-menopausal females from developing diabetic complications in spite of the presence of diabetes.

Overexpression of cathepsin D in malignant melanoma.

Cathepsin D is an aspartic lysosomal endopeptidase present in most mammalian cells. Overexpression of cathepsin D is associated with the progression of several human cancers including melanoma. We examined the expression levels of cathepsin D in 20 primary malignant melanomas, 20 metastatic malignant melanomas, 20 benign nevus pigmentosus and 10 normal skin samples in Japanese. In normal skin, granular or dotted pattern of positive staining was observed along the granular layer of epidermis and hair follicle with apparent moderate to strong staining in sebaceous and eccrine glands. The percent positivity and staining intensity of cathepsin D in primary and metastatic malignant melanomas were significantly higher than that of nevus pigmentosus. Moreover, the expression levels of cathepsin D in metastatic malignant melanomas were significantly higher than those of primary malignant melanomas. Data from our and previous reports strongly supports a notion that the upregulation of cathepsin D may be critically involved in the malignant transformation and progression of melanocytic tumors.

Corrigendum: Citrate synthase insufficiency leads to specific metabolic adaptations in the heart and skeletal muscles upon low-carbohydrate diet feeding in mice.

[This corrects the article DOI: 10.3389/fnut.2022.925908.].

Antigen-independent development of Foxp3+ regulatory T cells suppressing autoantibody production in experimental pemphigus vulgaris.

The CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) play suppressive roles in various types of autoimmunity. It has been reported that Tregs develop in the thymus after high-affinity interaction of their TCR with self-peptide/MHC ligands mostly utilizing TCR-transgenic system. In this study, we examined whether the specific antigen is involved in the development of polyclonal Tregs in pemphigus vulgaris (PV), an autoimmune blistering disease caused by anti-desmoglein 3 (Dsg3) IgG antibodies, as a model system. Adoptive transfer of splenocytes of Dsg3(-)(/-) mice immunized with recombinant mouse Dsg3 to Rag2(-)(/-) recipient mice expressing Dsg3 resulted in the stable production of anti-Dsg3 IgG and the development of PV phenotypes. We show here that Tregs control anti-Dsg3 antibody production in PV model mice: the adoptive transfer of Tregs and the depletion of endogenous Tregs suppressed and augmented, respectively, the anti-Dsg3 antibody production. To examine whether the endogenous expression of Dsg3 is involved in the generation of these PV-relevant Tregs, we compared the potential of wild-type Tregs with that of Tregs from Dsg3(-)(/-) mice. Polyclonal Tregs from Dsg3(-)(/-) mice were more potent than that of wild-type mice, in both adoptive transfer and Treg-depletion experiments, while suppressive activities against IgG production against an irrelevant antigen were similar between Tregs from wild-type and Dsg3(-)(/-) mice. Our observation implies that Tregs capable of suppressing T(h) cells that drive autoantibody production can develop in the absence of the target antigen.

Problem-solving therapy for psychological distress in Japanese early-stage breast cancer patients.

OBJECTIVE: The current report provides the result of a Phase II clinical trial regarding the effectiveness and feasibility of problem-solving therapy for psychological distress experienced by Japanese early-stage breast cancer patients. METHODS: Participants were 36 post-surgery Japanese breast cancer patients in a university hospital located in Osaka Prefecture, Japan. After screening for psychological distress using the Distress and Impact Thermometer and the Hospital Anxiety and Depression Scale, highly distressed patients were exposed to 5 weekly sessions of the problem-solving therapy program. RESULTS: Nineteen patients completed the intervention and follow-up. There was a significant difference between the pre-intervention and the 3-month follow-up in the total Hospital Anxiety and Depression Scale score (P = 0.02), and the mean change score from the pre-intervention to the follow-up was 6.05 (SD = 1.94). The intervention had a large effect size (d = 0.82). There were also significant changes in worry, self-efficacy and quality of life measures. CONCLUSIONS: The findings of our study suggest that the problem-solving therapy program has potential to be effective for alleviating psychological distress experienced by Japanese early-stage breast cancer patients. The true effectiveness of the program should be confirmed by a future randomized control trial.

Isonitrogenous low-carbohydrate diet elicits specific changes in metabolic gene expression in the skeletal muscle of exercise-trained mice.

With the renewed interest in low-carbohydrate diets (LCDs) in the sports field, a few animal studies have investigated their potential. However, most rodent studies have used an LCD containing low protein, which does not recapitulate a human LCD, and the muscle-specific adaptation in response to an LCD remains unclear. Therefore, we investigated the effects of two types of LCDs, both containing the same proportion of protein as a regular diet (isonitrogenous LCD; INLCD), on body composition, exercise performance, and metabolic fuel selection at the genetic level in the skeletal muscles of exercise-trained mice. Three groups of mice (n = 8 in each group), one fed a regular AIN-93G diet served as the control, and the others fed either of the two INLCDs containing 20% protein and 10% carbohydrate (INLCD-10%) or 20% protein and 1% carbohydrate (INLCD-1%) had a regular exercise load (5 times/week) for 12 weeks. Body weight and muscle mass did not decrease in either of the INLCD-fed groups, and the muscle glycogen levels and endurance capacity did not differ among the three groups. Only in the mice fed INLCD-1% did the plasma ketone concentration significantly increase, and gene expression related to glucose utilization significantly declined in the muscles. Both INLCD-1% and INLCD-10% consumption increased gene expression related to lipid utilization. These results suggest that, although INLCD treatment did not affect endurance capacity, it helped maintain muscle mass and glycogen content regardless of the glucose intake restrictions in trained mice. Moreover, an INLCD containing a low carbohydrate content might present an advantage by increasing lipid oxidation without ketosis and suppressing muscle glucose utilization.

Citrate Synthase Insufficiency Leads to Specific Metabolic Adaptations in the Heart and Skeletal Muscles Upon Low-Carbohydrate Diet Feeding in Mice.

A decrease in TCA cycle activity may lead to impaired nutrition metabolism and cellular energy shortage. Herein, we aimed to characterize the detailed metabolic changes that compensate for energy shortages in energy-consuming organs (heart and skeletal muscles) in mice with knockout of citrate synthase (CS), an important enzyme in the TCA cycle. CS hetero knockout (CS +/-) mice and wild-type mice were fed a low-carbohydrate ketogenic diet (LCKD) or high-fat, high-carbohydrate diet (HFHCD) to induce metabolic changes. Body weight, blood serum parameters, metabolic gene expression, and adenosine triphosphate (ATP) levels were measured in the heart and skeletal muscles. Glycogen content, anabolic and catabolic biomarkers, and morphological changes were also assessed in the skeletal muscles. After diet feeding, there were no differences observed in the body weight and blood serum parameters between wild-type and CS +/- mice. The cardiac expression of genes related to the utilization of fatty acids, monocarboxylates, and branched amino acids increased in LCKD-fed CS +/- mice. In contrast, no significant differences in gene expression were observed in the muscles of LCKD-fed mice or the heart and muscles of HFHCD-fed mice. ATP levels decreased only in the skeletal muscles of LCKD-fed CS +/- mice. Additionally, the decrease in glycogen content, suppression of p70 S6 kinase, and presence of type I fiber atrophy were observed in the muscles of LCKD-fed CS +/- mice. These results suggest that the energy-consuming organs with CS insufficiency may undergo tissue-specific adaption to compensate for energy shortages when the carbohydrate supply is limited.

TNF-α induces Claudin-1 expression in renal tubules in Alport mice.

Claudin-1 (CL-1) is responsible for the paracellular barrier function of glomerular parietal epithelial cells (PEC) in kidneys, but the role of CL-1 in proximal tubules remains to be elucidated. In this study, to evaluate CL-1 as a potential therapeutic drug target for chronic kidney disease, we investigated change of CL-1 expression in the proximal tubules of diseased kidney and elucidated the factors that induced this change. We established Alport mice as a kidney disease model and investigated the expression of CL-1 in diseased kidney using quantitative PCR and immunohistochemistry (IHC). Compared to wild type mice, Alport mice showed significant increases in plasma creatinine, urea nitrogen and urinary albumin excretion. CL-1 mRNA was increased significantly in the kidney cortex and CL-1 was localized on the adjacent cell surfaces of PECs and proximal tubular epithelial cells. The infiltration of inflammatory cells around proximal tubules and a significant increase in TNF-α mRNA were observed in diseased kidneys. To reveal factors that induce CL-1, we analyzed the induction of CL-1 by albumin or tumor necrosis factor (TNF)-α in human proximal tubular cells (RPTEC/TERT1) using quantitative PCR and Western blotting. TNF-α increased CL-1 expression dose-dependently, though albumin did not affect CL-1 expression in RPTEC/TERT1. In addition, both CL-1 and TNF-α expression were significantly increased in UUO mice, which are commonly used as a model of tubulointerstitial inflammation without albuminuria. These results indicate that CL-1 expression is induced by inflammation, not by albuminuria in diseased proximal tubules. Moreover, we examined the localization of CL-1 in the kidney of IgA nephropathy patients by IHC and found CL-1 expression was also elevated in the proximal tubular cells. Taken together, CL-1 expression is increased in the proximal tubular epithelial cells of diseased kidney. Inflammatory cells around the tubular epithelium may produce TNF-α which in turn induces CL-1 expression.

Inorganic phosphate homeostasis in sodium-dependent phosphate cotransporter Npt2b⁺/⁻ mice.

An inorganic phosphate (P(i))-restricted diet is important for patients with chronic kidney disease and patients on hemodialysis. Phosphate binders are essential for preventing hyperphosphatemia and ectopic calcification. The sodium-dependent P(i) (Na/P(i)) transport system is involved in intestinal P(i) absorption and is regulated by several factors. The type II sodium-dependent P(i) transporter Npt2b is expressed in the brush-border membrane in intestinal epithelial cells and transports P(i). In the present study, we analyzed the phenotype of Npt2b(-/-) and hetero(+/-) mice. Npt2b(-/-) mice died in utero soon after implantation, indicating that Npt2b is essential for early embryonic development. At 4 wk of age, Npt2b(+/-) mice showed hypophosphatemia and low urinary P(i) excretion. Plasma fibroblast growth factor 23 levels were significantly decreased and 1,25(OH)(2)D(3) levels were significantly increased in Npt2b(+/-) mice compared with Npt2b(+/+) mice. Npt2b mRNA levels were reduced to 50% that in Npt2b(+/+) mice. In contrast, renal Npt2a and Npt2c transporter protein levels were significantly increased in Npt2b(+/-) mice. At 20 wk of age, Npt2b(+/-) mice showed hypophosphaturia and reduced Na/P(i) cotransport activity in the distal intestine. Npt2b(+/+) mice with adenine-induced renal failure had hyperphosphatemia and high plasma creatinine levels. Npt2b(+/-) mice treated with adenine had significantly reduced plasma P(i) levels compared with Npt2b(+/+) mice. Intestinal Npt2b protein and Na(+)/P(i) transport activity levels were significantly lower in Npt2b(+/-) mice than in the Npt2b(+/+) mice. The findings of the present studies suggest that Npt2b is an important target for the prevention of hyperphosphatemia.