Protective role of protease-activated receptor-2 in anaphylaxis model mice.

Anaphylaxis is a severe life-threatening hypersensitivity reaction induced by mast cell degranulation. Among the various mediators of mast cells, little is known about the role of tryptase. Therefore, we aimed to elucidate the role of protease-activating receptor-2 (PAR-2), a receptor activated by tryptase, in murine anaphylactic models using PAR-2-deficient mice and newly generated tryptase-deficient mice. Anaphylaxis was induced by IgE-dependent and IgE-independent mast cell degranulation in mice. PAR-2 deficiency exacerbated the decrease in body temperature and hypotension during anaphylaxis; however, the number of skin mast cells, degree of mast cell degranulation, and systemic and local vascular hyperpermeability were comparable in PAR-2 knockout and wild-type mice. Nitric oxide, which is produced by endothelial nitric oxide synthase (eNOS), is an indispensable vasodilator in anaphylaxis. In the lungs of anaphylactic mice, PAR-2 deficiency promoted eNOS expression and phosphorylation, suggesting a protective effect of PAR-2 against anaphylaxis by downregulating eNOS activation and expression. Based on the hypothesis that the ligand for PAR-2 in anaphylaxis is mast cell tryptase, tryptase-deficient mice were generated using CRISPR-Cas9. In wild-type mice, the PAR-2 antagonist exacerbated the body temperature drop due to anaphylaxis; however, the effect of the PAR-2 antagonist was abolished in tryptase-deficient mice. These results suggest that tryptase is a possible ligand of PAR-2 in anaphylaxis and that the tryptase/PAR-2 pathway attenuates the anaphylactic response in mice.

Mutation of the Thap4 gene causes dwarfism and testicular anomalies in rats and mice.

The petit (pet) locus is associated with dwarfism, testicular anomalies, severe thymic hypoplasia, and high postnatal lethality, which are inherited in autosomal recessive mode of inheritance in rats with a Wistar strain genetic background. Linkage analysis localized the pet locus between 98.7 Mb and 101.2 Mb on rat chromosome 9. Nucleotide sequence analysis identified 2 bp deletion in exon 2 of the Thap4 gene as the causative mutation for pet. This deletion causes a frameshift and premature termination codon, resulting in a truncated THAP4 protein lacking approximately two-thirds of the C-terminal side. Thap4 is expressed in various organs, including the testis and thymus in rats. To elucidate the biological function of THAP4 in other species, we generated Thap4 knockout mice lacking exon 2 of the Thap4 gene through genome editing. Thap4 knockout mice also exhibited dwarfism and small testis but did not show high postnatal lethality. Thymus weights of adult Thap4 knockout male mice were significantly higher compared to wild-type male mice. Although Thap4 knockout male mice were fertile, their testis contained seminiferous tubules with spermatogenesis and degenerative seminiferous tubules lacking germ cells. Additionally, we observed vacuoles in seminiferous tubules, and clusters of cells in the lumen in seminiferous tubules in Thap4 knockout male mice. These results demonstrate that spontaneous mutation of Thap4 gene in rats and knockout of Thap4 gene in mice both cause dwarfism and testicular anomalies. Thap4 gene in rats and mice is essential for normal testicular development, maintaining spermatogenesis throughout the entire region of seminiferous tubules.

Effectiveness of intravenous methylprednisolone pulse in patients with severe microscopic polyangiitis and granulomatosis with polyangiitis.

OBJECTIVES: To evaluate the effectiveness and safety of two different intravenous methylprednisolone (IVMP) pulse doses in patients with severe microscopic polyangiitis (MPA) and granulomatosis with polyangiitis (GPA). METHODS: We emulated a target trial using observational data from the nationwide registry in Japan. Patients with severe glomerulonephritis or diffuse alveolar haemorrhage were selected and pseudo-randomised into three groups using propensity score-based overlap weighting as follows: non-IVMP, IVMP 0.5 g/day, and IVMP 1.0 g/day. The primary outcome was all-cause death, and the secondary outcomes were composite all-cause death and kidney failure, severe relapse, and serious infection from 2 to 48 weeks after treatment initiation. To estimate the treatment effects, the Cox proportional hazard model and Fine-Gray subdistribution hazard model were used. RESULTS: In this emulated target trial, of 201 eligible patients (MPA, 175; GPA, 26), 6 (2.8%) died, 4 (2.0%) had kidney failure, 11 (5.3%) had severe relapse, and 40 (19.8%) had severe infections. Hazard ratios (HR) for IVMP 0.5 g/day and IVMP 1.0 g/day pulse groups compared with non-IVMP pulse were as follows: all-cause death = 0.46 (95% confidence interval [95%CI]: 0.07-2.81) and 0.07 (95%CI: 0.01-0.41); all-cause death/kidney failure = 1.18 (95%CI: 0.26-5.31) and 0.59 (95%CI: 0.08-4.52); subdistribution HRs for severe relapse = 1.26 (95%CI: 0.12-13.70) and 3.36 (95%CI: 0.49-23.29); and serious infection = 1.88 (95%CI: 0.76-4.65) and 0.94 (95%CI: 0.28-3.13). CONCLUSIONS: IVMP 1.0 g/day pulse may improve 48-week mortality in patients with severe MPA/GPA.

Dynamics of extracellular vesicle uptake by mural granulosa cells in mice.

Extracellular vesicles (EVs) play an important role in the development and function of mammalian ovarian follicles. However, the mechanisms by which they are taken up by the follicular granulosa cells remain unclear. In addition, while oocytes play a pivotal role in follicular development, the possible interactions between oocyte-derived paracrine factors (ODPFs) and EV signals are unknown. Therefore, this study aimed to elucidate the mechanism of EV uptake and the effects of ODPFs on EV uptake by follicular somatic mural granulosa cells in mice. Fluorescence-labeled transferrin (TRF) and cholera toxin B (CTB), substrates for clathrin- and caveolae-mediated endocytosis, respectively, were taken up by mural granulosa cells in vitro. Their uptake was inhibited by Pitstop 2 and genistein, inhibitors of clathrin and caveolae pathways, respectively. Mural granulosa cells took up EVs, and this uptake was suppressed by Pitstop 2 and genistein. Moreover, ODPFs promoted the uptake of EVs and CTB, but not TRF, by mural granulosa cells. These results suggest that mural granulosa cells take up EVs through both clathrin- and caveolae-mediated endocytosis and that oocytes may promote caveolae-mediated endocytosis to facilitate the uptake of EVs.

Inflammatory CD11b+ Macrophages Produce BAFF in Spleen of Mice Infected with Leishmania donovani.

Visceral leishmaniasis (VL) is an infectious disease caused by parasitic protozoa of the genus Leishmania and manifests clinical symptoms such as splenomegaly, hepatomegaly, anemia, and fever. It has previously been shown that B-cell-activating factor (BAFF) is involved in splenomegaly during VL. Although BAFF is known to be expressed by a variety of cells, the mechanism of elevated BAFF expression in VL is not clear. In this study, we aimed to identify BAFF-producing cells in the spleens of mice infected with Leishmania donovani. Splenocytes of L. donovani-infected mice showed elevated BAFF expression compared to that of naive mice. In the infected spleen, the number of both CD11b+ and F4/80+ cells increased, and the major BAFF-producing cells were CD11b+ cells, which did not serve as host cells of Leishmania. Immunohistochemical/immunofluorescent staining of spleens of infected mice revealed that the increased CD11b+ cells were primarily MRP14+ mononuclear cells. Together, these results suggest the increased BAFF expression in the spleen of L. donovani-infected mice involves a recruitment of inflammatory macrophages distinct from host macrophages for the parasites.

Establishment of African pygmy mouse induced pluripotent stem cells using defined doxycycline inducible transcription factors.

Mus minutoides is one of the smallest mammals worldwide; however, the regulatory mechanisms underlying its dwarfism have not been examined. Therefore, we aimed to establish M. minutoides induced pluripotent stem cells (iPSCs) using the PiggyBac transposon system for applications in developmental engineering. The established M. minutoides iPSCs were found to express pluripotency markers and could differentiate into neurons. Based on in vitro differentiation analysis, M. minutoides iPSCs formed embryoid bodies expressing marker genes in all three germ layers. Moreover, according to the in vivo analysis, these cells contributed to the formation of teratoma and development of chimeric mice with Mus musculus. Overall, the M. minutoides iPSCs generated in this study possess properties that are comparable to or closely resemble those of naïve pluripotent stem cells (PSCs). These findings suggest these iPSCs have potential utility in various analytical applications, including methods for blastocyst completion.

Effective Management of Hypertensive Emergencies with Aliskiren Treatment in a Patient Before and After Introducing Hemodialysis Secondary to Scleroderma Renal Crisis-like Condition Under Corticosteroid Treatment for Sjögren Syndrome-associated Multiple Mononeuropathy.

A middle-aged woman presented with hypertensive emergency after corticosteroid treatment for Sjögren syndrome-associated multiple mononeuropathy with suspected systemic sclerosis. Hypertensive heart failure with hyperreninemia improved with antihypertensives, including aliskiren; however, she became hemodialysis-dependent. Clinical findings and biopsy-proven thrombotic microangiopathy indicated conditions resembling scleroderma renal crisis (SRC). Severe hypertension and heart failure with hyperreninemia occurred after stopping aliskiren for hypotension due to diverticular bleeding, which improved after the reintroduction of aliskiren. Aliskiren appears to be effective in managing hypertensive heart failure in patients with SRC. Nevertheless, hemodialysis remained necessary in our case, and whether or not aliskiren can restore the renal function is unclear.

Correlation between attitude towards dental care for older adults and ageism in dental students and clinical trainees: Cross-sectional study.

INTRODUCTION: Since the needs for dental care among older adults in Japan are expected to increase in the future, this study aimed to examine the influence of ageism among dental students and clinical trainees on the attitude towards the older adults in dental care. MATERIALS AND METHODS: In 2022, we conducted a questionnaire survey of 9th-, 11th-semester dental students and first-year clinical trainees enrolled at Kyushu Dental University, using the Japanese version of Ageism Scale for Dental Students (ASDS-J), Japanese version of Fraboni Scale of Ageism (FSA-J). Questions on intention to provide dental care to the older adults, relationships with the older adults and living with the older adults were also surveyed. RESULTS: There were 162 participants (84 males and 78 females), and the response rate was 71.7%. The participants were divided into the positive group for dental care to older adults (68 respondents) and the negative group (94 respondents). The ASDS-J score of the negative group was significantly higher than that of the positive group [49 (36-66) vs. 52 (34-88), p < .001]. The binomial logistic regression analysis revealed that the odds ratio of being negative attitude was 1.1 per one point of ASDS-J (95% confidence interval: 1.0-1.2) in the model adjusted for student grade, interaction with the older adults, gender, experience living with the older adults and FSA-J. CONCLUSION: These findings suggest that ageism specific to dental students and clinical trainees (as assessed using the ASDS-J) was independently correlated with negative attitudes towards dental care for older adults.

Association between tongue pressure and oral status and activities of daily living in stroke patients admitted to a convalescent rehabilitation unit.

BACKGROUND: Clarifying how tongue pressure in convalescent stroke patients affects oral condition and activities of daily living (ADL) is important for developing oral rehabilitation programs and for rehabilitation care to reacquire ADL. OBJECTIVE: To clarify how tongue pressure is associated with oral status, ADL, and nutritional status in stroke patients. METHODS: Sixty-eight patients aged 77.9 ± 10.0 years participated. The Japanese version of the Oral Health Assessment Tool was used to evaluate oral status. Data such as the ADL index functional independence measure (FIM), nutritional intake method, serum albumin, and body mass index were extracted from medical records. To examine factors associated with tongue pressure, multiple regression analysis was performed adjusting for confounding factors. The level of statistical significance was set at p < .05. RESULTS: In recovery phase stroke patients, tongue pressure was significantly lower in the total assistance group than in the partial assistance/independent group. In addition, tongue pressure was significantly lower in tube feeding patients than in oral feeding patients. FIM cognition score was an independent factor that had a significant effect on tongue pressure. CONCLUSION: These findings suggest that ADL status also affects tongue pressure, thus patients' ADL including the FIM cognition subscale should also be evaluated while measuring tongue pressure.

Decreased serum creatinine to cystatin C ratio is associated with low tongue pressure.

AIM: Tongue strength plays an important role in the process of food intake, and low tongue pressure has been associated with aspiration pneumonia, cognitive decline, and mortality. However, special equipment for tongue pressure measurement is uncommon in general practice. Recently, the serum creatinine-to-cystatin C (Cr/CysC) ratio has been validated as a marker of muscle volume mass. Thus, we aimed to investigate the association of the serum Cr/CysC ratio with tongue pressure in a cross-sectional study. METHODS: This single-center, cross-sectional study enrolled 73 participants (mean age, 71.7 years; men, 49.3%) who regularly attended the hospital for treatment of chronic disease. A tongue pressure of <30 kPa was defined as low tongue pressure. We evaluated the relationships between the serum Cr/CysC ratio and tongue pressure using multiple regression analysis. RESULTS: The serum Cr/CysC ratio was correlated with tongue pressure (R2 = 0.25, P < 0.0001). In multiple regression analyses adjusted for confounders including age, sex, body mass index, and serum albumin, the association remained significant (P = 0.0001). In logistic analyses, the multivariable-adjusted odds ratios of the Cr/CysC ratio for tertiles 1 and 2 compared with tertile 3 for low tongue pressure were 7.81 (95% confidence interval, 1.45-51.73) and 2.71 (95% confidence interval, 0.60-13.19), respectively. CONCLUSIONS: We demonstrated that a decreased serum Cr/CysC ratio was associated with a higher risk of low tongue pressure. Our findings suggest that this simple serum surrogate marker may be a first step toward an intervention for oral function by general practitioners. Geriatr Gerontol Int 2024; 24: 102-108.

Structural analysis of polyglycerol fatty acid ester-coenzyme Q10 aggregates in solution.

Polyglycerol fatty acid esters (PGFEs) are common food additives. PGFE-based formulations exhibit high structural stability, however, the stability mechanism of the micellar structures has not been yet elucidated. In this study, nanostructural analysis was performed using small-angle neutron and X-ray scattering (SANS and SAXS) measurements to reveal the mechanism of the structural stability of PGFE-coenzyme Q10 (CoQ10) mixtures as a CoQ10 formulation. Pure PGFE formed multilamellar vesicles, whereas PGFE-CoQ10 formed spherical micelles. Furthermore, when the amount of added water increased, the PGFE-CoQ10 micellar size and the amount of water in the micelles remained unchanged. A model-fitting analysis of the SANS results suggested that the CoQ10 molecules were introduced between the surfactants, forming a palisade-type structure. The high structural stability of the PGFE-CoQ10 micelles was attributed to two factors: proper spreading of the hydrophilic head chains and inhibition of the change of the amount of water inside the micelles by the PGFE heads and quinone ring of CoQ10. This indicates that PGFE-CoQ10 can function in water while maintaining the micellar structure formed in the storage solution. The findings of this study are important for the safety and nano-hazard aspects of PGFE-CoQ10 formulations.

Ivabradine ameliorates cardiomyopathy progression in a Duchenne muscular dystrophy model rat.

Duchenne muscular dystrophy (DMD) is an X-linked recessive myopathy caused by dystrophin mutations. Inevitable progressive cardiomyopathy is a current leading cause of premature death although respiratory management has improved the prognosis of patients with DMD. Recent evidence shows that reducing the heart rate is expected as one of the promising strategies for heart failure treatment, but administering a sufficient dose of ß-blocker for patients with DMD with tachycardia is difficult because of their low blood pressure (BP). Thus, this study aimed to clarify the role of ivabradine, which suppresses cardiac sinus node pacemakers without decreasing BP, in ameliorating cardiomyopathy progression in a rat model with DMD. A trans-oral single ivabradine administration demonstrated a declined dose-dependent heart rate without any significant BP reduction. Trans-gastric repeated administrations of 5 mg/kg of ivabradine twice a day for 3 months showed ameliorated cardiomyopathy in DMD rats based on echocardiography and histopathological observations (left ventricular dysfunction, right ventricular dysfunction, and myocardial fibrosis) as compared with vehicle administration. Our finding indicates that ivabradine is expected as another treatment choice for patients with DMD having tachycardia.

Factors affecting the number of bacteria in saliva and oral care methods for the recovery of bacteria in contaminated saliva after brushing: a randomized controlled trial.

BACKGROUND: Oral care is important in preventing aspiration pneumonia in older adults. However, it is not clear what kind of oral care can reduce the number of bacteria in saliva. The purposes of this study are to clarify whether there is a relationship between plaque amounts and salivary bacterial counts, and how bacteria dispersed into the oral cavity by brushing can be reduced. METHODS: First, saliva samples were collected from 10 healthy adult volunteers after 30 h of unbrushing and after thorough brushing, and the total bacterial count was determined by real-time PCR. Next, 40 older adults attending an outpatient dental clinic were randomly assigned into two groups: a wiping group (20 patients) and a mouthwashing group (20 patients). Saliva was collected before and after brushing, and after wiping in the wiping group and after mouthwashing in the mouthwashing group, and the total bacterial count was quantified by real-time PCR. RESULTS: In a study of volunteers, there was no association between plaque amounts and salivary bacterial counts. In a study of older adult patients, salivary bacterial counts were significantly higher in patients with higher oral hygiene index and fewer remaining teeth. Brushing increased salivary bacterial counts. Wiping did not significantly reduce the number of bacteria, while mouthwash returned the increased number of bacteria after brushing to the pre-brushing level. CONCLUSIONS: There is no direct relationship between the amount of plaque and the number of bacteria in saliva. Brushing disperses bacteria into the oral cavity, resulting in a marked increase in the number of bacteria in saliva. Wiping does not collect the dispersed bacteria, and it seems essential to rinse the mouth after brushing. TRIAL REGISTRATION: UMIN000045854.

Accumulation of senescent cells in the stroma of aged mouse ovary.

Senescent cells play a detrimental role in age-associated pathogenesis by producing factors involved in senescence-associated secretory phenotype (SASP). The present study was conducted to examine the possibility that senescent cells are present in aged ovaries and, if so, to determine the tissue region where senescent cells accumulate using a mouse model. Female mice at 2-4 and 8-10 months were used as reproductively young and aged models, respectively; the latter included mice with and without reproductive experience. Cells positive for senescence-associated ß-galactosidase (SA-ß-Gal) staining, one of the markers of cellular senescence, were detected in the stromal region of aged, but not young, ovaries regardless of reproductive experience. Likewise, the localization of cells expressing CDKN2A (cyclin dependent kinase inhibitor 2A), another senescence marker, in the stromal region of aged ovaries was detected with immunohistochemistry. CDKN2A expression detected by western blotting was significantly higher in the ovaries of aged mice with reproductive experience than in those without the experience. Moreover, cells positive for both γH2AX (a senescence marker) and fluorescent SA-ß-Gal staining were present in those isolated from aged ovaries. In addition, the transcript levels of several SASP factors were significantly increased in aged ovaries. These results suggest that senescent cells accumulate in the ovarian stroma and may affect ovarian function in aged mice. Additionally, reproductive experience may promote accumulation.

Optimal Dose of Intravenous Cyclophosphamide during remission induction therapy in ANCA-associated vasculitis: a retrospective cohort study of J-CANVAS.

OBJECTIVES: To identify the optimal dose of intravenous cyclophosphamide (IVCY) for induction therapy for anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV). METHODS: We retrospectively assessed patients with AAV who received IVCY every 2-3 weeks during the remission induction phase. The associations of the IVCY dose with infection-free survival and relapse-free survival were analysed using a Cox regression model. We compared patients in three categories: very low-dose (VLD), low-dose (LD), and conventional dose (CD) (<7.5 mg/kg, 7.5-12.5 mg/kg, and >12.5 mg/kg, respectively). The non-linear association between IVCY dose and the outcomes were also evaluated. RESULTS: Of the 80 patients (median age 72 years), 12, 42, and 26 underwent the VLD, LD, and CD regimens, respectively, of whom 4, 3, and 7 developed infection or died. The adjusted hazard ratios for infection or death were 4.3 (95% confidence interval (CI) 0.94-19.8) for VLD and 5.1 (95% CI 1.21-21.3) for CD, compared with LD. We found the hazard ratio for infection or death increased when the initial IVCY dose exceeded 9 mg/kg. Relapse-free survival did not differ clearly. CONCLUSION: Low-dose IVCY (7.5-12.5 mg/kg) may result in fewer infections and similar relapse rates compared with the conventional regimen (>12.5 mg/kg).

Renin inhibition and the long-term renal function in patients with hypertensive emergency: a retrospective cohort study.

BACKGROUND: The rehospitalization rate in hypertensive emergency is high, indicating the necessity for optimizing its long-term management. The role of the renin-angiotensin system (RAS) blockade in this disorder remains uncertain. METHODS: We conducted a retrospective analysis involving 20 admitted patients who received aliskiren, a direct renin inhibitor (DRI), for the management of hypertensive emergency associated with elevated plasma renin activity (PRA). We analyzed the changes in blood pressure (BP), kidney function, and RAS activity in the subacute and chronic phases. RESULTS: The use of DRI was associated with a marked reduction in PRA (median, from 25.0 to 1.2 ng/mL/hr) and serum aldosterone levels (from 404 to 130 pg/mL) during the index admission. BP also decreased from 226/143 to 142/80 mmHg. A comparison of clinical characteristics according to the renal function indicated that dialysis-dependent patients had higher aldosterone levels than non-dialysis-dependent patients at admission, despite comparable BP levels. After a median follow-up of 567 days in non-dialysis-dependent patients with DRI, median eGFR levels were significantly increased from 14.3 to 23.1 mL/min/1.73 m2. PRA levels were consistently suppressed at 0.8 ng/mL/hr. We found a significant correlation between the degree of PRA suppression and changes in eGFR (r = -0.58), indicating that the effective blockade of RAS is associated with the preservation of eGFR in the study subjects. CONCLUSIONS: DRI can successfully suppress PRA in patients with high-renin hypertensive emergency in both subacute and chronic phases. An efficient RAS blockade is associated with preserved renal function in these patients.

The effect of ACVR1B/TGFBR1/ACVR1C signaling inhibition on oocyte and granulosa cell development during in vitro growth culture.

The signals of the transforming growth factor ß (TGF-ß) superfamily play a critical role in follicular development in mammals. ACVR1B/TGFBR1/ACVR1C receptors mediate the signaling of several TGF-ß superfamily ligands in granulosa cells. Although the requirement for ACVR1B/TGFBR1/ACVR1C receptor signaling in follicular development has been confirmed using mutant mouse models, the detailed roles of the signaling in granulosa cell and oocyte development have not been clearly defined. In the present study, we examined the requirement for ACVR1B/TGFBR1/ACVR1C receptor signaling in granulosa cells using an in vitro growth culture of oocyte-granulosa cell complexes (OGCs) and SB431542, a potent inhibitor of the receptor signaling. Although cumulus-oocyte complexes isolated from the control OGCs were able to undergo cumulus expansion, those isolated from OGCs grown with the inhibitor were not competent, even in the presence of in vivo-grown oocytes. The diameter of the oocytes in the SB431542-treated OGCs was comparable with that of the control; however, these oocytes were not competent for complete meiotic maturation or preimplantation development. Therefore, ACVR1B/TGFBR1/ACVR1C receptor signaling is not required for oocytes to increase their volume but is essential for the normal development of cumulus cells and oocyte developmental competence.

The UFMylated ribosome-recognition protein SAYSD1 is predominantly expressed in spermatids but is dispensable for fertility in mice.

SAYSVFN domain-containing protein 1 (SAYSD1) is an evolutionarily conserved membrane protein that has recently been identified as a ubiquitin-fold modifier 1 (UFM1)-conjugated ribosome-recognition protein that plays a critical role in translocation-associated quality control (TAQC). However, its expression and roles in mammals in vivo remain largely unknown. We found that SAYSD1 is predominantly expressed in round and elongating spermatids and localizes in the endoplasmic reticulum (ER) of mouse testes, but not in differentiated spermatozoa. Mice deficient in Saysd1 developed normally post-partum. Furthermore, Saysd1-deficient mice were fertile, with no apparent differences in sperm morphology or motility compared with wild-type sperm, although the cauda epididymis contained slightly less sperm. Expression of the ER stress markers spliced X-box binding protein 1s (XBP1s) and CCAAT/enhancer binding protein (C/EBP)-homologous protein (CHOP) in the testes was comparable between Saysd1-deficient and wild-type mice. These results suggested that SAYSD1 is involved in sperm production in mice but is dispensable for their development and fertility.

Ascorbic acid enhances chondrocyte differentiation of ATDC5 by accelerating insulin receptor signaling.

Chondrogenesis is strictly regulated by several factors, including cytokines, hormones, and extracellular matrix proteins. Mouse teratocarcinoma-derived lineage cells, differentiate into chondrocytes in the presence of insulin. Although ascorbic acid promotes chondrogenic differentiation, the detailed regulative mechanisms underlying its role in chondrogenesis remain unclear. Therefore, in this study, we evaluated the effects of ascorbic acid on insulin-induced chondrogenic differentiation of ATDC5 cells and the underlying intracellular signaling. The results revealed that insulin-stimulated collagen deposition, matrix formation, calcification, and expression of chondrogenic differentiation marker genes in ATDC5 cells. This enhancement by insulin was amplified with the addition of ascorbic acid. Molecular analysis revealed that the activation of insulin-induced phosphoinositide 3-kinase (PI3K)/Akt signaling was enhanced in the presence of ascorbic acid. In contrast, Wnt/ß-catenin signaling was suppressed during chondrocyte differentiation via upregulation of the Wnt agonist, secreted Frizzled-related protein 1 (sFRP-1) and 3 (sFRP-3). Notably, ascorbic acid upregulated the expression of insulin receptors and their substrates (IRS-1 and IRS-2). Furthermore, ascorbic acid reversed the suppression of IRS-1 and IRS-2 protein by insulin. These results indicate that ascorbic acid positively regulates the chondrogenic differentiation of ATDC5 cells via enhancement of insulin signaling. Our findings provide a substantial basis for further elucidation of the regulatory mechanisms of chondrocyte differentiation and the pathophysiology of OA, thus aiding in development of effective treatment strategies.

Mineralocorticoid Receptor Antagonists for Preventing Chronic Kidney Disease Progression: Current Evidence and Future Challenges.

Regulation and action of the mineralocorticoid receptor (MR) have been the focus of intensive research over the past 80 years. Genetic and physiological/biochemical analysis revealed how MR and the steroid hormone aldosterone integrate the responses of distinct tubular cells in the face of environmental perturbations and how their dysregulation compromises fluid homeostasis. In addition to these roles, the accumulation of data also provided unequivocal evidence that MR is involved in the pathophysiology of kidney diseases. Experimental studies delineated the diverse pathological consequences of MR overactivity and uncovered the multiple mechanisms that result in enhanced MR signaling. In parallel, clinical studies consistently demonstrated that MR blockade reduces albuminuria in patients with chronic kidney disease. Moreover, recent large-scale clinical studies using finerenone have provided evidence that the non-steroidal MR antagonist can retard the kidney disease progression in diabetic patients. In this article, we review experimental data demonstrating the critical importance of MR in mediating renal injury as well as clinical studies providing evidence on the renoprotective effects of MR blockade. We also discuss areas of future investigation, which include the benefit of non-steroidal MR antagonists in non-diabetic kidney disease patients, the identification of surrogate markers for MR signaling in the kidney, and the search for key downstream mediators whereby MR blockade confers renoprotection. Insights into these questions would help maximize the benefit of MR blockade in subjects with kidney diseases.