Citrate supplementation is well known to alleviate muscle fatigue. Furthermore, our previous clinical study revealed that citrate supplementation prevents renal oxidative stress and dysfunction. We hypothesize that an interaction between muscle and kidney tissues underlies the effects of citrate supplementation. This study investigated the effects of a citrate agent, potassium citrate/sodium citrate (PCSC), on muscle and renal functions. Male Sprague-Dawley rats were randomly assigned to two groups (control and PCSC groups). PCSC (2000 mg/kg body weight) was administered orally administrated for one week. Kidney weight, vastus lateralis muscle weight, and renal function were compared between the groups. Subsequently, the kidney and muscle tissues were analyzed using metabolomics. The PCSC group showed a significant increase in muscle mass relative to the weight gain (p = 0.0472). Renal function development with growth was more pronounced in the PCSC group (p < 0.0001). Metabolomic analysis of muscle tissue in the PCSC group revealed increased alanine levels and decreased levels of sarcosine, creatinine, and NADPH/NADP+ ratio. In the kidney tissue, PCSC supplementation led to elevated N,N-dimethylglycine, urea, and the ratio of malic acid to aspartate, while betaine aldehyde, carnitine, and Fisher's ratio decreased. The study concluded that PCSC supplement facilitated muscle growth metabolically through an alanine-associated pathway and renal function development by increasing intrarenal urea and accelerating the malate-shuttle and the betaine pathway. These findings indicate PCSC's potential impact of PCSCs on muscle-kidney interactions.

Long COVID, a major post-acute infection syndrome (PAIS), characterized by prolonged or new-onset symptoms following acute COVID-19, critically affects patients' quality of life. Establishing easily measurable and objective biomarkers that reflect disease severity is essential for clinical management. This retrospective cohort study evaluated serum levels of growth differentiation factor 15 (GDF-15), a mitochondrial stress marker, and vascular cell adhesion molecule-1 (VCAM-1), a vascular endothelial stress marker, in 32 patients with Long COVID who presented with persistent systemic or neurological symptoms and had not required acute-phase hospitalization. Serum GDF-15, VCAM-1, and inflammatory cytokine levels were measured at the initial visit and at 3 and 6 months. Differences between patients who recovered at 6 months and those who did not (non-improved group) were analyzed using linear mixed-effects models (LMMs). Most inflammatory cytokines remained near their lower detection limits and were excluded from the longitudinal analysis. The LMMs revealed sustained elevation of both GDF-15 (p = 0.02) and VCAM-1 (p = 0.03) levels in the non-improved group. These findings suggest that longitudinal tracking of these two markers may offer clinically relevant insights into disease activity and treatment-resistant pathophysiology in mild acute-phase Long COVID.

BACKGROUND:Some patients recovered from COVID-19 may experience cognitive and psychological symptoms, such as "brain fog" or neuropsychological long COVID, and its mechanism is unclear. OBJECTIVE:This study aimed to investigate the mechanism of brain damage in neuropsychological long COVID using imaging and blood biomarkers. METHODS:Patients who met the criteria on the "brain fog" screening questionnaire and provided informed consent were enrolled in this study (n = 33; mean age 38.5 years). All participants were examined using magnetic resonance imaging, single-photon emission computed tomography (assessment of regional cerebral blood flow [rCBF]), and blood biomarkers. Neuropsychological tests (Montreal Cognitive Assessment-Japanese version [MoCA-J], Trail Making Test [TMT], Frontal Assessment Battery, Digital Symbol Coding [DSC] test, State-Trait Anxiety Inventory [STAI], and Self-Rating Depression Scale [SDS]) were performed simultaneously. RESULTS:Significant correlations were observed between the MoCA-J score and decreased rCBF in the left occipital lobe and increased rCBF in the right occipital lobe (p < 0.05), between the STAI score and decreased rCBF in the right parietal lobe (p < 0.05), and between the SDS score and decreased rCBF in the right parietal lobe (p < 0.05). The MoCA-J and DSC scores were correlated with plasma levels of neurofilament light chain (p < 0 0.05). The TMT time correlated with plasma glial fibrillary acidic protein levels (p < 0.01). CONCLUSION:This study was a cross-sectional and could not distinguish pathological abnormalities. However, as correlations of neuropsychological long COVID with specific brain regions and plasma biomarkers have been elucidated, conducting a case-control analysis may be worthwhile.

Background:Long COVID symptoms, especially brain fog, significantly impair patient quality of life (QOL); however, effective treatments remain elusive. Japanese traditional medicine (JTM, usually called Kampo medicine) is often used adjunctively to treat patients with diverse manifestations of long COVID. Objective:To evaluate the effectiveness of JTM in treating long COVID using a comprehensive QOL assessment. Methods:This single-center, prospective observational study evaluated QOL changes in patients with symptoms persisting beyond 28 days from the onset of COVID-19 (long COVID) who visited our general medicine outpatient department between October 2021 and August 2024. The treatment plan was determined by the attending physician based on the patient's condition. The health-related QOL (HR-QOL) was comprehensively assessed using EuroQol-5 demensions-5levels (EQ-5D-5L) scores (ranging from -0.025 to 1.000, with higher values indicating better HR-QOL) at baseline and 3 months after the first visit. The formulations and factors associated with QOL changes were analyzed using multivariate logistic regression analyses. Results:We analyzed 112 patients. The most common symptoms were fatigue (83.9%). The median (interquartile range) HR-QOL of the entire cohort significantly increased from 0.711 (0.561-0.711) at baseline to 0.833 (0.671-0.890) at 3 months (p < 0.0001); the proportion of patients exceeding the national standard significantly increased from 7.1% to 20.4% (p = 0.0037). The brain fog patients group (50.0%, N = 56), the median (interquartile range) HR-QOL of the entire cohort significantly increased from 0.677 (0.551-0.770) at baseline to 0.750 (0.623-0.846) at 3 months (p < 0.005). However, the proportion of patients achieving the Japanese average HR-QOL did not show improvement significantly. A total of 101 patients (90.2%) were treated with JTM, and a combination of kamikihito and saikokeishito was administered to 20 patients. Multivariate regression analysis revealed that the combination usage was associated with greater improvements in the HR-QOL in all patients (odds ratio 5.4) and brain fog patients' group (odds ratio 6.1). Conclusion:Long COVID treatment involving JTM improved the patients' QOL at 3 months. The combination of kamikihito with saikokeishito may be a potential treatment option for long COVID. However, a randomized controlled trial is required to confirm its efficacy.

The beneficial effects of oral supplements with alkalinizing agents in patients with chronic kidney disease (CKD) have been limited to the severe stages. We investigated whether two types of supplements, sodium bicarbonate (SB) and potassium citrate/sodium citrate (PCSC), could maintain renal function in patients with mild-stage CKD. This was a single-center, open-labeled, randomized cohort trial. Study participants with CKD stages G2, G3a, and G3b were enrolled between March 2013 and January 2019 and randomly assigned by stratification according to age, sex, estimated glomerular filtration rate (eGFR), and diabetes. They were followed up for 6 months (short-term study) for the primary endpoints and extended to 2 years (long-term study) for the secondary endpoints. Supplementary doses were adjusted to achieve an early morning urinary pH of 6.8–7.2. We observed renal dysfunction or new-onset cerebrovascular disease and evaluated urinary surrogate markers for renal injury. Overall, 101 participants were registered and allocated to three groups: standard (n = 32), SB (n = 34), and PCSC (n = 35). Two patients in the standard group attained the primary endpoints (renal stones and overt proteinuria) but were not statistically significant. There was one patient in the standard reduced eGFR during the long-term study (p = 0.042 by ANOVA). SB increased proteinuria (p = 0.0139, baseline vs. 6 months), whereas PCSC significantly reduced proteinuria (p = 0.0061, baseline vs. 1 year, or p = 0.0186, vs. 2 years) and urinary excretion of 8-hydroxy-2′-deoxyguanosine (p = 0.0481, baseline vs. 6 months). This study is the first to report supplementation of PCSC reduced intrarenal oxidative stress in patients with mild-stage CKD.

Introduction Urine contains various cells derived from the kidney, including renal tubular epithelial cells, glomerular epithelial cells, and mesenchymal stem cells. Urinary exfoliated mesenchymal stem cells can be differentiated into adipocytes, osteoblasts, chondrocytes, muscle cells, or neuronal cells, if grown in specialized culture conditions. In this study, however, we identified the development of multiple mesodermal cell lineages from urinary exfoliated cells despite using a single culture system not designed for these cell differentiations. Methods Urinary exfoliated cells used for culturing were obtained from pediatric patients with kidney disease. All cultures were maintained using an identical, non-specifically supplemented, rich, DMEM/F12-based growth medium, refreshed regularly throughout the experiments. Cells of different mesodermal lineages—adipocytes, osteoblasts, and chondrocytes—were identified by staining with Oil Red O, Alizarin Red, and Alcian Blue solutions. Moreover, the expression of mRNA markers specific for adipocytes, osteoblasts, and chondrocytes, as well as for stem cells and—more specifically—mesenchymal stem cells, was analyzed by RT-PCR. Results Although the abundances varied, in all 11 cultures of urinary exfoliated cells from six patients, dye staining revealed multiple cell clusters of mesodermal lineages. All cultures contained clusters of the adipocyte and chondrocyte lineages, and seven also contained clusters of the osteoblast lineage. Morphology differences were found even between clusters stained with the same dye. RT-PCR confirmed the presence of the three lineages, while also revealing expression of genes specific to pluripotent stem cells ( Nanog, Oct3/4, SOX2 , and LIF ) and genes more specific for mesenchymal stem cells ( CD73, CD90 , and CD105 ). Conclusions Among urinary exfoliated cells cultured using our method, clusters of cells of mesodermal lineages were observed without needing to selectively adjust the culture medium. The cells seemed to differentiate and proliferate from mesenchymal stem cells in the urine. We envision that these initial findings may eventually contribute to a deeper understanding of urinary stem cell differentiation and to optimizing lineage-specific differentiation for research and therapeutic applications. ### Competing Interest Statement The authors have declared no competing interest. * ACS : Acetyl-CoA synthetase ALP : Alkaline phosphatase ANCA : anti-neutrophil cytoplasmic antibody associated glomerulonephritis BSA : bovine serum albumin C/EBP : CCAAT/enhancer-binding protein DEX : dexamethasone DMEM/F12 : Dulbecco’s modified Eagle’s medium/Hams F12 FABP4 : Fatty acid-binding protein 4 FBS : fetal bovine serum GAPDH : Glyceraldehyde-3-phosphate dehydrogenase HSPN : Henoch-Schönlein purpura nephritis IBMX : isobutylmethylxanthine IgAN : IgA nephropathy ITS : insulin, transferrin, and selenium LIF : Leukemia inhibitory factor LN : Lupus nephritis LPL : Lipoprotein lipase MEM : minimum essential medium MN : membranous nephropathy PBS : phosphate-buffered saline PPAR : Peroxisome proliferator-activated receptor RUX2 : Runt-related transcription factor 2 SOX2 : SRY-box transcription factor 2 SOX9 : SRY-box transcription factor 9 Japan Society for the Promotion of Science, JP21790966

Background Metabolic acidosis is a result and a risk factor of chronic kidney disease (CKD). Alkali loads can neutralize acidosis, but the reno-protective effects of the supplementation on CKD have been limited. Here we evaluated the reno-protective effect of the alkali supplement, potassium citrate/sodium citrate tablet (PCSC), on an adenine-induced-CKD animal model. Methods To evaluate the reno-protective effects of PCSC, mice (C57BL/6J, female, 5 weeks) orally administrated with adenine for 18 days were compared between the PCSC group and the vehicle. The weights of the body, heart, bilateral kidneys, quadriceps femoris, renal function, and histological changes were statistically analyzed Results Renal functions of both groups were not different, however, increments of kidney weights were significantly different (p=0.047). It was considered that PCSC suppressed adenine-induced acute injury. In the histological analysis, enlargement of the papilla area and tubular dilatation of the cortex were diminished in the PCSC group. Conclusion This is the first report demonstrating that PCSC prevented the development of renal tubulointerstitial injury of the murine adenine-induced CKD model.

The gastrointestinal (GI) tract harbors trillions of microorganisms known to influence human health and disease, and next-generation sequencing (NGS) now enables the in-depth analysis of their diversity and functions. Although a significant amount of research has been conducted on the GI microbiome, comprehensive metagenomic datasets covering the entire tract are scarce due to cost and technical challenges. Despite the widespread use of fecal samples, integrated datasets encompassing the entire digestive process, beginning at the mouth and ending with feces, are lacking. With this study, we aimed to fill this gap by analyzing the complete metagenome of the GI tract, providing insights into the dynamics of the microbiota and potential therapeutic avenues. In this study, we delved into the complex world of the GI microbiota, which we examined in five healthy Japanese subjects. While samples from the whole GI flora and fecal samples provided sufficient bacteria, samples obtained from the stomach and duodenum posed a challenge. Using a principal coordinate analysis (PCoA), clear clustering patterns were identified; these revealed significant diversity in the duodenum. Although this study was limited by its small sample size, the flora in the overall GI tract showed unwavering consistency, while the duodenum exhibited unprecedented phylogenetic diversity. A visual heat map illustrates the discrepancy in abundance, with Fusobacteria and Bacilli dominating the upper GI tract and Clostridia and Bacteroidia dominating the fecal samples. Negativicutes and Actinobacteria were found throughout the digestive tract. This study demonstrates that it is possible to continuously collect microbiome samples throughout the human digestive tract. These findings not only shed light on the complexity of GI microbiota but also provide a basis for future research.

Abstract Background Proximal renal tubular dysfunction occurs during diabetic ketoacidosis (DKA) in type 1 diabetes. However, only a few studies have reported on the multiple proximal renal tubular functions simultaneously. Moreover, to the best of our knowledge, distal renal tubular function has not yet been investigated. Methods Patients with newly diagnosed type 1 diabetes mellitus were classified into those with DKA and those without DKA, and their proximal and distal renal tubular functions were investigated. The diagnostic criteria for DKA were blood glucose > 200 mg/dL, blood pH < 7.3 or HCO3– < 15 mEq/L, and urine ketone body positivity. Results Six patients with DKA and five patients without DKA were included. In patients with DKA, urinary β2-microglobulin levels were significantly higher, while blood pH, HCO3–, and tubular reabsorption of phosphorus were significantly lower than in those without DKA. There were no significant differences in blood glucose, HbA1c, serum phosphorus, urinary N-acetyl-beta-glucosaminidase, and urinary amino acid excretion between patients with and without DKA. Elevated NH3 levels and impaired urinary acidification were not observed in patients with and without DKA. Conclusions In patients with newly diagnosed type 1 diabetes mellitus complicated with DKA, multiple proximal renal tubular dysfunctions occur simultaneously, suggesting transient Fanconi syndrome. Distal renal tubular acidosis was unlikely. The diagnostic criteria for DKA are appropriate also in the view of proximal renal tubular dysfunction and are considered suggestive of pathophysiological factors that may cause proximal renal tubular dysfunction.

Mitochondrial dysfunction is associated with various diseases. Mitochondria plays a regulatory role during infection. The association between mitokines and subsequent COVID progression has not been previously studied. The retrospective cohort study aimed to investigate the potential of serum mitokines as long COVID biomarkers in non-hospitalized patients. Patients with confirmed SARS-CoV-2 infection and blood test reports between January 2021 and April 2023 were included. Patients were categorized into two groups, the recovered and long COVID groups, based on fatigue, decline in focus, and pain. Serum levels of growth differentiation factor 15 (GDF-15) and fibroblast growth factor-21 (FGF-21), which are affected by mitochondrial function, along with inflammatory and vascular endothelium markers, were measured using enzyme-linked immunosorbent assays (ELISA). A receiver operating characteristic curve was used to screen the biomarkers. The threshold value of GDF-15 in the acute phase was 965 pg/mL (sensitivity: 71.4%, specificity: 83.3%), indicating that GDF-15 may be associated with the presence of symptoms three months post onset. No association with inflammatory markers and vascular structures was observed. Therefore, elevated GDF-15 levels in the acute phase may act as a predictive biomarker of long COVID.

Table S1. Patients' characteristics analyzed in this study

Table S2. Nucleotide sequences of the primers and PCR conditions

Supplementary Figure S1. Genetic alterations of TP53 (exons 5 through 9) and KRAS (exon 2 containing codons 12 and 13) in Cases #1, 6, and 9 were Sanger sequenced; both strands were analyzed.

<p>Table S2. Nucleotide sequences of the primers and PCR conditions</p>

The coronavirus disease 2019 (COVID-19) pandemic remains a global public health concern. The clinical course and risk of developing severe illness among patients with COVID-19 who are at low-risk of severe COVID-19 remain uncertain. This retrospective cohort study from an isolation facility for low-risk COVID-19 patients in Japan evaluated the potential risks for severe disease with hypoxia (SpO2 ≤ 93%) or experiencing prolonged isolation period longer than 14 days with persistent acute symptoms. The study was performed before the spread of the alpha variant in the country and before the start of a nationwide mass vaccination campaign against COVID-19. Among the 929 participants with reliable outcome data regarding the development of hypoxia, 63 (6.8%) developed severe disease with hypoxia during their stays at the facility. Higher age [adjusted odds ratio (aOR), 1.08; 95% confidence interval (CI), 1.06-1.10] and male sex (aOR, 4.70; 95% CI, 2.39-9.22) were associated with this outcome. As for the experience of prolonged isolation period, higher age (aOR, 1.02; 95% CI, 1.01-1.04), atopic diseases (aOR, 1.69, 95% CI, 1.09-2.64), presence of cough at onset (aOR, 1.64; 95% CI, 1.09-2.48), and prescription of oral antibiotics before positive test results for COVID-19 (aOR, 2.37; 95% CI, 1.33-4.22) were associated with this outcome. In summary, 5-10% of low-risk COVID-19 patients later develop hypoxia. Older age and male sex were associated with both the development of hypoxia and prolonged acute symptoms. The unnecessary prescription of antibiotics before COVID-19 diagnosis may prolong COVID-19 symptoms.

Table S2. Nucleotide sequences of the primers and PCR conditions

Background and aim The prevalence and characteristics of physical complaints related to bad weather among the general population remain poorly understood. This study aimed to elucidate the characteristics of subjective physical symptoms related to bad weather. Methods A cross-sectional survey was conducted by using self-reported health-related questionnaires obtained from individuals undergoing annual medical check-ups at a municipal hospital in Japan. Participants were asked about the presence and details of physical symptoms related to bad weather, together with other health-related questions. Results Among the 133 participants, 42 (32%) (95%CI 24-40) reported experiencing physical conditions related to bad weather. Among these 42 patients, the most common ailment was headache (67%; n=28), followed by low back pain (21%; n=9), fatigue (19%; n=8), and stiff neck/shoulder discomfort (12%; n=5). Comparison between individuals with and without bad weather-related conditions revealed that those affected were younger (p=0.0014) and exhibited higher numerical rating scale scores for gastrointestinal problems (p=0.0027), irritability/agitation (p<0.0001), and sleep disorders (p=0.0295). These associations were confirmed even after adjusting for age and sex. Conclusions Physical conditions related to bad weather, represented by headache, fatigue, and back pain, can be seen in 25-40% of the general population, especially in younger age groups. Individuals with these conditions are more likely to experience irritability/agitation, gastrointestinal problems, and sleep disorders.