Malt1-induced cleavage of regnase-1 in CD4(+) helper T cells regulates immune activation.

Regnase-1 (also known as Zc3h12a and MCPIP1) is an RNase that destabilizes a set of mRNAs, including Il6 and Il12b, through cleavage of their 3' UTRs. Although Regnase-1 inactivation leads to development of an autoimmune disease characterized by T cell activation and hyperimmunoglobulinemia in mice, the mechanism of Regnase-1-mediated immune regulation has remained unclear. We show that Regnase-1 is essential for preventing aberrant effector CD4(+) T cell generation cell autonomously. Moreover, in T cells, Regnase-1 regulates the mRNAs of a set of genes, including c-Rel, Ox40, and Il2, through cleavage of their 3' UTRs. Interestingly, T cell receptor (TCR) stimulation leads to cleavage of Regnase-1 at R111 by Malt1/paracaspase, freeing T cells from Regnase-1-mediated suppression. Furthermore, Malt1 protease activity is critical for controlling the mRNA stability of T cell effector genes. Collectively, these results indicate that dynamic control of Regnase-1 expression in T cells is critical for controlling T cell activation.

HKDC1, a target of TFEB, is essential to maintain both mitochondrial and lysosomal homeostasis, preventing cellular senescence.

Mitochondrial and lysosomal functions are intimately linked and are critical for cellular homeostasis, as evidenced by the fact that cellular senescence, aging, and multiple prominent diseases are associated with concomitant dysfunction of both organelles. However, it is not well understood how the two important organelles are regulated. Transcription factor EB (TFEB) is the master regulator of lysosomal function and is also implicated in regulating mitochondrial function; however, the mechanism underlying the maintenance of both organelles remains to be fully elucidated. Here, by comprehensive transcriptome analysis and subsequent chromatin immunoprecipitation-qPCR, we identified hexokinase domain containing 1 (HKDC1), which is known to function in the glycolysis pathway as a direct TFEB target. Moreover, HKDC1 was upregulated in both mitochondrial and lysosomal stress in a TFEB-dependent manner, and its function was critical for the maintenance of both organelles under stress conditions. Mechanistically, the TFEB-HKDC1 axis was essential for PINK1 (PTEN-induced kinase 1)/Parkin-dependent mitophagy via its initial step, PINK1 stabilization. In addition, the functions of HKDC1 and voltage-dependent anion channels, with which HKDC1 interacts, were essential for the clearance of damaged lysosomes and maintaining mitochondria-lysosome contact. Interestingly, HKDC1 regulated mitophagy and lysosomal repair independently of its prospective function in glycolysis. Furthermore, loss function of HKDC1 accelerated DNA damage-induced cellular senescence with the accumulation of hyperfused mitochondria and damaged lysosomes. Our results show that HKDC1, a factor downstream of TFEB, maintains both mitochondrial and lysosomal homeostasis, which is critical to prevent cellular senescence.

Self-Supervised Learning for Feature Extraction from Glomerular Images and Disease Classification with Minimal Annotations.

BACKGROUND: Deep learning has great potential in digital kidney pathology. However, its effectiveness depends heavily on the availability of extensively labeled datasets, which are often limited due to the specialized knowledge and time required for their creation. This limitation hinders the widespread application of deep learning for the analysis of kidney biopsy images. METHODS: We applied self-distillation with no labels (DINO), a self-supervised learning method, to a dataset of 10,423 glomerular images obtained from 384 PAS-stained kidney biopsy slides. Glomerular features extracted from the DINO-pretrained backbone were visualized using principal component analysis (PCA). We then performed classification tasks by adding either k-nearest neighbor (kNN) classifiers or linear head layers to the DINO-pretrained or ImageNet-pretrained backbones. These models were trained on our labeled classification dataset. Performance was evaluated using metrics such as the area under the receiver operating characteristic curve (ROC-AUC). The classification tasks encompassed four disease categories (minimal change disease, mesangial proliferative glomerulonephritis, membranous nephropathy, and diabetic nephropathy) as well as clinical parameters such as hypertension, proteinuria, and hematuria. RESULTS: PCA visualization revealed distinct principal components corresponding to different glomerular structures, demonstrating the capability of the DINO-pretrained backbone to capture morphological features. In disease classification, the DINO-pretrained transferred model (ROC-AUC = 0.93) outperformed the ImageNet-pretrained fine-tuned model (ROC-AUC = 0.89). When the labeled data were limited, the ImageNet-pretrained fine-tuned model's ROC-AUC dropped to 0.76 (95% confidence interval [CI], 0.72-0.80), whereas the DINO-pretrained transferred model maintained superior performance (ROC-AUC 0.88, 95% CI 0.86-0.90). The DINO-pretrained transferred model also exhibited higher AUCs for the classification of several clinical parameters. External validation using two independent datasets confirmed DINO pre-training's superiority, particularly when labeled data were limited. CONCLUSIONS: The application of DINO to unlabeled PAS-stained glomerular images facilitated the extraction of histological features that can be effectively utilized for disease classification.

Endogenous activation of peroxisome proliferator-activated receptor-α in proximal tubule cells in counteracting phosphate toxicity.

Increased dietary phosphate consumption intensifies renal phosphate burden. Several mechanisms for phosphate-induced renal tubulointerstitial fibrosis have been reported. Considering the dual nature of phosphate as both a potential renal toxin and an essential nutrient for the body, kidneys may possess inherent protective mechanisms against phosphate overload, rather than succumbing solely to injury. However, there is limited understanding of such mechanisms. To identify these mechanisms, we conducted single-cell RNA sequencing (scRNA-seq) analysis of the kidneys of control and dietary phosphate-loaded (Phos) mice at a time point when the Phos group had not yet developed tubulointerstitial fibrosis. scRNA-seq analysis identified the highest number of differentially expressed genes in the clusters belonging to proximal tubular epithelial cells (PTECs). Based on these differentially expressed genes, in silico analyses suggested that the Phos group activated peroxisome proliferator-activated receptor-α (PPAR-α) and fatty acid ß-oxidation (FAO) in the PTECs. This activation was further substantiated through various experiments, including the use of an FAO activity visualization probe. Compared with wild-type mice, Ppara knockout mice exhibited exacerbated tubulointerstitial fibrosis in response to phosphate overload. Experiments conducted with cultured PTECs demonstrated that activation of the PPAR-α/FAO pathway leads to improved cellular viability under high-phosphate conditions. The Phos group mice showed a decreased serum concentration of free fatty acids, which are endogenous PPAR-α agonists. Instead, experiments using cultured PTECs revealed that phosphate directly activates the PPAR-α/FAO pathway. These findings indicate that noncanonical metabolic reprogramming via endogenous activation of the PPAR-α/FAO pathway in PTECs is essential to counteract phosphate toxicity.NEW & NOTEWORTHY This study revealed the activation of peroxisome proliferator-activated receptor-α and fatty acid ß-oxidation in proximal tubular epithelial cells as an endogenous mechanism to protect the kidney from phosphate toxicity. These findings highlight noncanonical metabolic reprogramming as a potential target for suppressing phosphate toxicity in the kidneys.

Enhanced fatty acid oxidation by selective activation of PPARα alleviates autoimmunity through metabolic transformation in T-cells.

While fatty acid oxidation (FAO) in mitochondria is a primary energy source for quiescent lymphocytes, the impact of promoting FAO in activated lymphocytes undergoing metabolic reprogramming remains unclear. Here, we demonstrate that pemafibrate, a selective PPARα modulator used clinically for the treatment of hypertriglyceridemia, transforms metabolic system of T-cells and alleviates several autoimmune diseases. Pemafibrate suppresses Th17 cells but not Th1 cells, through the inhibition of glutaminolysis and glycolysis initiated by enhanced FAO. In contrast, a conventional PPARα agonist fenofibrate significantly inhibits cell growth by restraining overall metabolisms even at a dose insufficient to induce fatty acid oxidation. Clinically, patients receiving pemafibrate showed a significant decrease of Th17/Treg ratio in peripheral blood. Our results suggest that augmented FAO by pemafibrate-mediated selective activation of PPARα restrains metabolic programs of Th17 cells and could be a viable option for the treatment of autoimmune diseases.

Evaluation of Individual Variation of d-Amino Acids in Human Plasma by a Two-Dimensional LC-MS/MS System and Application to the Early Diagnosis of Chronic Kidney Disease.

For the discovery of sensitive biomarkers of kidney function focusing on chiral amino acids, a multiple heart-cutting two-dimensional (2D) liquid chromatography-mass spectrometry (LC-MS)/MS system has been designed/developed. As the target analytes, alanine (Ala), aspartic acid, glutamic acid (Glu), leucine (Leu), lysine, methionine, phenylalanine (Phe), proline (Pro), serine (Ser), and valine were selected considering the presence of their d-forms in mammals. The 2D LC-MS/MS system consisted of the nonenantioselective reversed-phase separation of the target amino acids, the separations of the d- and l-enantiomers, and detection using MS/MS. Using the method, the plasma chiral amino acids, precolumn derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole, were isolated from other intrinsic substances, then determined without losing sensitivity by the fully automated whole-peak volume transfer operation from first to second dimension. In all of the tested plasma samples obtained from five healthy individuals and 15 patients with chronic kidney disease (CKD), the target chiral amino acids were determined without interference. In healthy individuals, the levels of all the tested d-amino acids were regulated in the low ranges. In contrast, the % d values of Glu, Leu, and Phe significantly increased with the progress of kidney dysfunction, besides the previously reported values of d-Ala, Pro, and Ser. Concerning Phe, the significant increase of the % d values (p < 0.05) was reported for the first time even in the mild CKD group compared to those of the healthy group; d-Phe might be a more sensitive marker than the previously reported d-forms. These results demonstrated the potential of these d-forms as the sensitive biomarkers of kidney function for the early diagnosis of CKD.

Long-Term Efficacy and Safety of Upacicalcet in Japanese Hemodialysis Patients with Secondary Hyperparathyroidism: Open-Label 52-Week Study.

INTRODUCTION: Upacicalcet is a novel injectable calcimimetic. This phase 3 multicenter open-label study aimed to assess the long-term efficacy and safety of upacicalcet in hemodialysis (HD) patients with secondary hyperparathyroidism (SHPT). METHODS: Japanese HD patients with serum intact parathyroid hormone (iPTH) levels >240 pg/mL and corrected calcium (cCa) levels ≥8.4 mg/dL were enrolled. Upacicalcet with a dose range of 25-300 µg was administered after each dialysis for 52 weeks. The main efficacy endpoint was the percentage of patients achieving the target iPTH level (60-240 pg/mL). RESULTS: A total of 157 patients were enrolled, of whom 138 completed the study. Overall, 94.2% of patients achieved the target serum iPTH level at week 52. Neither symptomatic hypocalcemia nor cCa level <7.5 mg/dL occurred despite the negligible increase of concomitant vitamin D receptor activators and calcium carbonate. Upacicalcet improved the control of serum phosphate (P) and calcium levels regardless of baseline PTH levels and decreased intact fibroblast growth factor-23 levels. The largest parathyroid glands shrank, irrespective of their baseline volume or prior calcimimetic usage. Upacicalcet was well tolerated, with no adverse events requiring dose reduction. CONCLUSION: This is the first study to show that a calcimimetic improves serum P and cCa control without inducing severe hypocalcemia in patients with iPTH levels ≤300 pg/mL. Upacicalcet is efficacious in HD patients with mild-to-severe SHPT, with few safety concerns.

Kinetic analysis of D-Alanine upon oral intake in humans.

D-Alanine, a rare enantiomer of alanine, can potentially alleviate the worsening of viral infections and maintain circadian rhythm. This study aimed to analyze the kinetics of D-Alanine upon oral intake. Five healthy volunteers were administered D-Alanine as a single oral dose at 11,236 or 33,708 µmoL (1-3 g). Upon intake of the lower dose, the plasma level of D-Alanine reached its peak concentration of 588.4 ± 40.9 µM with a peak time of 0.60 ± 0.06 h. The compartment model estimated the clearance of D-Alanine at 12.5 ± 0.3 L/h, or 208 ± 5 mL/min, distribution volume of 8.3 ± 0.7 L and half-life of 0.46 ± 0.04 h, suggesting a rapid clearance of D-Alanine. The peak concentration and area under the curve increased proportionally upon intake of the higher dose, while the clearance, distribution volume and half-life did not. The urinary ratio of D-Alanine per sum of D- and L-Alanine reached its peak of nearly 100%, followed by a slow decline. The peak time of the urinary ratio was 1.15 ± 0.15 h, showing a time lag of blood to urine excretion. Fractional excretion, a ratio of the clearance of a substance per a standard molecule in kidney, of D-Alanine increased from 14.0 ± 5.8% to 64.5 ± 10.3%; the latter corresponded to the urinary clearance of D-Alanine as about 77 mL/min for an adult, with a peak time of 1.90 ± 0.56 h. D-Alanine was quickly absorbed and appeared in blood, followed by urinary excretion. This kinetic analysis increases our fundamental knowledge of the oral intake of D-Alanine for the chronic dosing. Trial number: #UMIN000050865. Date of registration: 2023/6/30.

Factors affecting the sodium-glucose cotransporter 2 inhibitors-related initial decline in glomerular filtration rate and its possible effect on kidney outcome in chronic kidney disease with type 2 diabetes: The Japan Chronic Kidney Disease Database.

AIM: Sodium-glucose cotransporter 2 (SGLT2) inhibitors often cause a transient decrease in glomerular filtration rate (GFR) shortly after the initiation, referred to as the 'initial drop'. However, the clinical significance of this initial drop in real-world practice remains unclear. MATERIALS AND METHODS: Using the nationwide Japan Chronic Kidney Disease Database, we examined factors that affected the initial drop, in patients with chronic kidney disease (CKD) and type 2 diabetes mellitus (T2DM). We also evaluated the effects of the initial drop on a composite kidney outcome (a decline in GFR of ≥50% or progression to end-stage kidney disease). RESULTS: Data from 2053 patients with CKD and T2DM newly prescribed an SGLT2 inhibitor were analysed. The follow-up period after SGLT2 inhibitor administration was 1015 days (interquartile range: 532, 1678). Multivariate linear regression models revealed that the concomitant use of the renin-angiotensin system inhibitors and diuretics, urinary protein levels ≥2+, and changes in GFR before the initiation of the SGLT2 inhibitor were associated with a larger initial GFR decline (ß = -0.609, p = .039; ß = -2.298, p < .001; ß = -0.936, p = .048; ß = -0.079, p < .001, respectively). Patients in the quartile with the largest initial GFR decline experienced a higher incidence of the subsequent composite kidney outcome than those in the other quartiles (p < .001). CONCLUSIONS: The concomitant use of renin-angiotensin system inhibitors and diuretics, higher urine protein levels and pre-treatment GFR changes were associated with a larger initial GFR decline. Of these factors, the use of a diuretic had the largest effect. Furthermore, patients with CKD and T2DM experiencing an excessive initial GFR drop might be at a higher risk of adverse kidney outcomes.

Clinical Impacts of Allograft Biopsy in Renal Transplant Recipients 10 Years or Longer After Transplantation.

We aimed to investigate the clinical value of allograft biopsy performed long after renal transplantation. We retrospectively evaluated 99 allograft biopsies in recipients with transplantation vintages of 10 years or longer. Mixed-effects model showed that 1-year estimated glomerular filtration rate (eGFR) slopes after biopsy were significantly greater than those before biopsy [-3.13, -4.42 mL/min/1.73 m2/year, p = 0.01]. Renal biopsy changed the treatment strategies in more than half of the patients. Improvement in eGFR slopes was pronounced in 51 patients with treatment modification based on the biopsy results [2.27 (95% confidence interval (CI): 0.66, 3.89) mL/min/1.73 m2/year], whereas no improvement was observed in those without [0.33 (95% CI: -1.05, 1.71) mL/min/1.73 m2/year, Pinteraction = 0.001]. Among the treatment modifications, enhancement of immunosuppression (IS) led to the most remarkable improvement in eGFR slope. Patients with g scores ≥2 were more likely to receive IS enhancement than those with g scores = 0 [odds ratio; 15.0 (95% CI: 1.65, 136)]. Patients with active glomerulitis (g ≥ 1) without chronicity (cg ≤ 1) showed the most significant improvement in eGFR slope. Given the prevalence of active glomerulitis (g ≥ 1, 21%), which is responsive to treatment even long after transplantation, and the observed magnitude of eGFR slope improvement, renal biopsy can indeed improve allograft prognosis.

The association between embedded catheter implantation and hospitalization costs for peritoneal dialysis initiation: a retrospective cohort study.

BACKGROUND: Compared with the conventional peritoneal dialysis (PD) catheter insertion, embedding PD catheter implantation is one of the procedures for planned PD initiation. However, facilities where embedded PD catheter implantation is available are limited, and the impact of embedded PD catheter implantation on hospitalization cost and length of hospitalization is unknown. METHODS: This retrospective single-center cohort study included 132 patients with PD initiation between 2005 and 2020. The patients were divided into two groups: 64 patients in the embedding group and 68 patients in the conventional insertion group. We created a multivariable generalized linear model (GLM) with the gamma family and log-link function to evaluate the association among catheter embedding, the duration and medical costs of hospitalization for PD initiation. We also evaluated the effect modification between age and catheter embedding. RESULTS: Catheter embedding (ß coefficient - 0.13 [95% confidence interval - 0.21, - 0.05]) and age (per 10 years 0.08 [0.03, 0.14]) were significantly associated with hospitalization costs. Catheter embedding (- 0.21 [- 0.32, - 0.10]) and age (0.11 [0.03, 0.19]) were also identified as factors significantly associated with length of hospitalization. The difference between the embedding group and the conventional insertion group in hospitalization costs for PD initiation (P for interaction = 0.060) and the length of hospitalization (P for interaction = 0.027) was larger in young-to-middle-aged patients than in elderly patients. CONCLUSIONS: Catheter embedding was associated with lower hospitalization cost and shorter length of hospitalization for PD initiation than conventional PD catheter insertion, especially in young-to-middle-aged patients.

Effects of sodium-glucose co-transporter 2 inhibitors on ultrafiltration in patients with peritoneal dialysis: a protocol for a randomized, double-blind, placebo-controlled, crossover trial (EMPOWERED).

BACKGROUND: Volume overload is common and associated with high mortality in patients on peritoneal dialysis (PD). Traditional strategies including diuretics, water/salt restriction, and icodextrin-based solutions cannot always fully correct this condition, necessitating novel alternative strategies. Recent studies confirmed the expression of sodium-glucose cotransporter 2 (SGLT2) in the human peritoneum. Experimental data suggest that SGLT2 inhibitors decrease glucose absorption from the PD solution, thereby increasing the ultrafiltration volume. This trial aims to assess whether SGLT2 inhibitors increase the ultrafiltration volume in patients on PD. METHODS: The EMPOWERED trial (trial registration: jRCTs051230081) is a multicenter, randomized, double-blind, placebo-controlled, crossover trial. Patients with clinically diagnosed chronic heart failure are eligible regardless of the presence of diabetes if they use at least 3 L/day glucose-based PD solutions. Participants will be randomly assigned (1:1) to receive empagliflozin 10 mg once daily and then placebo or vice versa. Each treatment period will last 8 weeks with a 4-week washout period. This study will recruit at least 36 randomized participants. The primary endpoint is the change in the daily ultrafiltration volume from baseline to week 8 in each intervention period. The key secondary endpoints include changes in the biomarkers of drained PD solutions, renal residual function, and anemia-related parameters. CONCLUSIONS: This trial aims to assess the benefit of SGLT2 inhibitors in fluid management with a novel mechanism of action in patients on PD. It will also provide insights into the effects of SGLT2 inhibitors on solute transport across the peritoneal membrane and residual renal function.

Cigarette smoking and progression of kidney dysfunction: a longitudinal cohort study.

INTRODUCTION: Cigarette smoking is one of the most important life-modifiable risk factors for CVD events. The effect on CKD progression caused by smoking remained uncertain, while the effect on CVD had been established. METHOD: The study population included participants from the specific health check and specific health guidance, an annual health check-up for all inhabitants of Japan who were aged between 40 and 74 years. 149,260 subjects (male, 37.1%; female, 62.9%) were included in this analysis. RESULTS: The relationship between smoking status along with new-onset proteinuria and eGFR deterioration more than 15 mL/min/1.73 m2 was examined. Median observation periods were 1427 days [738, 1813] in males and 1437 days [729, 1816] in females. In male participants, the strongest factor upon kidney dysfunction was new-onset proteinuria (1.41 [1.31 1.51], P < 0.001). The second strongest factor on kidney deterioration was smoking (1.24 [1.16 1.31], P < 0.001). In female participants, strongest factor upon kidney dysfunction was smoking (1.27 [1.16-1.39], P < 0.001). The second strongest factor on kidney deterioration was new-onset proteinuria (1.26 [1.17 1.36], P < 0.001). To reveal the relationship of effects from new-onset proteinuria and smoking on the kidney function, the participants were divided into four groups with and without new-onset proteinuria and smoking. The group with both proteinuria and smoking had significantly worst renal prognosis (P for trend < 0.001). CONCLUSION: Large longitudinal observation study revealed smoking has an evil effect on the progression of CKD. This evil effect could be observed in CKD patients with proteinuria as well as in general population without new-onset proteinuria.

Guidelines for clinical evaluation of chronic kidney disease in early stages : AMED research on regulatory science of pharmaceuticals and medical devices.

BACKGROUND: For the development of pharmaceutical products in kidney field, appropriate surrogate endpoints which can predict long-term prognosis are needed as an alternative to hard endpoints, such as end-stage kidney disease. Though international workshop has proposed estimated glomerular filtration rate (GFR) slope reduction of 0.5-1.0 mL/min/1.73 m /year and 30% decrease in albuminuria/proteinuria as surrogate endpoints in early and advanced chronic kidney disease (CKD), it was not clear whether these are applicable to Japanese patients. METHODS: We analyzed J-CKD-DB and CKD-JAC, Japanese databases/cohorts of CKD patients, and J-DREAMS, a Japanese database of patients with diabetes mellitus to investigate the applicability of eGFR slope and albuminuria/proteinuria to the Japanese population. Systematic review on those endpoints was also conducted including the results of clinical trials published after the above proposal. RESULTS: Our analysis showed an association between eGFR slope and the risk of end-stage kidney disease. A 30% decrease in albuminuria/proteinuria over 2 years corresponded to a 20% decrease in the risk of end-stage kidney disease patients with baseline UACR ≥ 30 mg/gCre or UPCR ≥ 0.15 g/gCre in the analysis of CKD-JAC, though this analysis was not performed on the other database/cohort. Those results suggested similar trends to those of the systematic review. CONCLUSION: The results suggested that eGFR slope and decreased albuminuria/proteinuria may be used as a surrogate endpoint in clinical trials for early CKD (including diabetic kidney disease) in Japanese population, though its validity and cutoff values must be carefully considered based on the latest evidence and other factors.

Protocol for the nationwide registry of patients with polycystic kidney disease: japanese national registry of PKD (JRP).

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) and autosomal recessive polycystic kidney disease (ARPKD) are major genetic polycystic kidney diseases that can progress to end-stage kidney disease (ESKD). Longitudinal data on the clinical characteristics associated with clinical outcomes in polycystic kidney disease (PKD), including the development of ESKD and cardiovascular disease (CVD) are lacking in Japan. To address this unmet need the authors are establishing a novel, web-based, Nationwide Cohort Registry Study-the Japanese Registry of PKD (JRP). METHODS: The JRP is a prospective cohort study for ADPKD (aim to recruit n = 1000 patients), and both a retrospective and prospective study for ARPKD (aim to recruit n = 100). In the prospective registry, patients will be followed-up for 10 years every 6 months and 12 months for patients with ADPKD and ARPKD, respectively. Data collection will be recorded on Research Electronic Data Capture (REDCap) starting on April 1, 2024, with recruitment ending on March 31, 2029. (jRCT 1030230618). RESULTS: Data to be collected include: baseline data, demographics, diagnostic and genetic information, radiological and laboratory findings, and therapeutic interventions. During follow-up, clinical events such as development of ESKD, hospitalization, occurrence of extra kidney complications including CVD events, and death will be recorded, as well as patient-reported health-related quality of life for patients with ADPKD. CONCLUSIONS: The JRP is the first nationwide registry study for patients with ADPKD and ARPKD in Japan, providing researchers with opportunities to advance knowledge and treatments for ADPKD and ARPKD, and to inform disease management and future clinical practice.

Spatiotemporally quantitative in vivo imaging of mitochondrial fatty acid ß-oxidation at cellular-level resolution in mice.

Mitochondrial fatty acid ß-oxidation (FAO) plays a key role in energy homeostasis. Several FAO evaluation methods are currently available, but they are not necessarily suitable for capturing the dynamics of FAO in vivo at a cellular-level spatial resolution and seconds-level time resolution. FAOBlue is a coumarin-based probe that undergoes ß-oxidation to produce a fluorescent substrate, 7-hydroxycoumarin-3-(N-(2-hydroxyethyl))-carboxamide (7-HC). After confirming that 7-HC could be specifically detected using multiphoton microscopy at excitation/emission wavelength = 820/415-485 nm, wild-type C57BL/6 mice were randomly divided into control, pemafibrate, fasting (24 or 72 h), and etomoxir groups. These mice received a single intravenous injection of FAOBlue. FAO activities in the liver of these mice were visualized using multiphoton microscopy at 4.2 s/frame. These approaches could visualize the difference in FAO activities between periportal and pericentral hepatocytes in the control, pemafibrate, and fasting groups. FAO velocity, which was expressed by the maximum slope of the fluorescence intensity curve, was accelerated in the pemafibrate and 72-h fasting groups both in the periportal and the pericentral hepatocytes in comparison with the control group. Our approach revealed differences in the FAO activation mode by the two stimuli, i.e., pemafibrate and fasting, with pemafibrate accelerating the time of first detection of FAO-derived fluorescence. No increase in the fluorescence was observed in etomoxir-pretreated mice, confirming that FAOBlue specifically detected FAO in vivo. Thus, FAOBlue is useful for visualizing in vivo liver FAO dynamics at the single-cell-level spatial resolution and seconds-level time resolution.NEW & NOTEWORTHY Fatty acid ß-oxidation (FAO) plays a key role in energy homeostasis. Here, the authors established a strategy for visualizing FAO activity in vivo at the cellular-level spatial resolution and seconds-level time resolution in mice. Quantitative analysis revealed spatiotemporal heterogeneity in hepatic FAO dynamics. Our method is widely applicable because it is simple and uses a multiphoton microscope to observe the FAOBlue-injected mice.

THOC4 regulates energy homeostasis by stabilizing TFEB mRNA during prolonged starvation.

TFEB, a basic helix-loop-helix transcription factor, is a master regulator of autophagy, lysosome biogenesis and lipid catabolism. Compared to posttranslational regulation of TFEB, the regulation of TFEB mRNA stability remains relatively uncharacterized. In this study, we identified the mRNA-binding protein THOC4 as a novel regulator of TFEB. In mammalian cells, siRNA-mediated knockdown of THOC4 decreased the level of TFEB protein to a greater extent than other bHLH transcription factors. THOC4 bound to TFEB mRNA and stabilized it after transcription by maintaining poly(A) tail length. We further found that this mode of regulation was conserved in Caenorhabditiselegans and was essential for TFEB-mediated lipid breakdown, which becomes over-represented during prolonged starvation. Taken together, our findings reveal the presence of an additional layer of TFEB regulation by THOC4 and provide novel insights into the function of TFEB in mediating autophagy and lipid metabolism.

Association of Longitudinal B-Type Natriuretic Peptide Monitoring With Kidney Failure in Patients With CKD: A Cohort Study.

RATIONALE & OBJECTIVE: Both hypervolemia and hypovolemia are associated with chronic kidney disease (CKD) progression. Although longitudinal monitoring of B-type natriuretic peptide (BNP) may aid physicians' decision making about the optimization of volume status, its clinical benefit remains uncertain in CKD. This study assessed the association between BNP monitoring and the risk of incident kidney replacement therapy (KRT). STUDY DESIGN: Retrospective cohort study. SETTING & PARTICIPANTS: A total of 2,998 outpatients with stages 3-5 of nondialyzed CKD referred to the department of nephrology at an academic hospital. EXPOSURE: BNP monitoring. OUTCOME: KRT, acute kidney injury (AKI), and heart failure hospitalization. ANALYTICAL APPROACH: Marginal structural models, which create a balanced pseudo population at each time point, were applied to account for potential time-dependent confounders. Inverse probability weighted pooled logistic regression models were employed to estimate hazard ratios. RESULTS: At baseline, the median age and estimated glomerular filtration rate were 66 years and 38.1mL/min/1.73m2, respectively. During the follow-up period (median, 5.9 [IQR, 2.8-9.9] years), 449 patients required KRT, 765 had AKI, and 236 were hospitalized for heart failure. After adjustment for time-updated clinical characteristics and physician-specific practice styles, BNP monitoring was associated with lower risks of KRT (HR, 0.44 [95% CI, 0.21-0.92]), AKI (HR, 0.36 [95% CI, 0.18-0.72]), and heart failure hospitalization (HR, 0.37 [95% CI, 0.14-0.95]). The association between BNP monitoring and KRT was attenuated after additional adjustment for AKI or heart failure hospitalization as a time-varying covariate. LIMITATIONS: Residual confounding by measured and unmeasured variables or indications for BNP measurements. CONCLUSIONS: BNP monitoring was associated with a lower risk of KRT among patients with CKD that did not require dialysis. This association is potentially mediated through a reduced risk of AKI or heart failure hospitalization. PLAIN-LANGUAGE SUMMARY: Both volume overload and volume depletion are deleterious to kidney function. B-type natriuretic peptide (BNP) is a biomarker that reflects volume status not only in heart failure but also in nondialysis chronic kidney disease (CKD). Although longitudinal BNP monitoring may aid physicians' decision making about the optimization of volume status, its clinical benefit remains uncertain in CKD. In this cohort study analyzing 2,998 patients with nondialyzed CKD, BNP monitoring was associated with a lower risk of kidney replacement therapy, acute kidney injury, and heart failure hospitalization over the follow-up period. The association with kidney replacement therapy may be mediated through a reduced risk of acute kidney injury or heart failure hospitalization. BNP monitoring may aid physicians in optimal fluid management, potentially conferring better kidney outcomes.

Association between serum iron markers, iron supplementation and cardiovascular morbidity in pre-dialysis chronic kidney disease.

BACKGROUND: The optimal range of serum iron markers and usefulness of iron supplementation are uncertain in patients with pre-dialysis chronic kidney disease (CKD). We investigated the association between serum iron indices and risk of cardiovascular disease (CVD) events and the effectiveness of iron supplementation using Chronic Kidney Disease Japan Cohort data. METHODS: We included 1416 patients ages 20-75 years with pre-dialysis CKD. The tested exposures were serum transferrin saturation and serum ferritin levels and the outcome measures were any cardiovascular event. Fine-Gray subdistribution hazard models were used to examine the association between serum iron indices and time to events. The multivariable fractional polynomial interaction approach was used to evaluate whether serum iron indices were effect modifiers of the association between iron supplementation and cardiovascular events. RESULTS: The overall incidence rate of CVD events for a median of 4.12 years was 26.7 events/1000 person-years. Patients with serum transferrin saturation <20% demonstrated an increased risk of CVD [subdistribution hazard ratio (HR) 2.13] and congestive heart failure (subdistribution HR 2.42). The magnitude of reduction in CVD risk with iron supplementation was greater in patients with lower transferrin saturations (P = .042). CONCLUSIONS: Maintaining transferrin saturation >20% and adequate iron supplementation may effectively reduce the risk of CVD events in patients with pre-dialysis CKD.

Biopsy-proven CKD etiology and outcomes: the Chronic Kidney Disease Japan Cohort (CKD-JAC) study.

BACKGROUND: The Kidney Disease: Improving Global Outcomes guidelines advocate the cause-glomerular filtration rate (GFR)-albuminuria (CGA) classification for predicting outcomes. However, there is a dearth of data supporting the use of the cause of chronic kidney disease. This study aimed to address how to incorporate a prior biopsy-proven diagnosis in outcome prediction. METHODS: We examined the association of biopsy-proven kidney disease diagnoses with kidney failure with replacement therapy (KFRT) and all-cause death before KFRT in patients with various biopsy-proven diagnoses (n = 778, analysis A) and patients with diabetes mellitus labeled with biopsy-proven diabetic nephropathy (DN), other biopsy-proven diseases and no biopsy (n = 1117, analysis B). RESULTS: In analysis A, adding biopsy-proven diagnoses to the GFR-albuminuria (GA) classification improved the prediction of 8-year incidence of KFRT and all-cause death significantly regarding integrated discrimination improvement and net reclassification index. Fine-Gray (FG) models with KFRT as a competing event showed significantly higher subdistribution hazard ratios (SHRs) for all-cause death in nephrosclerosis {4.12 [95% confidence interval (CI) 1.11-15.2)], focal segmental glomerulosclerosis [3.77 (95% CI 1.09-13.1)]} and membranous nephropathy (MN) [2.91 (95% CI 1.02-8.30)] than in immunoglobulin A nephropathy (IgAN), while the Cox model failed to show significant associations. Crescentic glomerulonephritis had the highest risk of all-cause death [SHR 5.90 (95% CI 2.05-17.0)]. MN had a significantly lower risk of KFRT than IgAN [SHR 0.45 (95% CI 0.24-0.84)]. In analysis B, other biopsy-proven diseases had a lower risk of KFRT than biopsy-proven DN in the FG model, with death as a competing event [SHR 0.62 (95% CI 0.39-0.97)]. CONCLUSIONS: The CGA classification is of greater value in predicting outcomes than the GA classification.