Straightforward and affordable agroinfiltration with RUBY accelerates RNA silencing research.

Transient expression and induction of RNA silencing by agroinfiltration is a fundamental method in plant RNA biology. Here, we introduce a new reporter assay using RUBY, which encodes three key enzymes of the betalain biosynthesis pathway, as a polycistronic mRNA. The red pigmentation conferred by betalains allows visual confirmation of gene expression or silencing levels without tissue disruption, and the silencing levels can be quantitatively measured by absorbance in as little as a few minutes. Infiltration of RUBY in combination with p19, a well-known RNA silencing suppressor, induced a fivefold higher accumulation of betalains at 7 days post infiltration compared to infiltration of RUBY alone. We demonstrated that co-infiltration of RUBY with two RNA silencing inducers, targeting either CYP76AD1 or glycosyltransferase within the RUBY construct, effectively reduces RUBY mRNA and betalain levels, indicating successful RNA silencing. Therefore, compared to conventional reporter assays for RNA silencing, the RUBY-based assay provides a simple and rapid method for quantitative analysis without the need for specialized equipment, making it useful for a wide range of RNA silencing studies.

Endogenous activation of peroxisome proliferator-activated receptor alpha 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 (Ctrl) 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 (DEGs) in the clusters belonging to proximal tubular epithelial cells (PTECs). Based on these DEGs, in silico analyses suggested that the Phos group activated peroxisome proliferator-activated receptor alpha (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 to 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.

NSPlex: an efficient method to analyze non-specific peaks amplified using commercial STR kits.

Commercial short tandem repeat (STR) kits exclusively contain human-specific primers; however, various non-human organisms with high homology to the STR kit's primer sequences can cause cross-reactivity. Owing to the proprietary nature of the primers in STR kits, the origins and sequences of most non-specific peaks (NSPs) remain unclear. Such NSPs can complicate data interpretation between the casework and reference samples; thus, we developed "NSPlex", an efficient method to discover the biological origins of NSPs. We used leftover STR kit amplicons after capillary electrophoresis and performed advanced bioinformatics analyses using next-generation sequencing followed by BLAST nucleotide searches. Using our method, we could successfully identify NSP generated from PCR amplicons of a sample mixture of human DNA and DNA extracted from matcha powder (finely ground powder of green tea leaves and previously known as a potential source of NSP). Our results showed our method is efficient for NSP analysis without the need for the primer information as in commercial STR kits.

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.

Children's and adults' evaluations of self-enhancement and self-deprecation depend on the usual performance of the self-presenter.

This study examined how evaluations of self-presentation vary with age depending on the self-presenter's usual performance. People's usual performance is a key factor because it generally influences the social evaluations and judgments that others make about them. Children aged 7 and 8 years (second graders) and 10 and 11 years (fifth graders), as well as adults, were presented with scenarios in which protagonists responded to praise after a good performance using either self-enhancement or self-deprecation. The other person in scenarios knew that the protagonist's usual performance on the task was either good or poor. After the protagonist responded to praise in a self-enhancing or self-deprecating way, the participants judged how the other person would evaluate the protagonist's ability (good/poor) and character (nice/mean). For ability evaluations specifically concerning protagonists who usually performed poorly, the results showed that by around 10 years of age children no longer tended to give more positive ability evaluations for self-enhancement than for self-deprecation. Adults gave less positive ability evaluations for self-deprecation than for self-enhancement, but only when the protagonists usually performed well. In relation to the character evaluations, by around 10 years of age self-enhancement led to less positive character evaluations than self-deprecation, but only when the protagonists usually performed poorly. Overall, second graders evaluated self-presenters as more competent and nicer. These results indicate that the expected evaluation of self-enhancement and self-deprecation is influenced by the usual level of performance but that there are developmental changes in this aspect of social cognition.

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.

Investigation of the efficacy and adverse effects of lacosamide over 36 months.

OBJECTIVE: To evaluate the efficacy and retention rate of lacosamide (LCM) over 36 months as a treatment for children and adolescents with focal and generalized epilepsy based on a retrospective study. METHODS: All patients prescribed LCM as monotherapy and add-on therapy between October 2016 and September 2019 at Jichi Children's Medical Center Tochigi were included in the study. The response rate, retention rate, and adverse effects were calculated. RESULTS: A total of 126 (female, n = 73) patients of 1.3 to 34.9 years old (median age: 12.8 years; mean ± SD 13.2 ± 6.6 years) received LCM as monotherapy or add-on treatment for focal, generalized, and combined focal and generalized epilepsy. The response rate was 40.5% at 3 months, 40.5% at 6 months, 38.1% at 9 months, 35.7% at 12 months, 25.9% at 24 months, and 29.4% at 36 months. For 34 patients who were observable for 36 months, the retention rate was 70.6% at 3 months, but then gradually declined to 34.8% at 36 months. According to the number of concomitant anti-seizure medications (ASMs), the retention rate was higher in patients receiving <3 ASMs than in those receiving ≥3 ASMs at all observation points. The most common adverse effects were somnolence in 21 patients (16.7%) and dizziness in 5 patients (39.7%). CONCLUSION: Our response rate was lower and our retention rate was higher in comparison to a previous study that observed patients over 36 months. Further prospective studies in children are required to confirm the response rate and retention rate in patients treated with LCM over 36 months.

An Adolescent Patient with Sick Sinus Syndrome Complicated by Hypothyroidism Carrying an SCN5A Variant.

Previous studies have reported that hypothyroidism can lead to sick sinus syndrome (SSS) or other rhythm disturbances. Variants in the alpha subunit of the cardiac sodium channel (SCN5A) are known to be among the genetic causes of SSS. We encountered an adolescent patient with SSS and hypothyroidism who also harbored an SCN5A variant. The patient was a 13-year-old girl who was referred to our hospital because of bradycardia identified during a school electrocardiography screening. Clinical examination revealed severe hypothyroidism due to Hashimoto thyroiditis and SSS. After levothyroxine supplementation, her symptoms of hypothyroidism improved; however, the SSS did not. Genetic testing revealed a heterozygous variant (c.1066 G>A, p.Asp356Asn) in SCN5A. This is the first report of the coexistence of SSS due to an SCN5A variant and severe hypothyroidism in an adolescent patient. While patients with SCN5A variants exhibit phenotypic heterogeneity due to the presence of various modifiers, the presence of severe hypothyroidism may affect the development of SSS. This case highlights the importance of genetic analysis, including testing for SCN5A variants, in patients with hypothyroidism complicated by SSS or cardiac conduction disorders.

Maxacalcitol (22-Oxacalcitriol (OCT)) Retards Progression of Left Ventricular Hypertrophy with Renal Dysfunction Through Inhibition of Calcineurin-NFAT Activity.

PURPOSE: Left ventricular hypertrophy (LVH) is a cardiovascular complication highly prevalent in patients with chronic kidney disease (CKD). Previous studies analyzing 1α-hydroxylase or vitamin D receptor (Vdr) knockout mice revealed active vitamin D as a promising agent inhibiting LVH progression. Paricalcitol, an active vitamin D analog, failed to suppress the progression of LV mass index (LVMI) in pre-dialysis patients with CKD. As target genes of activated VDR differ depending on its agonists, we examined the effects of maxacalcitol (22-oxacalcitriol: OCT), a less calcemic active vitamin D analog, on LVH in hemodialysis patients and animal LVH models with renal insufficiency. METHODS: In retrospective cohort study, patients treated with OCT who underwent hemodialysis were enrolled. Using cardiac echocardiography, LV mass was evaluated by the area-length method. In animal study, angiotensin II (Ang II)-infused Wister rats with heminephrectomy or Ang II-stimulated neonatal rat ventricular myocytes (NRVM) were treated with OCT. RESULTS: OCT significantly inhibited the progression of LVMI in hemodialysis patients. In Ang II-infused heminephrectomized rats, OCT suppressed the progression of LVH in a blood pressure-independent manner. OCT also suppressed the activity of calcineurin in the left ventricle of model rats. Specifically, OCT reduced the protein levels of calcineurin A, but not the mRNA levels of Ppp3ca (calcineurin Aα). Luciferase assays showed that OCT increased the promoter activity of Fbxo32 (atrogin1), an E3 ubiquitin ligase targeting calcineurin A. Finally, OCT promoted ubiquitination and degradation of calcineurin A. CONCLUSION: Our works indicated that OCT retards progression of LVH through calcineurin-NFAT pathway, which reveal a novel aspect of OCT in attenuating pathological LVH.

Recurrent membranous nephropathy with a possible alteration in the etiology: a case report.

BACKGROUND: Phospholipase A2 receptor 1 (PLA2R1) and thrombospondin type-1 domain-containing 7A (THSD7A) are the two major pathogenic antigens for membranous nephropathy (MN). It has been reported that THSD7A-associated MN has a higher prevalence of comorbid malignancy than PLA2R1-associated MN. Here we present a case of MN whose etiology might change from idiopathic to malignancy-associated MN during the patient's clinical course. CASE PRESENTATION: A 68-year-old man with nephrotic syndrome was diagnosed with MN by renal biopsy. Immunohistochemistry showed that the kidney specimen was negative for THSD7A. The first course of corticosteroid therapy achieved partial remission; however, nephrotic syndrome recurred 1 year later. Two years later, his abdominal echography revealed a urinary bladder tumor, but he did not wish to undergo additional diagnostic examinations. Because his proteinuria increased consecutively, corticosteroid therapy was resumed, but it failed to achieve remission. Another kidney biopsy was performed and revealed MN with positive staining for THSD7A. PLA2R1 staining levels were negative for both first and second biopsies. Because his bladder tumor had gradually enlarged, he agreed to undergo bladder tumor resection. Pathological examination indicated that the tumor was THDS7A-positive bladder cancer. Subsequently, his proteinuria decreased and remained in remission. CONCLUSIONS: This case suggests that the etiology of MN might be altered during the therapeutic course. Intensive screening for malignancy may be preferable in patients with unexpected recurrence of proteinuria and/or change in therapy response.

Lithocholic acid increases intestinal phosphate and calcium absorption in a vitamin D receptor dependent but transcellular pathway independent manner.

Phosphate/calcium homeostasis is crucial for health maintenance. Lithocholic acid, a bile acid produced by intestinal bacteria, is an agonist of vitamin D receptor. However, its effects on phosphate/calcium homeostasis remain unclear. Here, we demonstrated that lithocholic acid increases intestinal phosphate/calcium absorption in an enterocyte vitamin D receptor-dependent manner. Lithocholic acid was found to increase serum phosphate/calcium levels and thus to exacerbate vascular calcification in animals with chronic kidney disease. Lithocholic acid did not affect levels of intestinal sodium-dependent phosphate transport protein 2b, Pi transporter-1, -2, or transient receptor potential vanilloid subfamily member 6. Everted gut sac analyses demonstrated that lithocholic acid increased phosphate/calcium absorption in a transcellular pathway-independent manner. Lithocholic acid suppressed intestinal mucosal claudin 3 and occludin in wild-type mice, but not in vitamin D receptor knockout mice. Everted gut sacs of claudin 3 knockout mice showed an increased permeability for phosphate, but not calcium. In patients with chronic kidney disease, serum 1,25(OH)2 vitamin D levels are decreased, probably as an intrinsic adjustment to reduce phosphate/calcium burden. In contrast, serum and fecal lithocholic acid levels and fecal levels of bile acid 7α-dehydratase, a rate-limiting enzyme involved in lithocholic acid production, were not downregulated. The effects of lithocholic acid were eliminated by bile acid adsorptive resin in mice. Thus, lithocholic acid and claudin 3 may represent novel therapeutic targets for reducing phosphate burden.

Prognostic value of hypochloremia versus hyponatremia among patients with chronic kidney disease-a retrospective cohort study.

BACKGROUND: Serum chloride (Cl) levels confer better prognostic value than serum sodium (Na) levels among patients with heart failure. Little is known about the relationship between serum Cl levels and clinical outcomes among patients with chronic kidney disease (CKD). METHODS: This was a retrospective cohort study enrolling patients with Stages G3-G5 CKD who visited the nephrology outpatient department of Osaka University Hospital from April 2005 to December 2014. The main exposure was time-varying serum Cl levels categorized as quartiles. The study outcome was a composite of all-cause death and cardiovascular events. RESULTS: A total of 2661 patients with CKD were included in the analysis. During a median follow-up of 4.0 years, 284 deaths and 416 cardiovascular events occurred. Compared with patients in the third Cl quartile, those in the first Cl quartile showed a significantly higher risk of the outcome after adjustment for demographics and clinical factors including time-varying serum Na, serum albumin and bicarbonate levels, and use of diuretics and sodium bicarbonate [hazard ratio (HR) 2.13; 95% confidence interval (CI) 1.20-3.81; P = 0.01] and, additionally, anion gap (HR 2.13; 95% CI 1.26-3.57; P = 0.004). Adding serum Cl levels, but not serum Na levels, to the multivariable model significantly improved net reclassification index (0.335; P < 0.001) and integrated discrimination improvement (0.0113; P = 0.01). CONCLUSIONS: Lower serum Cl levels are an independent predictor of death and cardiovascular events. The incremental prognostic value of Cl was superior to that of Na in patients with CKD.

Gene therapy improves motor and mental function of aromatic l-amino acid decarboxylase deficiency.

In patients with aromatic l-amino acid decarboxylase (AADC) deficiency, a decrease in catecholamines and serotonin levels in the brain leads to developmental delay and movement disorders. The beneficial effects of gene therapy in patients from 1 to 8 years of age with homogeneous severity of disease have been reported from Taiwan. We conducted an open-label phase 1/2 study of population including adolescent patients with different degrees of severity. Six patients were enrolled: four males (ages 4, 10, 15 and 19 years) and one female (age 12 years) with a severe phenotype who were not capable of voluntary movement or speech, and one female (age 5 years) with a moderate phenotype who could walk with support. The patients received a total of 2 × 1011 vector genomes of adeno-associated virus vector harbouring DDC via bilateral intraputaminal infusions. At up to 2 years after gene therapy, the motor function was remarkably improved in all patients. Three patients with the severe phenotype were able to stand with support, and one patient could walk with a walker, while the patient with the moderate phenotype could run and ride a bicycle. This moderate-phenotype patient also showed improvement in her mental function, being able to converse fluently and perform simple arithmetic. Dystonia disappeared and oculogyric crisis was markedly decreased in all patients. The patients exhibited transient choreic dyskinesia for a couple of months, but no adverse events caused by vector were observed. PET with 6-[18F]fluoro-l-m-tyrosine, a specific tracer for AADC, showed a persistently increased uptake in the broad areas of the putamen. In our study, older patients (>8 years of age) also showed improvement, although treatment was more effective in younger patients. The genetic background of our patients was heterogeneous, and some patients suspected of having remnant enzyme activity showed better improvement than the Taiwanese patients. In addition to the alleviation of motor symptoms, the cognitive and verbal functions were improved in a patient with the moderate phenotype. The restoration of dopamine synthesis in the putamen via gene transfer provides transformative medical benefit across all patient ages, genotypes, and disease severities included in this study, with the most pronounced improvements noted in moderate patients.10.1093/brain/awy331_video1awy331media15991361892001.

A Randomized Trial of Magnesium Oxide and Oral Carbon Adsorbent for Coronary Artery Calcification in Predialysis CKD.

BACKGROUND: Developing strategies for managing coronary artery calcification (CAC) in patients with CKD is an important clinical challenge. Experimental studies have demonstrated that magnesium inhibits vascular calcification, whereas the uremic toxin indoxyl sulfate aggravates it. METHODS: To assess the efficacy of magnesium oxide (MgO) and/or the oral carbon adsorbent AST-120 for slowing CAC progression in CKD, we conducted a 2-year, open-label, randomized, controlled trial, enrolling patients with stage 3-4 CKD with risk factors for CAC (diabetes mellitus, history of cardiovascular disease, high LDL cholesterol, or smoking). Using a two-by-two factorial design, we randomly assigned patients to an MgO group or a control group, and to an AST-120 group or a control group. The primary outcome was percentage change in CAC score. RESULTS: We terminated the study prematurely after an interim analysis with the first 125 enrolled patients (of whom 96 completed the study) showed that the median change in CAC score was significantly smaller for MgO versus control (11.3% versus 39.5%). The proportion of patients with an annualized percentage change in CAC score of ≥15% was also significantly lower for MgO compared with control (23.9% versus 62.0%). However, MgO did not suppress the progression of thoracic aorta calcification. The MgO group's dropout rate was higher than that of the control group (27% versus 17%), primarily due to diarrhea. The percentage change in CAC score did not differ significantly between the AST-120 and control groups. CONCLUSIONS: MgO, but not AST-120, appears to be effective in slowing CAC progression. Larger-scale trials are warranted to confirm these findings.

VEGF-A Links Angiolymphoid Hyperplasia With Eosinophilia (ALHE) to THSD7A Membranous Nephropathy: A Report of 2 Cases.

Autoantibodies against thrombospondin type 1 domain-containing 7A (THSD7A) cause membranous nephropathy (MN); however, the mechanisms involved in THSD7A expression and immunization are uncertain. We present 2 cases of THSD7A-associated MN accompanied by angiolymphoid hyperplasia with eosinophilia (ALHE), a benign tumor characterized by proliferation of plump endothelial cells. Prednisolone therapy, but not surgical resection of ALHE tumors, successfully suppressed eosinophilia and proteinuria in both cases. Because ALHE is characterized by the proliferation of plump endothelial cells, we focused on the roles of vascular endothelial growth factor A (VEGF-A) in MN pathogenesis. We found that plump endothelial cells in ALHE modestly expressed THSD7A in both cases. We also found that eosinophils in ALHE expressed VEGF-A, which upregulated THSD7A expression, especially under T-helper type 2-prone conditions in cultured endothelial cells. Furthermore, double-positive cells for THSD7A and CD83 surrounded the proliferated small vessels. Our results suggest that VEGF-A-induced THSD7A expression outside the kidney may be important for MN pathogenesis.

Low magnesium diet aggravates phosphate-induced kidney injury.

BACKGROUND: Magnesium is known to protect against phosphate-induced tubular cell injuries in vitro. We investigated in vivo effects of magnesium on kidney injuries and phosphate metabolism in mice exposed to a high phosphate diet. METHODS: Heminephrectomized mice were maintained on a high phosphate/normal magnesium diet or a high phosphate/low magnesium diet for 6 weeks. We compared renal histology, phosphaturic hormones and renal α-Klotho expression between the two diet groups. RESULTS: High phosphate diet-induced tubular injuries and interstitial fibrosis were remarkably aggravated by the low-magnesium diet. At 1 week after high phosphate feeding when serum creatinine levels were similar between the two groups, the low magnesium diet suppressed not only fecal phosphate excretion but also urinary phosphate excretion, resulting in increased serum phosphate levels. Parathyroid hormone (PTH) levels were not appropriately elevated in the low magnesium diet group despite lower 1,25-dihydroxyvitamin D and serum calcium levels compared with the normal magnesium diet group. Although fibroblast growth factor 23 (FGF23) levels were lower in the low magnesium diet group, calcitriol-induced upregulation of FGF23 could not restore the impaired urinary phosphate excretion. The low magnesium diet markedly downregulated α-Klotho expression in the kidney. This downregulation of α-Klotho occurred even when mice were fed the low phosphate diet. CONCLUSIONS: A low magnesium diet aggravated high phosphate diet-induced kidney injuries. Impaired PTH secretion and downregulation of renal α-Klotho were likely to be involved in the blunted urinary phosphate excretion by the low magnesium diet. Increasing dietary magnesium may be useful to attenuate phosphate-induced kidney injury.

Proteinuria-associated renal magnesium wasting leads to hypomagnesemia: a common electrolyte abnormality in chronic kidney disease.

BACKGROUND: Hypomagnesemia (Hypo-Mg) predicts mortality and chronic kidney disease (CKD) progression. However, in CKD, its prevalence, kidney-intrinsic risk factors, and the effectiveness of oral magnesium (Mg) therapy on serum Mg levels is uncertain. METHODS: In a cross-sectional study enrolling pre-dialysis outpatients with CKD, the prevalence of electrolyte abnormalities (Mg, sodium, potassium, calcium and phosphorus) was compared. In an open-label randomized controlled trial (RCT), we randomly assigned CKD patients to either the magnesium oxide (MgO) or control arm. The outcome was serum Mg levels at 1 year. RESULTS: In 5126 patients, Hypo-Mg was the most common electrolyte abnormality (14.7%) with similar prevalence across stages of CKD. Positive proteinuria was a risk factor of Hypo-Mg (odds ratio 2.2; 95% confidence interval 1.2-4.0). However, stratifying the analyses by diabetes mellitus (DM), it was not significant in DM (Pinteraction = 0.04). We enrolled 114 patients in the RCT. Baseline analyses showed that higher proteinuria was associated with higher fractional excretion of Mg. This relationship between proteinuria and renal Mg wasting was mediated by urinary tubular markers in mediation analyses. In the MgO arm, higher proteinuria or tubular markers predicted a significantly lower 1-year increase in serum Mg. In patients with a urinary protein-to-creatinine ratio (uPCR) <0.3 g/gCre, serum Mg at 1 year was 2.4 and 2.0 mg/dL in the MgO and control arms, respectively (P < 0.001), with no significant between-group difference in patients whose uPCR was ≥0.3 g/gCre (Pinteraction=0.001). CONCLUSIONS: Proteinuria leads to renal Mg wasting through tubular injuries, which explains the high prevalence of Hypo-Mg in CKD.

Cardiac hypertrophy elevates serum levels of fibroblast growth factor 23.

Several experimental studies have shown that fibroblast growth factor 23 (FGF23) induces left ventricular hypertrophy (LVH). However, the opposite directional relationship, namely a potential effect of LVH on FGF23, remains uncertain. Here we evaluated the effects of LVH on FGF23 using cardiomyocyte-specific calcineurin A transgenic mice. At six weeks, these mice showed severe LVH, with elevated levels of serum intact FGF23. FGF23 levels were elevated in cardiomyocytes, but not osteocytes, of the transgenic animals. Moreover, transverse aortic constriction also upregulated myocardial FGF23 expression in wild type mice. The promoter region of the FGF23 gene contains two putative nuclear factors of activated T cells (NFAT)-binding sites, with NFAT1 activating the promoter in a proximal NFAT-binding site dependent manner. Neither serum, urinary, or fractional excretion values of calcium and phosphate nor serum levels of 1,25(OH)2 vitamin D were different between wild type and transgenic mice. Moreover, the renal expression of FGF receptors and α-Klotho was comparable. However, plasma levels of antidiuretic hormone were significantly increased in the transgenic mice, and aquaporin-2 immunohistochemical staining was mainly positive in the apical membrane of the collecting duct, compared to a primarily cytoplasmic staining in wild type mice. Real-time PCR analyses of kidney CYP27B1 and CYP24A1 expression in wild type mice showed that exogenous antidiuretic hormone blocked FGF23's actions on these vitamin D activating or inactivating enzymes. Finally, the renal resistance of transgenic mice to FGF23 was partly overcome by tolvaptan. Thus, LVH in transgenic mice is associated with an increase in myocardial and serum intact FGF23, with the kidneys being protected against FGF23 excess by elevated antidiuretic hormone levels.