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Adenosine 5'-triphosphate (ATP) is the major energy currency of cells and is involved in many cellular processes. However, there is no method for real-time monitoring of ATP levels inside individual living cells. To visualize ATP levels, we generated a series of fluorescence resonance energy transfer (FRET)-based indicators for ATP that were composed of the ε subunit of the bacterial FoF₁-ATP synthase sandwiched by the cyan- and yellow-fluorescent proteins. The indicators, named ATeams, had apparent dissociation constants for ATP ranging from 7.4 μM to 3.3 mM. By targeting ATeams to different subcellular compartments, we unexpectedly found that ATP levels in the mitochondrial matrix of HeLa cells are significantly lower than those of cytoplasm and nucleus. We also succeeded in measuring changes in the ATP level inside single HeLa cells after treatment with inhibitors of glycolysis and/or oxidative phosphorylation, revealing that glycolysis is the major ATP-generating pathway of the cells grown in glucose-rich medium. This was also confirmed by an experiment using oligomycin A, an inhibitor of FoF₁-ATP synthase. In addition, it was demonstrated that HeLa cells change ATP-generating pathway in response to changes of nutrition in the environment.

Background In 2009, the Oxford classification of IgA nephropathy was published. However, its validity has not been fully examined in children. This study aimed to assess this system in an independent large-scale cohort of children. Methods We analyzed 161 consecutive children with newly diagnosed IgA nephropathy from 1977 to 1989 retrospectively. We examined the ability of each variable in the Oxford classification as a predictor of renal outcome defined as ≥ stage III chronic kidney disease (CKD) (estimated glomerular filtration rate (eGFR) <60 mL/min/1.73 m 2 ) using Cox regression analysis. Results The mean mesangial score, and ratios of segmental glomerulosclerosis, endocapillary hypercellularity, tubular atrophy, and crescents were 0.49, 0.8%, 13.1%, 3.3%, and 9.2% respectively. Seven cases reached ≥ stage III CKD. In univariate analyses, mesangial hypercellularity score, endocapillary hypercellularity, tubular atrophy, and crescents were significant predictors of renal outcome. In a multivariate analysis, only mesangial hypercellularity score, tubular atrophy, and crescents were significant though, depending on models. Segmental glomerulosclerosis was not a significant predictor of renal outcome. Although the significance of crescents was not addressed in the Oxford classification, crescents were important predictors of outcome. Conclusions The Oxford classification appears to be valid for predicting renal outcome in children.

Background Although the Oxford classification of IgA nephropathy appears valid, we found crescents were significantly related to renal outcome in our cohort, whereas segmental glomerulosclerosis (S) was not. The timing of renal biopsy may significantly affect the variables in the Oxford classification. Method The relationship between biopsy timing and pathological variables (mesangial hypercellularity score [M], endocapillary hypercellularity [E], S, tubular atrophy/interstitial fibrosis [T], crescents, and global glomerulosclerosis [G]) was analyzed retrospectively in 250 children with IgA nephropathy. Results The median time from disease onset to renal biopsy was 5.1 months (interquartile range, 2.7–15.4). M (ρ = −0.26, P  < 0.0001), E (ρ = −0.34, P  < 0.0001), and crescents (ρ = −0.14, P  = 0.023) showed significant negative correlations, and S (ρ = 0.15, P  = 0.018) and G (ρ = 0.25, P  < 0.0001) showed significant positive correlations with time to biopsy (Spearman test). M, E, and crescents differed significantly in renal biopsies obtained before and after 3 years from onset (Wilcoxon test). Most crescents (92.9 %) were cellular/fibrocellular and were acute lesions. As crescents formed early after disease onset and decreased over time, they may be prognostic for acute phase, but not for chronic phase disease. Conclusions Renal biopsy timing may alter the significance of variables used in the Oxford classification.

Background Hereditary hypomagnesemia is difficult to diagnose accurately because of its rarity and the variety of causative genes. We established a flowchart for identifying responsible genes for hypomagnesemia, and we confirmed its diagnostic efficacy in patients with suspected inherited hypomagnesemia. Methods We established a flowchart and applied it to five index cases with suspected inherited hypomagnesemia. Direct sequence analysis was used to detect the causative gene variants in four cases, and targeted sequencing analysis using next-generation sequencing (NGS) of all causative genes for hypomagnesemia was used in one. Results Expected pathogenic variants were detected in the HNF1B, TRPM6, CLDN16, CASR , or SLC12A3 gene in all five cases. The results of all genetic analyses were consistent with the clinical diagnostic results using the flowchart. Conclusions Accurate genetic diagnosis is crucial for estimating the prognosis, detecting complications in organs other than the kidneys, and for directing genetic counseling. The developed flowchart for identifying responsible genes for hypomagnesemia was useful for diagnosing inherited hypomagnesemia. In addition, NGS analysis will help to resolve clinical difficulties in making an accurate diagnosis and thus improve the diagnostic strategy for inherited hypomagnesemia.

Background Despite a low incidence, nephrotic syndrome (NS) can present with IgA nephropathy (IgAN). The clinical characteristics and long-term outcomes of pediatric patients with IgAN presenting with NS (NS-IgAN) at onset have not been fully elucidated. Methods We retrospectively analyzed 426 patients, and compared clinical and pathological (Oxford) findings between those with NS-IgAN and those with non-NS-IgAN. Results Among 426 patients, 30 (7.0 %) had NS-IgAN. Logistic analyses showed that male sex (OR: 7.6, p  = 0.0002), M1 (OR: 10.3, p  = 0.002), and E1 (OR: 15.2, p  = 0.0001) were significantly related to NS. The mean observation period was 6.2 ± 3.2 years. Although NS-IgAN was associated with significantly lower renal survival than non-NS-IgAN according to Kaplan–Meier analysis ( p  = 0.02), renal survival of NS-IgAN was good (92.4 % at 10 years). The most significant prognostic factor for renal survival was remission of proteinuria after treatment, and NS at onset is also a significant prognostic factor for renal survival after adjusting for remission of proteinuria. Twenty children with NS-IgAN were treated with prednisolone alone, or prednisolone and immunosuppressant. Remission of proteinuria occurred in 21 patients. Three cases of NS-IgAN progressed to stage III–V chronic kidney disease at the most recent observation. They all demonstrated heavy proteinuria after the 2-year initial treatment. The significant factor for persistent proteinuria at 5 years was S1 in NS-IgAN. Conclusions The most significant factor for renal survival was responsiveness to treatment, not NS itself. As modifiable acute lesions are the dominant pathological findings in NS-IgAN, histological improvements achieved by appropriate treatments can result in a favorable prognosis.

Background Some patients with childhood immunoglobulin A nephropathy (IgAN) progress to end-stage renal disease within 20 years, while others achieve spontaneous remission even without medication. Prognosis of IgAN with minimal proteinuria (MP-IgAN, <0.5 g/day/1.73 m 2 ) at diagnosis seems to be generally good. However, the long-term outcome for patients with childhood MP-IgAN has not yet been determined. Methods We retrospectively analyzed 385 children newly diagnosed with biopsy-proven IgAN between June 1976 and July 2009 whose renal biopsy specimens could be evaluated by the Oxford classification criteria. Of these 385 children with IgAN, 106 (27.5 %) were diagnosed with MP-IgAN. We compared clinical and pathological findings between the 106 patients with MP-IgAN and the remaining 279 patients to elucidate the characteristics of MP-IgAN in children. Results Patients with MP-IgAN were identified through a school screening program (73.6 %) or upon presentation with gross hematuria (26.4 %). Patients with MP-IgAN had significantly milder pathological symptoms than those with IgAN. The most frequently used therapeutic regimes were angiotensin converting enzyme inhibitors (30.2 %) and no therapy (36.8 %). None of the patients with MP-IgAN reached stage III chronic kidney disease within 15 years after onset. Four patients with MP-IgAN (3.8 %) received immunosuppressive therapy during the course of the disease. Conclusion Our results indicate that the outcome of patients with a diagnosis of childhood MP-IgAN is good, but that careful long-term observation is required.

Background Some patients with IgA nephropathy (IgAN) achieve spontaneous remission even when not receiving medication. However, details on such remissions remain unknown. The aim of our study was to clarify this information in the clinical setting of childhood IgAN with minor glomerular abnormalities or focal mesangial proliferation (MGA/FMP). Methods This study was a retrospective analysis of 96 children with MGA/FMP who did not receive medication from among the 555 patients with newly diagnosed childhood IgAN treated between January 1972 and December 2000. The Kaplan–Meier method and Cox proportional hazard model were used for the analysis. Results Of the 96 pediatric patients who did not receive medication, 57 (59.4 %) achieved spontaneous remission. The cumulative spontaneous remission rates among these patients were 57.5  and 77.4 % at 5 and 10 years, respectively, from onset. The mean time from onset to remission was 5.9 ± 0.4 years. Clinical and histological findings were similar between the remission and non-remission groups. Of the 57 patients with spontaneous remissions, ten (17.5 %) also developed a recurrence of urinary abnormalities. The cumulative recurrence-free rates were 79.9 and 67.9 % at 5 and 10 years, respectively, after remission. Conclusions The spontaneous remission rate in childhood IgAN with MGA/FMP was higher than expected. Our results suggest that physicians should consider the potential for spontaneous remission and refrain from very aggressive treatment in IgAN patients with MGA/FMP.

Background The criterion for performing a renal biopsy in children with idiopathic nephrotic syndrome (NS) showing microscopic hematuria at onset remains controversial. Methods To determine an adequate renal biopsy criterion in children with NS showing hematuria, the optimal cutoff for the maximum red blood cell (RBC) range in urine sediment to separate minimal change disease (MCD) from other glomerular changes was obtained by receiver operating characteristic analysis. We studied 29 children with NS showing hematuria who were screened from 1,320 patients who underwent renal biopsies between January 2001 and September 2011. Patients were divided into two groups according to the cutoff value to verify its validity. Results The optimal maximum RBC range was 30–49/high-power field (HPF). In group 1 (RBC ≤29/HPF, n  = 14), 3 patients showed nephritis and the other 11 patients showed MCD. In group 2 (RBC ≥30/HPF, n  = 15), 1 patient showed focal segmental glomerulosclerosis, 12 showed nephritis, and the other 2 showed MCD. These findings indicated that the ratio of non-MCD/MCD was significantly higher in group 2 than in group 1 ( P  < 0.01). Conclusions The use of maximum RBC range (30–49/HPF) for a criterion of renal biopsy in patients with NS showing hematuria may be reasonable for clinical practice.

Background A possible mechanism of cyclosporine (CsA) nephrotoxicity is tubular apoptosis. Endoplasmic reticulum (ER) stress has been shown to be an apoptosis activator. Glucose-regulated proteins 78 and 94 (GRP78, GRP94, respectively) are ER stress-induced chaperones. Eukaryotic translation initiation factor 2α (EIF2α) attenuates protein synthesis. If stress is prolonged, cells undergo apoptosis, inducing the production of GADD153, a transcription factor, which in turn downregulates anti-apoptotic protein B-cell lymphoma 2 (Bcl-2). Methods Endoplasmic reticulum stress-related molecules were evaluated by real-time polymerase chain reaction (PCR) using renal biopsy tissues from 17 children with frequently relapsing nephrotic syndrome before and after 2 years of CsA therapy. Results GRP78, GRP94, eIF2α, and Bcl-2 were significantly upregulated in renal biopsy tissues from children 2 years post-CsA treatment. However, there was almost no change in GADD153. Mean ratios of post- to pre-CsA expression of GRP78, GRP94, eIF2α and Bcl-2 were 2.53, 1.80, 2.38 and 1.92, respectively. Post-CsA administration, GRP78 and eIF2α were upregulated by up to sixfold, and GRP94 and Bcl-2 were upregulated by up to fourfold compared with the respective pre-CsA levels. There were significant correlations between GRP78, GRP94, eIF2α, and Bcl-2 levels. These findings suggest that CsA induced an unfolded protein response due to ER stress, but did not cause apoptosis. Conclusions An unfolded protein response due to ER stress induced by CsA may function in a defensive manner, with less apoptosis occurring under low-dose conditions. This finding is important for the rationale for CsA administration.

Since the beginning of the 1990s, Japanese medical practitioners have extensively prescribed angiotensin-converting enzyme (ACE) inhibitors for children with mild IgA nephropathy (IgA-N) and steriods for those with severe IgA-N. We have performed a retrospective cohort study to clarify whether the long-term outcome has improved in Japanese children with IgA-N. Renal survival was defined as the time from onset to end-stage renal disease (ESRD). We divided the study period into two time periods based on the occurrence of the initial renal biopsy:1976–1989 and 1990–2004. Actuarial survivals were calculated by Kaplan–Meier method, and comparisons were made with the logrank test. The Cox proportional hazard model was used for multivariate analysis. Between 1976 and 2004, 500 children were diagnosed as having IgA-N in our hospitals. The actuarial renal survival from the time of apparent disease onset was 96.4% at 10 years, 84.5% at 15 years and 73.9% at 20 years. Renal survival in the 1990–2004 period was significantly better than that in 1976–1989 ( p  = 0.008), and a marked improvement in renal survival in patients with severe IgA-N was also observed ( p  = 0.0003). Multivariate analysis indicated that diagnosis year was a significant factor for ESRD-free survival independently of baseline characteristics. The results of this study show that there has been an improvement in terms of renal survival in Japanese children with IgA-N.