Aberrant Smad3 phosphoisoforms in cyst-lining epithelial cells in the cpk mouse, a model of autosomal recessive polycystic kidney disease.

Cystic epithelia acquire mesenchymal-like features in polycystic kidney disease (PKD). In this phenotypic alteration, it is well known that transforming growth factor (TGF)-ß/Smad3 signaling is involved; however, there is emerging new data on Smad3 phosphoisoforms: Smad3 phosphorylated at linker regions (pSmad3L), COOH-terminal regions (pSmad3C), and both (pSmad3L/C). pSmad3L/C has a pathological role in colorectal cancer. Mesenchymal phenotype-specific cell responses in the TGF-ß/Smad3 pathway are implicated in carcinomas. In this study, we confirmed mesenchymal features and examined Smad3 phosphoisoforms in the cpk mouse, a model of autosomal recessive PKD. Kidney sections were stained with antibodies against mesenchymal markers and domain-specific phospho-Smad3. TGF-ß, pSmad3L, pSmad3C, JNK, cyclin-dependent kinase (CDK) 4, and c-Myc were evaluated by Western blotting. Cophosphorylation of pSmad3L/C was assessed by immunoprecipitation. α-Smooth muscle actin, which indicates mesenchymal features, was expressed higher in cpk mice. pSmad3L expression was increased in cpk mice and was predominantly localized in the nuclei of tubular epithelial cells in cysts; however, pSmad3C was equally expressed in both cpk and control mice. Levels of pSmad3L, JNK, CDK4, and c-Myc protein in nuclei were significantly higher in cpk mice than in controls. Immunoprecipitation showed that Smad3 was cophosphorylated (pSmad3L/C) in cpk mice. Smad3 knockout/cpk double-mutant mice revealed amelioration of cpk abnormalities. These findings suggest that upregulating c-Myc through the JNK/CDK4-dependent pSmad3L pathway may be key to the pathophysiology in cpk mice. In conclusion, a qualitative rather than a quantitative abnormality of the TGF-ß/Smad3 pathway is involved in PKD and may be a target for disease-specific intervention.

IgA nephropathy with presentation of nephrotic syndrome at onset in children.

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.

Diagnostic strategy for inherited hypomagnesemia.

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.

Renal biopsy criterion in idiopathic nephrotic syndrome with microscopic hematuria at onset.

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.

Long-term outcome of childhood IgA nephropathy with minimal proteinuria.

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.

Biopsy timing and Oxford classification variables in childhood/adolescent IgA nephropathy.

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.

Spontaneous remission in children with IgA nephropathy.

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.

Endoplasmic reticulum stress with low-dose cyclosporine in frequently relapsing nephrotic syndrome.

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.

First Japanese case of Pierson syndrome with mutations in LAMB2.

Pierson syndrome (OMIM 609049) is typically characterized by congenital nephritic syndrome and peculiar ocular anomalies with microcoria. It is caused by mutations in LAMB2, which encodes laminin ß2. Approximately 50 mutations of LAMB2 from approximately 40 unrelated families have been identified; however, most of them were from Western countries. Although three patients in Asia with mutations of LAMB2 have been reported, they were not typical cases. We report the first Japanese case of Pierson syndrome with proven causative LAMB2 mutations. She presented with congenital nephrotic syndrome and bilateral microcoria at birth, and developed end-stage renal disease at 2 months of age. This is the first report of a typical case from Asia. LAMB2 analysis by direct sequencing revealed the compound heterozygous mutations c.3974_3975insA (p.N1325KfsX1331, maternal, novel) in exon 25 and c.4519C>T (p.Q1507X, paternal) in exon 27. The phenotype due to LAMB2 mutations appears to be similar between different ethnic groups.

Renal biopsy criterion in children with asymptomatic constant isolated proteinuria.

BACKGROUND: The criterion of a renal biopsy in children with asymptomatic persistent isolated proteinuria is controversial. METHODS: To determine an adequate renal biopsy criterion in children with asymptomatic constant isolated proteinuria, the optimal cutoff maximum urinary protein/creatinine ratio (uP/Cr) to separate minor glomerular abnormalities (MGA) and other significant glomerular changes was obtained by receiver operating characteristic analysis in 44 children with asymptomatic constant isolated proteinuria (uP/Cr ≥ 0.2 g/g) screened from 1167 patients who underwent a renal biopsy between September 2000 and April 2010. Patients were divided into two groups according to the cutoff value to verify its validity. RESULTS: The optimal uP/Cr was 0.5 g/g. In Group 1 (uP/Cr <0.5 g/g, n = 15), only one patient (6.7%) showed focal segmental glomerulosclerosis (FSGS) and the other 14 patients (93.3%) had MGA. In Group 2 (uP/Cr ≥ 0.5 g/g at least once before biopsy, n = 29), 5 patients showed FSGS and 7 patients had nephritis such as IgA nephropathy (41.4%, n = 12) and the other 17 patients (58.6%) showed MGA. These findings indicated that the ratio of non-MGA/MGA was significantly higher in Group 2 than that in Group 1 (P = 0.016) and that if renal biopsies were performed with a criterion of a maximum uP/Cr ≥ 0.5 g/g (criterion for Group 2), renal biopsies could be avoided in 45.2% of patients with MGA. One patient with FSGS in Group 1 showed proteinuria with uP/Cr ≥ 0.5 g/g in the clinical course. CONCLUSIONS: An adequate renal biopsy criterion in children with asymptomatic constant isolated proteinuria is uP/Cr ≥ 0.5 g/g.

Validity of the Oxford classification of IgA nephropathy in children.

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.

Visualization of ATP levels inside single living cells with fluorescence resonance energy transfer-based genetically encoded indicators.

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 epsilon subunit of the bacterial F(o)F(1)-ATP synthase sandwiched by the cyan- and yellow-fluorescent proteins. The indicators, named ATeams, had apparent dissociation constants for ATP ranging from 7.4 muM 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 F(o)F(1)-ATP synthase. In addition, it was demonstrated that HeLa cells change ATP-generating pathway in response to changes of nutrition in the environment.

Epithelial-to-mesenchymal transition in cyst lining epithelial cells in an orthologous PCK rat model of autosomal-recessive polycystic kidney disease.

In polycystic kidney disease (PKD), cyst lining cells show polarity abnormalities. Recent studies have demonstrated loss of cell contact in cyst cells, suggesting induction of epithelial-to-mesenchymal transition (EMT). Recently, EMT has been implicated in the pathogenesis of PKD. To explore further evidence of EMT in PKD, we examined age- and segment-specific expression of adhesion molecules and mesenchymal markers in PCK rats, an orthologous model of human autosomal-recessive PKD. Kidneys from 5 male PCK and 5 control rats each at 0 days, 1, 3, 10, and 14 wk, and 4 mo of age were serially sectioned and stained with segment-specific markers and antibodies against E-cadherin, Snail1, ß-catenin, and N-cadherin. mRNAs for E-cadherin and Snail1 were quantified by real-time PCR. Vimentin, fibronectin, and α-smooth muscle actin (α-SMA) expressions were assessed as mesenchymal markers. E-cadherin expression pattern was correlated with the disease pathology in that tubule segments showing the highest expression in control had much severer cyst formation in PCK rats. In PCK rats, E-cadherin and ß-catenin in cystic tubules was attenuated and localized to lateral areas of cell-cell contact, whereas nuclear expression of Snail1 increased in parallel with cyst enlargement. Some epithelial cells in large cysts derived from these segments, especially in adjacent fibrotic areas, showed positive immunoreactivity for vimentin and fibronectin. In conclusion, these findings suggest that epithelial cells in cysts acquire mesenchymal features in response to cyst enlargement and participate in progressive renal fibrosis. Our study clarified the nephron segment-specific cyst profile related to EMT in PCK rats. EMT may play a key role in polycystic kidney disease.

Disappearance of glomerular IgA deposits in childhood IgA nephropathy showing diffuse mesangial proliferation after 2 years of combination/prednisolone therapy.

BACKGROUND: The prognosis of children with severe IgA nephropathy showing diffuse mesangial proliferation is poor. However, the prognosis can be improved by combination therapy (prednisolone + azathioprine or mizoribine + warfarin + dipyridamole) or prednisolone alone over a 2-year period, and disappearance of glomerular IgA deposits is often observed. Details of the incidence and clinicopathological significance of glomerular IgA disappearance remain unclear. METHODS: To investigate this phenomenon, we retrospectively screened and analysed 124 consecutive children (age ≤ 18 years at first biopsy) with newly diagnosed severe IgA nephropathy showing diffuse mesangial proliferation, who received combination therapy or prednisolone alone for 2 years and underwent repeat biopsies. RESULTS: Among these patients, 90 received combination therapy, and 34 received prednisolone alone. After 2 years of treatment, 27 of the patients (21.8%) showed disappearance of glomerular IgA. Logistic analysis showed that IgA disappearance was associated with less severe urinary protein excretion at the end of treatment. Kaplan-Meier analysis of the long-term course revealed a significant difference in proteinuria-free survival after the 2-year treatment period between the patients with IgA disappearance and those without (P = 0.008; log-rank test). The Cox proportional hazards model showed that disappearance of glomerular IgA after the treatment was a factor significantly associated with proteinuria-free survival in both univariate and multivariate analyses. CONCLUSIONS: The present results suggest that disappearance of IgA after 2 years of treatment indicates milder disease severity, even in patients with diffuse mesangial proliferation, and is a prognostic factor related to proteinuria-free survival.

Increased chymase-positive mast cells in children with crescentic glomerulonephritis.

Mast cell-derived chymase is an angiotensin II-forming enzyme that appears to be involved in tubulointerstitial fibrosis in the kidneys. Previous studies have shown that the level of chymase increases in grafted kidneys after rejection and in adult patients with diabetic nephropathy. However, the significance of chymase in children with renal diseases has not been investigated. Using immunohistochemistry, we have investigated chymase expression in biopsy samples of renal tissue from 104 children with kidney diseases, including rapidly progressive crescentic glomerulonephritis (n = 3), diabetic nephropathy (n = 2), allografted kidney (n = 3), membranoproliferative glomerulonephritis (n = 6), immunoglobulin A nephropathy (n = 33) and Henoch-Schönlein purpura nephritis (n = 23). Increased numbers of chymase-positive mast cells were observed in the renal cortex of all three patients with crescentic glomerulonephritis (mean 26.0/mm(2); range 19.3-36.8/mm(2)). Chymase-positive cells were also observed in the renal biopsy of an allografted kidney and in those from children with diabetic nephropathy. The mean number of chymase-positive cells in renal tissue samples characterized by each renal disease was significantly correlated with the mean intensity of the interstitial fibrosis in that same tissue sample (Spearman's rank correlation test p = 0.0013; rank correlation coefficient 0.84). These findings suggest that mast cell-derived chymase plays an important role in juvenile crescentic glomerulonephritis.

EHBP1L1 coordinates Rab8 and Bin1 to regulate apical-directed transport in polarized epithelial cells.

The highly conserved Rab guanosine triphosphatase (GTPase) Rab8 plays a role in exocytosis toward the polarized plasma membrane in eukaryotic cells. In murine Rab8-deficient small intestine cells, apical proteins are missorted into lysosomes. In this study, we identified a novel Rab8-interacting protein complex containing an EH domain-binding protein 1-like 1 (EHBP1L1), Bin1/amphiphysin II, and dynamin. Biochemical analyses showed that EHBP1L1 directly bound to GTP-loaded Rab8 and Bin1. The spatial dependency of these complexes at the endocytic recycling compartment (ERC) was demonstrated through overexpression and knockdown experiments. EHBP1L1- or Bin1-depleted or dynamin-inhibited small intestine organoids significantly accumulated apical membrane proteins but not basolateral membrane proteins in lysosomes. Furthermore, in EHBP1L1-deficient mice, small intestine cells displayed truncated and sparse microvilli, suggesting that EHBP1L1 maintains the apical plasma membrane by regulating apical transport. In summary, our data demonstrate that EHBP1L1 links Rab8 and the Bin1-dynamin complex, which generates membrane curvature and excises the vesicle at the ERC for apical transport.

Recurrent EIARF and PRES with severe renal hypouricemia by compound heterozygous SLC2A9 mutation.

Renal hypouricemia (RHU) is a hereditary disease that predisposes affected people to exercise-induced acute renal failure (EIARF). In most patients with RHU, the disorder is caused by loss-of-function mutations in SLC22A12 (solute carrier family 22, member 12), which encodes urate transporter 1 (URAT1). Patients with RHU without any mutations in the URAT1 gene were recently found to have a mutation in the glucose transporter 9 (GLUT9) gene (SLC2A9 [solute carrier family 2, member 9]). Central nervous system complications seem to be rare in patients with RHU with SLC22A12 mutations. Here, we report the case of a girl with severe RHU (serum urate: 5.9 µmol/L [0.1 mg/dL]) associated with recurrent EIARF in whom the disease was caused by a compound heterozygous mutation in SLC2A9, a nonsense mutation in the paternal allele (p.G207X in exon 7), and a large duplication (c.1-2981_1204+16502) in the maternal allele detected by reverse-transcription polymerase chain reaction (PCR), semiquantitative PCR, long PCR, and direct sequencing. The episodes of EIARF were complicated by posterior reversible encephalopathy syndrome (PRES), which suggested a relationship between PRES and GLUT9 or severe hypouricemia. This is the second report of mutations of both alleles of SLC2A9 that resulted in severe hypouricemia. Our findings indicate that even a nonsense mutation responsible for the heterozygous status of SLC2A9 did not cause severe hypouricemia, and they lend support to previous speculation that mutations of both SLC2A9 alleles cause severe hypouricemia. Our case shows that GLUT9, unlike URAT1, may play a specific role in exercise-induced PRES.

Membranous nephropathy associated with thyroid-peroxidase antigen.

A 6-year-old previously healthy Japanese girl was found to have dipstick 2+ proteinuria and a goiter based on the results of a routine school medical examination. Her serum free-thyroxine level was 4.98 ng/dL (normal range 0.95-1.74 ng/dL), thyroid-stimulating hormone (TSH) was less than 0.003 microU/mL (0.34-3.88 microU/mL), anti-microsomal (anti-thyroid-peroxidase) antibody was 1600 T (up to 100), anti-thyroglobulin antibody was 400 T (up to 100), and TSH-receptor antibody was 84% (up to +/-10%). These results are consistent with a diagnosis of Graves' disease. Electron microscopy examination of a renal biopsy specimen revealed electron-dense deposits located in the subepithelial spaces, and immunofluorescence microscopy examination demonstrated bright granular stainings of immunoglobulin G along the glomerular capillary walls. These findings are characteristic of membranous nephropathy. To investigate the relationship between the membranous nephropathy and Graves' disease, we carried out a second immunofluorescence study, which revealed that the immunoglobulin G granular deposits corresponded to glomerular granular staining of thyroid-peroxidase, whereas staining for thyroglobulin was absent. It was therefore assumed that the deposition of immune complexes mediated by thyroid-peroxidase had caused the membranous nephropathy in this patient. This is the first report of membranous nephropathy associated with Graves' disease in which deposits of thyroid-peroxidase, rather than thyroglobulin, have been confirmed in the kidney.

Improved renal survival in Japanese children with IgA nephropathy.

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.