WT1 exon 10 missense variant in a pediatric patient with focal segmental glomerulosclerosis with embryonal hyperplasia.

A 6-year-old boy was diagnosed with chromosomal abnormalities (48,XYY, + 21[11]/46,XY[19]) at 4 months of age after a physical examination revealed an undescended testis and a dwarf penis. He also had mild renal dysfunction and severe proteinuria, and kidney biopsy at 2 years of age revealed focal segmental glomerulosclerosis. Genetic analysis to investigate suspected WT1 gene abnormalities revealed a novel variant in NM_024426.6:exon10:c.1506 T > A (p.(Asp502Glu)). His kidney function deteriorated rapidly, leading to the induction of peritoneal dialysis at 5 years of age. Although this variant had not been previously reported, bilateral nephrectomy was performed to prevent any progression of the tumor. Histopathology showed all the glomeruli observed within the observation area to be completely sclerotic, while also showing evidence of embryonal hyperplasia. This case was not a hot spot for Denys-Drash syndrome, but it had a similar phenotype and pathology that could have been derived from a WT1 gene abnormality.

Necessity and choice of therapy for Henoch-Schönlein purpura nephritis.

BACKGROUND: Henoch-Schönlein purpura nephritis often resolves spontaneously, without treatment, making decisions regarding therapeutic interventions difficult. METHODS: Fifty-four patients who were diagnosed as having Henoch-Schönlein purpura nephritis between April 2004 and March 2018, and developed hematuria and/or proteinuria, were studied retrospectively. The observation period ended at the disappearance of hematuria or proteinuria, or the last observation date before December 2019 for each patient. Twenty-four of the patients received no treatment (Group A), 19 underwent renin-angiotensin-aldosterone system inhibitors only (B), 4 experienced steroid pulse therapy and combination therapy only (C) and the remaining 7 received steroid pulse therapy and combination therapy following renin-angiotensin-aldosterone system inhibitors (C). Clinical characteristics were examined according to the treatment method. Survival analysis for persistent urinary abnormalities was performed according to treatment modality, with multiple treatment records created per subject, if necessary. RESULTS: The highest urine protein/creatinine levels were significantly higher in groups B and C than in group A. The lowest estimated glomerular filtration rate was not significantly different among the three groups. In groups A and B, proteinuria resolved in >90% of patients. Survival analysis showed that steroid pulse therapy and combination therapy was not related to the better resolution of hematuria or proteinuria than renin-angiotensin-aldosterone system inhibitors. CONCLUSIONS: Several patients with Henoch-Schönlein purpura nephritis went into remission either without treatment or with renin-angiotensin-aldosterone system inhibitors alone. The treatment plan for patients with Henoch-Schönlein purpura nephritis needs to be determined carefully.

A predictive score for detecting vesicoureteral reflux in children with their first Escherichia coli-induced urinary tract infection.

OBJECTIVES: In children with a first Escherichia coli-induced febrile urinary tract infection (fUTI), routine voiding cytourethrography (VCUG) is not recommended for detecting vesicoureteral reflux (VUR). Meanwhile, the sensitivity of renal and bladder ultrasound (RBUS) for detecting VUR is insufficient. Aiming to implement VCUG properly for children with a first E. coli-induced fUTI, we attempted to construct a predictive scoring system for the early screening of VUR. METHODS: This study enrolled patients aged <2 years of age hospitalized for their first E. coli-induced fUTI during the period when VCUG was implemented for all patients (2007-14, non-selective group [n = 111]) and only for those with VUR-suspected RBUS findings, bacteremia or acute focal bacterial nephritis (2016-19, selective group [n = 102]). We evaluated the accuracy of the current criteria and the VUR predictive score constructed using data from the non-selective group. RESULTS: In the non-selective group, 32 patients had VUR (29%). In the selective group, 20 of 45 VCUG-tested patients had VUR (44%). Among 57 patients not undergoing VCUG in the selective group, 8 had a recurrence of fUTI, 3 of whom were diagnosed with VUR. In the non-selective group, 9 patients with VUR did not fulfill the current criteria and the VUR predictive score consisting of young age, female sex, prolonged fever, hypoproteinemia, hyponatremia and hyperglycemia, showed higher sensitivity, specificity than the current criteria. CONCLUSIONS: The current imaging/bacteriological criteria were ineffective in screening for VUR in patients with their first E. coli-induced fUTI. The VUR predictive score can be an accurate indicator for implementing VCUG.

Efficacy of oral tolvaptan for severe edema and hyponatremia in a patient with refractory nephrotic syndrome.

Tolvaptan (TLV) is a vasopressin V2 receptor antagonist that increases free water excretion. However, there are few reports of the use of TLV in pediatric patients with nephrotic syndrome (NS). The efficacy of TLV for the management of edema and hyponatremia in a 12-year-old boy with refractory NS is demonstrated. In this patient, refractory NS developed at 5 years of age, and remission was maintained with several immunosuppressive agents. He was admitted to hospital due to relapse with oliguria, edema, hypoalbuminemia, and hyponatremia. Infusion of human albumin and furosemide did not increase his urine volume, and hyponatremia worsened. Administration of TLV increased urine volume and improved his edema and hyponatremia. There were no adverse effects except for slow elevation of the serum sodium level. Serum osmolality increased gradually, and urine osmolality remained at low levels during TLV treatment. Additionally, a decrease in the sum of the urinary sodium and potassium concentrations was useful to predict the response to TLV and to assess the therapeutic effect of TLV as a biomarker for monitoring. TLV is effective for the management of fluid and electrolyte balance in pediatric NS patients. Early administration of TLV is considered a useful therapy for hyponatremia and refractory edema resistant to other diuretics.

Involvement of activated cytotoxic T lymphocytes and natural killer cells in Henoch-Schönlein purpura nephritis.

OBJECTIVES: Immunoglobulin A vasculitis/Henoch-Schönlein purpura (IgAV/HSP) is a major cause of vasculitis in children. It is often accompanied by nephritis (HSPN) and could progress to chronic kidney disease. Galactose-deficient IgA1 was recently reported to be involved in the pathogenesis of HSPN, for which immunosuppressive drugs are considered key treatment. However, the involvement of immune cells in the development of HSPN remains unclear. METHODS: We compared gene expressions of peripheral blood mononuclear cells (PBMCs) among healthy controls (n = 10), IgAV/HSP patients (n = 21) and HSPN patients (n = 8), which required nephritis development within 3 months of IgAV/HSP onset. Immunohistochemistry analysis and flow cytometry were performed to assess renal biopsy specimens and PBMCs, respectively. Serum CX3CL1 levels were measured by ELISA. RESULTS: GNLY and GZMB expressions increased in HSPN patients, consistent with increased number of glomerular granulysin- and/or granzyme B-positive cells demonstrated by immunohistochemistry analysis. Additionally, circulating cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells were activated with the up-regulated surface expressions of human leucocyte antigen DR (HLA-DR) and CX3CR1 in HSPN patients with severe proteinuria. Renal biopsies demonstrated increased number of CD8+ cells and/or CD56+ cells and up-regulated expression of glomerular CX3CL1, a specific ligand for CX3CR1, along with increased serum CX3CL1 level. CONCLUSION: Activated CTLs and NK cells play roles in the development of nephritis in IgAV/HSP patients and can be used as novel biomarkers for HSPN.

Common risk variants in NPHS1 and TNFSF15 are associated with childhood steroid-sensitive nephrotic syndrome.

To understand the genetics of steroid-sensitive nephrotic syndrome (SSNS), we conducted a genome-wide association study in 987 childhood SSNS patients and 3,206 healthy controls with Japanese ancestry. Beyond known associations in the HLA-DR/DQ region, common variants in NPHS1-KIRREL2 (rs56117924, P=4.94E-20, odds ratio (OR) =1.90) and TNFSF15 (rs6478109, P=2.54E-8, OR=0.72) regions achieved genome-wide significance and were replicated in Korean, South Asian and African populations. Trans-ethnic meta-analyses including Japanese, Korean, South Asian, African, European, Hispanic and Maghrebian populations confirmed the significant associations of variants in NPHS1-KIRREL2 (Pmeta=6.71E-28, OR=1.88) and TNFSF15 (Pmeta=5.40E-11, OR=1.33) loci. Analysis of the NPHS1 risk alleles with glomerular NPHS1 mRNA expression from the same person revealed allele specific expression with significantly lower expression of the transcript derived from the risk haplotype (Wilcox test p=9.3E-4). Because rare pathogenic variants in NPHS1 cause congenital nephrotic syndrome of the Finnish type (CNSF), the present study provides further evidence that variation along the allele frequency spectrum in the same gene can cause or contribute to both a rare monogenic disease (CNSF) and a more complex, polygenic disease (SSNS).

Strong Association of the HLA-DR/DQ Locus with Childhood Steroid-Sensitive Nephrotic Syndrome in the Japanese Population.

Background Nephrotic syndrome is the most common cause of chronic glomerular disease in children. Most of these patients develop steroid-sensitive nephrotic syndrome (SSNS), but the loci conferring susceptibility to childhood SSNS are mainly unknown.Methods We conducted a genome-wide association study (GWAS) in the Japanese population; 224 patients with childhood SSNS and 419 adult healthy controls were genotyped using the Affymetrix Japonica Array in the discovery stage. Imputation for six HLA genes (HLA-A, -C, -B, -DRB1, -DQB1, and -DPB1) was conducted on the basis of Japanese-specific references. We performed genotyping for HLA-DRB1/-DQB1 using a sequence-specific oligonucleotide-probing method on a Luminex platform. Whole-genome imputation was conducted using a phased reference panel of 2049 healthy Japanese individuals. Replication was performed in an independent Japanese sample set including 216 patients and 719 healthy controls. We genotyped candidate single-nucleotide polymorphisms using the DigiTag2 assay.Results The most significant association was detected in the HLA-DR/DQ region and replicated (rs4642516 [minor allele G], combined Pallelic=7.84×10-23; odds ratio [OR], 0.33; 95% confidence interval [95% CI], 0.26 to 0.41; rs3134996 [minor allele A], combined Pallelic=1.72×10-25; OR, 0.29; 95% CI, 0.23 to 0.37). HLA-DRB1*08:02 (Pc=1.82×10-9; OR, 2.62; 95% CI, 1.94 to 3.54) and HLA-DQB1*06:04 (Pc=2.09×10-12; OR, 0.10; 95% CI, 0.05 to 0.21) were considered primary HLA alleles associated with childhood SSNS. HLA-DRB1*08:02-DQB1*03:02 (Pc=7.01×10-11; OR, 3.60; 95% CI, 2.46 to 5.29) was identified as the most significant genetic susceptibility factor.Conclusions The most significant association with childhood SSNS was detected in the HLA-DR/DQ region. Further HLA allele/haplotype analyses should enhance our understanding of molecular mechanisms underlying SSNS.

Combination therapy with or without warfarin and dipyridamole for severe childhood IgA nephropathy: an RCT.

BACKGROUND: Two previous randomized controlled trials showed that treatment of severe childhood immunoglobulin A (IgA) nephropathy using prednisolone with azathioprine, heparin-warfarin, or dipyridamole prevented the increase of sclerosed glomeruli. Prednisolone alone, however, did not prevent further increase. These studies indicated the importance of immunosuppressants in the treatment. An additional pilot study using mizoribine instead of azathioprine enabled us to complete 2 years of combined regimen. It showed non-numerical inferior effectiveness compared with the azathioprine regimen. Further examination of the additional efficacy of warfarin and dipyridamole was required. METHODS: A randomized control trial of prednisolone and mizoribine with (group 1) or without (group 2) warfarin and dipyridamole was administered for treatment of 71 children with severe IgA nephropathy to evaluate the efficacy of additional warfarin and dipyridamole. RESULTS: Thirty of 34 patients (88.2%) in group 1, and 27 of 36 patients (75.0%) showed the disappearance of proteinuria as defined by early morning urinary protein to creatinine ratio of < 0.2 during the 2-year treatment period. The cumulative disappearance rate of proteinuria determined by the Kaplan-Meier method showed that the disappearance rate of proteinuria was significantly higher in group 1 than in group 2 (log-rank P = 0.04). There was no significant difference in pathological findings, but there was a tendency of increase of global sclerosis in group1 which might be related to warfarin. Most of the adverse effects were related to prednisolone, but fortunately transient. CONCLUSIONS: The balance between minimal benefits of warfarin/dipyridamole and potential adverse effects may be in favor of avoiding them in children with IgA nephropathy.

A pure chloride channel mutant of CLC-5 causes Dent's disease via insufficient V-ATPase activation.

Dent's disease is characterized by defective endocytosis in renal proximal tubules (PTs) and caused by mutations in the 2Cl(-)/H(+) exchanger, CLC-5. However, the pathological role of endosomal acidification in endocytosis has recently come into question. To clarify the mechanism of pathogenesis for Dent's disease, we examined the effects of a novel gating glutamate mutation, E211Q, on CLC-5 functions and endosomal acidification. In Xenopus oocytes, wild-type (WT) CLC-5 showed outward-rectifying currents that were inhibited by extracellular acidosis, but E211Q and an artificial pure Cl(-) channel mutant, E211A, showed linear currents that were insensitive to extracellular acidosis. Moreover, depolarizing pulse trains induced a robust reduction in the surface pH of oocytes expressing WT CLC-5 but not E211Q or E211A, indicating that the E211Q mutant functions as a pure Cl(-) channel similar to E211A. In HEK293 cells, E211A and E211Q stimulated endosomal acidification and hypotonicity-inducible vacuolar-type H(+)-ATPase (V-ATPase) activation at the plasma membrane. However, the stimulatory effects of these mutants were reduced compared with WT CLC-5. Furthermore, gene silencing experiments confirmed the functional coupling between V-ATPase and CLC-5 at the plasma membrane of isolated mouse PTs. These results reveal for the first time that the conversion of CLC-5 from a 2Cl(-)/H(+) exchanger into a Cl(-) channel induces Dent's disease in humans. In addition, defective endosomal acidification as a result of insufficient V-ATPase activation may still be important in the pathogenesis of Dent's disease.

X-linked Alport syndrome caused by splicing mutations in COL4A5.

BACKGROUND AND OBJECTIVES: X-linked Alport syndrome is caused by mutations in the COL4A5 gene. Although many COL4A5 mutations have been detected, the mutation detection rate has been unsatisfactory. Some men with X-linked Alport syndrome show a relatively mild phenotype, but molecular basis investigations have rarely been conducted to clarify the underlying mechanism. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: In total, 152 patients with X-linked Alport syndrome who were suspected of having Alport syndrome through clinical and pathologic investigations and referred to the hospital for mutational analysis between January of 2006 and January of 2013 were genetically diagnosed. Among those patients, 22 patients had suspected splice site mutations. Transcripts are routinely examined when suspected splice site mutations for abnormal transcripts are detected; 11 of them showed expected exon skipping, but others showed aberrant splicing patterns. The mutation detection strategy had two steps: (1) genomic DNA analysis using PCR and direct sequencing and (2) mRNA analysis using RT-PCR to detect RNA processing abnormalities. RESULTS: Six splicing consensus site mutations resulting in aberrant splicing patterns, one exonic mutation leading to exon skipping, and four deep intronic mutations producing cryptic splice site activation were identified. Interestingly, one case produced a cryptic splice site with a single nucleotide substitution in the deep intron that led to intronic exonization containing a stop codon; however, the patient showed a clearly milder phenotype for X-linked Alport syndrome in men with a truncating mutation. mRNA extracted from the kidney showed both normal and abnormal transcripts, with the normal transcript resulting in the milder phenotype. This novel mechanism leads to mild clinical characteristics. CONCLUSIONS: This report highlights the importance of analyzing transcripts to enhance the mutation detection rate and provides insight into genotype-phenotype correlations. This approach can clarify the cause of atypically mild phenotypes in X-linked Alport syndrome.

Standardized peritoneal equilibration test in Japanese children and the influence of long-term peritoneal dialysis.

OBJECTIVE: To establish a standardized method and standard values of peritoneal equilibration test (PET) in children and to elucidate the factors influencing peritoneal permeability, we standardized the method of PET in the leading hospitals of the Japanese Study Group of Pediatric Peritoneal Dialysis. METHODS: Using 2.5% glucose dialysate and an infusion volume of 1,100 mL/m(2) body surface area (BSA), we performed 202 standardized PETs. The patients within 1 month of a peritonitis episode were excluded. RESULTS: The-1 standard deviation (SD), mean, and +1 SD values of end dialysate-to-initial dialysate ratio of (D/D(0)) glucose were 0.31, 0.41, and 0.51 respectively. The dialysate-to-plasma ratios (D/P) of creatinine were 0.52, 0.65, and 0.78. These values were similar to those of Twardowski's adult PET, although previous reports about standardized pediatric PET described more permeable values. Because the duration of PD was longer in the study patients, peritoneal permeability was increased significantly in patients with and without experience of peritonitis alike. The slope indices of the regression equations between the duration of PD and peritoneal permeability in the two groups were same. CONCLUSIONS: Standardized PET in Japanese children with 2.5% glucose dialysate and an infusion volume of 1,100 mL/m(2) BSA produces standard values similar to those of Twardowski's adult PET. This standardized method of PET is suitable for pediatric PD patients, and peritoneal permeability in children is not higher than that in adults. In addition, the study data show that long-term peritoneal dialysis worsens peritoneal function more than experience of peritonitis does.

Clinicopathologic correlation and outcome of C1q nephropathy.

BACKGROUND AND OBJECTIVES: The number of patients with C1q nephropathy (C1qN) in previous reports is small and the duration of follow-up is short. Our study describes the clinicopathologic correlation and clinical outcome through the mean follow-up period of 7.2 yr in 61 patients. DESIGN, SETTINGS, PARTICIPANTS, & MEASUREMENTS: Sixty-one patients, 1 to 67 yr of age, with C1qN were enrolled in this study. RESULTS: According to presentation at onset, patients were divided into two groups: asymptomatic urinary abnormalities (asymptomatic) (n = 36) and nephrotic syndrome (NS) (n = 25). Light microscopy showed minimal change disease (MCD) in 46 patients (75%), mesangial proliferative glomerulonephritis in 7 (12%), and focal segmental glomerulosclerosis (FSGS) in 8 (13%). The prevalence of MCD was higher in the NS group than in the asymptomatic group. Nine patients in the asymptomatic group and all patients in the NS group were treated with prednisolone and/or cyclosporine. Normal urinalysis was found in 10 patients in asymptomatic group and 8 in NS group during the follow-up. Thirteen patients in the NS group were frequent relapsers at the latest follow-up. Three patients with FSGS developed chronic renal failure 8 to 15 yr after the diagnosis. C1q deposits disappeared in 3 of 8 patients receiving repeat biopsy, and 2 of these 3 showed FSGS. CONCLUSIONS: The prognosis of C1qN is good, associated with MCD in a large number. In some patients, C1q deposits disappear through the follow-up period. FSGS may develop in some patients on repeat biopsies. Further investigation is critically needed to settle this issue.

Clinicopathologic correlation of C1q nephropathy in children.

BACKGROUND: Clinicopathologic correlation of C1q nephropathy is clarified poorly. The aim of our study is to clarify clinicopathologic correlation in childhood C1q nephropathy. METHODS: Thirty children aged 3 to 15 years who met criteria proposed by Jennette and Hipp were enrolled in this study. RESULTS: According to their presentation at onset, children were divided into 2 groups: the asymptomatic urinary abnormalities (asymptomatic) group (n = 18) and the nephrotic syndrome (NS) group (n = 12). Light microscopy showed minimal change disease (MCD) in 22 children (73%), mesangial proliferative glomerulonephritis in 6 children (20%), and focal segmental glomerulosclerosis (FSGS) in 2 children (7%). Four children in the asymptomatic group and all children in the NS group were administered prednisolone and/or cyclosporine. Normal urinalysis results were found in 8 children in the asymptomatic group and 3 children in the NS group during the follow-up period of 3 to 15 years. Eight children in the NS group were frequent relapsers at the latest follow-up. Two children with FSGS (1 child, asymptomatic group; 1 child, NS group) received dialysis 10 and 15 years after the diagnosis. There were no differences in histological findings and clinical outcomes between the 2 groups. Four children with MCD in the NS group underwent a second biopsy. C1q deposits disappeared in 2 children, and 1 of these 2 children showed FSGS. CONCLUSION: Childhood C1q nephropathy is found in a wide clinical spectrum. Some children showed disappearance of C1q deposits through the follow-up period. A large number of children with C1q nephropathy showed MCD. However, FSGS may develop in some children on repeated biopsy. Therefore, long-term follow-up is needed in children with C1q nephropathy.

Analysis of NPHS1, NPHS2, ACTN4, and WT1 in Japanese patients with congenital nephrotic syndrome.

BACKGROUND: Congenital nephrotic syndrome (CNS) causes significant renal failure, and is classified into two types: (1) Finnish type; and (2) other, including diffuse mesangial sclerosis. Mutations of NPHS1 and NPHS2, which encode the slit diaphragm components nephrin and podocin, cause CNS and autosomal-recessive familial steroid-resistant nephrotic syndrome, respectively. Most patients with Finnish-type CNS in Europe and the United States have NPHS1 mutations. However, NPHS2 mutations have been detected in some cases. Mutations in ACTN4, encoding alpha-actinin-4, cause an autosomal-dominant focal segmental glomerulosclerosis. alpha-actinin-4 stabilizes the podocyte cytoskeleton structure, connecting with actin filaments. WT1 mutations, causing Wilm's tumor, have been demonstrated in some CNS patients with diffuse mesangial sclerosis. Systematic investigation of genes for CNS in Japan has never been performed. METHODS: To clarify the role of mutations in these four genes, we used polymerase chain reaction (PCR) and direct sequencing to investigate all exons and exon-intron boundaries for these genes in 13 unrelated CNS patients from regional pediatric kidney disease centers in Japan. RESULTS: A novel homozygous nonsense mutation of NPHS1, E246X in exon 7, and a novel homozygous deletion mutation of NPHS1, 2156_2163del in exon 16 were detected in one patient each. A novel homozygous nonsense mutation of NPHS2, R196X in exon 5, was found in one patient, and the same heterozygous nonsense mutation was detected in another. No ACTN4 or WT1 mutations were detected. CONCLUSION: These studies demonstrate that mutation of NPHS1 is not a major cause of CNS in Japanese patients, and that mutation of NPHS2 can be responsible for CNS in this population.