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Establishing a molecular genetic diagnosis of focal segmental glomerulosclerosis (FSGS)/steroid-resistant nephrotic syndrome (SRNS) can be useful for predicting post-transplant recurrence. Monogenic causes are reportedly present in approximately 20-30% of patients with FSGS/SRNS. However, the characteristics of patients who are likely to have a monogenic cause remain to be determined.Pediatric recipients with SRNS and/or biopsy-proven FSGS who underwent their first kidney transplantation at our center between 1999 and 2019 were analyzed. Patients with secondary FSGS/SRNS were excluded. The recipients were divided into three groups: familial/syndromic, presumed primary, and undetermined FSGS/SRNS. Patients who met all of the following criteria were categorized as having presumed primary FSGS/SRNS: (i) nephrotic syndrome, (ii) complete or partial remission with initial steroid therapy and/or additional immunosuppressive therapies, and (iii) diffuse foot process effacement on electron microscopy in the native kidney biopsy. All patients underwent genetic testing using next-generation sequencing.Twenty-four patients from 23 families were analyzed in this study. Pathogenic or likely pathogenic variants in FSGS/SRNS-related genes were identified in four of four families, zero of eight families, and 10 of 11 families with familial/syndromic, presumed primary, and undetermined FSGS/SRNS, respectively. Post-transplant recurrence only occurred in patients with presumed primary FSGS/SRNS.Our systematic approach based on precise clinicopathological findings including nephrotic syndrome, treatment responses, and diffuse foot process effacement might be useful to differentiate pediatric kidney transplant recipients with FSGS/SRNS who are likely to have a monogenic cause from patients who are not, and to predict post-transplant recurrence. A higher resolution version of the Graphical abstract is available as Supplementary information.

Congenital anomalies of the kidney and urinary tract (CAKUT) are frequently associated with Mullerian anomalies. This can be explained by the fact that Mullerian duct elongation depends on the preformed Wolffian duct during embryogenesis. While CAKUT such as unilateral renal agenesis and multicystic dysplastic kidney are commonly identified prenatally by routine ultrasound, the diagnosis of Mullerian anomalies is often delayed, increasing the risk of complications such as endometriosis or pelvic inflammatory disease. Herein, we report a case of a premenarchal girl who had initially been diagnosed with right multicystic dysplastic kidney. She presented with continuous urinary incontinence at 4 years old and further evaluation by contrast-enhanced computed tomography, cystoscopy, colposcopy, ureterography, and hysterosalpingography led to the final diagnosis of right hypodysplastic kidney and ectopic ureter associated with bicornuate uterus. A strong family history of uterine malformations prompted the examination of the uterus. Genetic testing was suggested but the family declined. She is planned to be referred to a gynecologist at puberty for further assessment. The recognition and screening rate of concurrent Mullerian anomalies in CAKUT patients varies between institutions. Screening for Mullerian anomalies in prediagnosed CAKUT girls may enable to provide timely counseling and to prevent gynecological complications.

Renal hypoplasia (RH) is the most common cause of chronic kidney disease in children. In cases of RH, proteinuria is often induced by glomerular hypertrophy and hyperfiltration that is commonly associated with focal segmental glomerulosclerosis. This study reports the first case series of a possible association between RH and membranous nephropathy (MN). Of the 168 children with RH who visited our department between 1999 and 2017, five with overt proteinuria (≥ 1 g/gCr) underwent renal biopsy. We retrospectively reviewed the medical charts and analyzed biopsy specimens using light microscopy (LM), immunofluorescence (IF), and electron microscopy. The five children (four boys and one girl) had a median age of 5.5 years at the time of renal biopsy. The median proteinuria was 4.23 g/gCr (range 1.46–14.25), median serum albumin, 2.9 g/dL (range 2.3–3.7), and median estimated glomerular filtration rate, 59.7 mL/min/1.73 m2 (range 36.7–103.6). LM showed segmental spike formation and mesangial hypercellularity and IF study showed segmental granular immunoglobulin G (IgG) staining (IgG1 and IgG3 dominant) along the capillary loops in all five patients. Electron-dense deposits were observed in the subepithelial and mesangial areas. Thus, the pathological studies showed MN-like lesions in all patients. Our study suggests that RH can be the cause of MN-like lesions.

Mutations of OCRL cause Lowe syndrome, which is characterised by congenital cataracts, infantile hypotonia with mental retardation, and renal tubular dysfunction and Dent-2 disease, which only affects the kidney. While few patients with an intermediate phenotype between these diseases have been reported, the mechanism underlying variability in the phenotype is unclear. We identified an intronic mutation, c.2257-5G>A, in intron 20 of OCRL in an older brother with atypical Lowe syndrome without eye involvement and a younger brother with renal phenotype alone. This mutation created a splice acceptor motif that was accompanied by a cryptic premature termination codon at the junction of exons 20 and 21. The mutation caused incomplete alternative splicing, which created a small amount of wild-type transcript and a relatively large amount of alternatively spliced transcript with a premature termination codon. In the patients' cells, the alternatively spliced transcript was degraded by nonsense-mediated decay and the wild-type transcript was significantly decreased, but not completely depleted. These findings imply that an intronic mutation creating an incomplete alternative splicing acceptor site results in a relatively low level of wild-type OCRL mRNA expression, leading to partial phenotypes of Lowe syndrome.

Pneumocystis jirovecii pneumonia associated with primary immunodeficiency should be considered in infants with slowly progressing cyanosis, even without fever or respiratory symptoms. Genetic counseling is crucial for incontinentia pigmenti families in advance of pregnancy because lethal infections can occur before the diagnosis of X‐linked anhidrotic ectodermal dysplasia with immunodeficiency., Pneumocystis jirovecii pneumonia in primary immunodeficiency (ID) should be considered in slowly progressing cyanotic infants. Prenatal genetic counseling is crucial to prevent fatal infection in X‐linked anhidrotic ectodermal dysplasia with ID.

Congenital disorders characterized by the quantitative and qualitative reduction in the number of functional nephrons are the primary cause of chronic kidney disease (CKD) in children. We aimed to describe the alteration of urinary extracellular vesicles (uEVs) associated with decreased renal function during childhood. By nanoparticle tracking analysis and quantitative proteomics, we identified differentially expressed proteins in uEVs in bilateral renal hypoplasia, which is characterized by a congenitally reduced number of nephrons. This expression signature of uEVs reflected decreased renal function in CKD patients by congenital anomalies of the kidney and urinary tract or ciliopathy. As a proof-of-concept, we constructed a prototype ELISA system that enabled the isolation of uEVs and quantitation of expression of molecules representing the signature. The system identified decreased renal function even in its early stage. The uEVs signature could pave the way for non-invasive methods that can complement existing testing methods for diagnosing kidney diseases.

The precise mechanism of hyponatremia in Kawasaki disease (KD) remains elusive because assessment of volume status based on serial changes in body weight is lacking in previous reports.Seventeen patients who were diagnosed with KD and hyponatremia (serum sodium levels135 mmol/L) were analyzed. Volume status was assessed based on serial changes in body weight. Plasma arginine vasopressin (ADH), urine electrolytes, and serum cytokine levels were measured on diagnosis of hyponatremia. An increase in body weight by3% was defined as hypervolemia and a decrease in body weight by3% was defined as hypovolemia.The volume status was hypervolemic in three patients (18%), euvolemic in 14 (82%), and hypovolemic in none (0%). Five (29%) patients were diagnosed with "syndrome of inappropriate secretion of antidiuretic hormone" (SIADH) and no patients were diagnosed with hypotonic dehydration. The contribution of decreased total exchangeable cations (salt loss) to hyponatremia (5.9% [interquartile range, 4.3%, 6.7%]) was significantly larger than that of increased total body water (-0.7% [-1.8%, 3.1%]) (P = 0.004). Serum interleukin-6 levels were elevated in all of the nine patients who were evaluated. Among the 12 (71%) patients who did not meet the criteria of SIADH and hypotonic dehydration, plasma ADH levels were inappropriately high in ten patients. These patients were also characterized by euvolemic or hypervolemic hyponatremia and salt loss, which might be compatible with a diagnosis of SIADH.Our study shows that hyponatremia in KD is euvolemic or hypervolemic and is associated with nonosmotic secretion of ADH and salt loss in the majority of patients.

Background Researchers have identified about 40 genes with mutations that result in the most common cause of CKD in children, congenital anomalies of the kidney and urinary tract (CAKUT), but approximately 85% of patients with CAKUT lack mutations in these genes. The anomalies that comprise CAKUT are clinically heterogenous, and thought to be caused by disturbances at different points in kidney development. However, identification of novel CAKUT-causing genes remains difficult because of their variable expressivity, incomplete penetrance, and heterogeneity. Methods We investigated two generations of a family that included two siblings with CAKUT. Although the parents and another child were healthy, the two affected siblings presented the same manifestations, unilateral renal agenesis and contralateral renal hypoplasia. To search for a novel causative gene of CAKUT, we performed whole-exome and whole-genome sequencing of DNA from the family members. We also generated two lines of genetically modified mice with a gene deletion present only in the affected siblings, and performed immunohistochemical and phenotypic analyses of these mice. Results We found that the affected siblings, but not healthy family members, had a homozygous deletion in the Cobalamin Synthetase W Domain-Containing Protein 1 (CBWD1) gene. Whole-genome sequencing uncovered genomic breakpoints, which involved exon 1 of CBWD1, harboring the initiating codon. Immunohistochemical analysis revealed high expression of Cbwd1 in the nuclei of the ureteric bud cells in the developing kidneys. Cbwd1-deficient mice showed CAKUT phenotypes, including hydronephrosis, hydroureters, and duplicated ureters. Conclusions The identification of a deletion in CBWD1 gene in two siblings with CAKUT implies a role for CBWD1 in the etiology of some cases of CAKUT.

Introduction: Mutations in genes encoding nucleoporins (NUPs; components of nuclear pore complexes [NPCs]), such as NUP93, have been reported to cause steroid-resistant nephrotic syndrome (SRNS) or focal segmental glomerulosclerosis (FSGS), which often progresses to end-stage renal disease (ESRD) in childhood. The expression of NUP93 in renal or extrarenal tissues, and the mechanism by which NUP93 mutations cause this renal phenotype, remain unclear. Methods: The expression of NUP93 in normal control kidney and in a patient with FSGS carrying NUP93 mutations was examined by immunofluorescence analysis. The expression of NUP93 in blood cells was analyzed by Western blot analysis. Results: Immunofluorescence analysis detected NUP93 expression in nuclei of all glomerular and tubulointerstitial cells in human kidneys. Whole-exome sequencing identified a compound heterozygous NUP93 mutation comprising a novel missense mutation p.Arg525Trp, and a previously reported mutation, p.Tyr629Cys, in a patient with FSGS that developed ESRD at the age of 6 years. In the patient’s kidney, the intensity of NUP93 immunofluorescence was significantly decreased in the nuclei of both glomerular and extraglomerular cells. The expression of CD2-associated protein (CD2AP) and nephrin in the patient’s podocytes was relatively intact. The amount of NUP93 protein was not significantly altered in the peripheral blood mononuclear cells of the patient. Conclusion: NUP93 is expressed in the nuclei of all the cell types of the human kidney. Altered NUP93 expression in glomerular cells as well as extraglomerular cells by NUP93 mutations may underlie the pathogenic mechanism of SRNS or FSGS. Keywords: focal segmental glomerulosclerosis, nuclear pore complex, nucleoporin, NUP93, steroid-resistant nephrotic syndrome

Complex rearrangements of chromosomes 3 and 9 were found in a patient presenting with severe epilepsy, developmental delay, dysmorphic facial features, and skeletal abnormalities. Molecular cytogenetic analysis revealed 46,XX.ish der(9)(3qter→3q28::9p21.1→9p22.3::9p22.3→9qter)(RP11-368G14+,RP11-299O8-,RP11-905L2++,RP11-775E6++). Her dysmorphic features are consistent with 3q29 microduplication syndrome and inv dup del(9p). Trio-based WES of the patient revealed no pathogenic single nucleotide variants causing epilepsy, but confirmed a 3q28q29 duplication involving FGF12, which encodes fibroblast growth factor 12. FGF12 positively regulates the activity of voltage-gated sodium channels. Recently, only one recurrent gain-of-function variant [NM_021032.4:c.341G>A:p.(Arg114His)] in FGF12 was found in a total of 10 patients with severe early-onset epilepsy. We propose that the patient's entire FGF12 duplication may be analogous to the gain-of-function variant in FGF12 in the epileptic phenotype of this patient.

Determining the cause of focal segmental glomerulosclerosis (FSGS) has crucial implications for evaluating the risk of posttransplant recurrence. The degree of foot process effacement (FPE) on electron micrographs (EM) of native kidney biopsies can reportedly differentiate primary FSGS from secondary FSGS. However, no systematic evaluation of FPE in genetic FSGS has been performed. In this study, percentage of FPE and foot process width (FPW) in native kidney biopsies were analyzed in eight genetic FSGS patients and nine primary FSGS patients. All genetic FSGS patients showed segmental FPE up to 38% and FPW below 2000 nm, while all primary FSGS patients showed diffuse FPE above 88% and FPW above 3000 nm. We reviewed the literature which described the degree of FPE in genetic FSGS patients and identified 38 patients with a description of the degree of FPE. The degree of FPE in patients with mutations in the genes encoding proteins associated with slit diaphragm and cytoskeletal proteins was varied, while almost all patients with mutations in other FSGS genes showed segmental FPE. In conclusion, the present study suggests that the degree of FPE in native kidney biopsies may be useful for differentiating some genetic FSGS patients from primary FSGS patients.

The gene encoding hepatocyte nuclear factor 1β (HNF1B), a transcription factor involved in the development of the kidney and other organs, is located on chromosome 17q12. Heterozygous deletions of chromosome 17q12, which involve 15 genes including HNF1B, are known as 17q12 deletion syndrome and are a common cause of congenital anomalies of the kidneys and urinary tract (CAKUT) and may also present as a multisystem disorder. Autosomal recessive polycystic kidney disease (ARPKD), on the other hand, is a severe form of polycystic kidney disease caused by mutations in PKHD1 (polycystic kidney and hepatic disease 1). It is important to differentiate between these two diseases because they differ significantly in inheritance patterns, renal prognosis, and extrarenal manifestations. Here we report a case of 17q12 deletion syndrome that clinically mimicked ARPKD in which genetic testing was essential for appropriate genetic counseling and monitoring of possible extrarenal manifestations. The patient presented antenatally with markedly enlarged kidneys and showed bilaterally hyperechoic kidneys with poor corticomedullary differentiation and multiple cysts on ultrasonography. There was no family history of renal disease. ARPKD was clinically suspected and genetic testing was performed to confirm diagnosis, resulting in an unexpected finding of 17q12 deletion including HNF1B. While some research has been done to identify patients that should be tested for HNF1B anomalies, this case illustrates the difficulty of recognizing HNF1B-related disease and the importance of genetic testing in appropriately managing CAKUT cases.

Background Heterozygous truncating variants in the Tripartite motif containing 8 (TRIM8) gene have been reported to cause epileptic encephalopathy, both with and without proteinuria. A recent study showed a lack of TRIM8 protein expression, with suppressor of cytokine signaling 1 (SOCS1) overexpression, in podocytes and tubules from a patient with a TRIM8 variant, who presented with epileptic encephalopathy and focal segmental glomerulosclerosis (FSGS). To date, no patients with TRIM8 variants who presented with nephrotic syndrome but without neurological manifestations have been described. Case presentation An 8-year-old girl presented with nephrotic syndrome, without epilepsy or developmental delay. Her kidney biopsy specimens showed FSGS and cystic dilatations of the distal tubules. Whole-exome sequencing identified a novel de novo heterozygous variant in the C-terminal encoding portion of TRIM8 (c.1461C > A), resulting in a premature stop codon (p.Tyr487*). Reverse transcription-polymerase chain reaction using peripheral blood mononuclear cells identified the mRNA sequence of the mutant allele, which confirmed an escape from nonsense-mediated mRNA decay. Immunofluorescence studies showed a lack of TRIM8 expression in glomerular and tubular cells and cystic dilatation of distal tubules. Immunohistochemical studies showed overexpression of SOCS1 in glomerular and tubular cells. Conclusions We reported a patient with FSGS, associated with a de novo heterozygous TRIM8 variant, without any neurological manifestations. Our results expanded the clinical phenotypic spectrum of TRIM8 variants.

Currently, there are no treatments for Alport syndrome, which is the second most commonly inherited kidney disease. Here we report the development of an exon-skipping therapy using an antisense-oligonucleotide (ASO) for severe male X-linked Alport syndrome (XLAS). We targeted truncating variants in exon 21 of the COL4A5 gene and conducted a type IV collagen α3/α4/α5 chain triple helix formation assay, and in vitro and in vivo treatment efficacy evaluation. We show that exon skipping enabled trimer formation, leading to remarkable clinical and pathological improvements including expression of the α5 chain on glomerular and the tubular basement membrane. In addition, the survival period was clearly prolonged in the ASO treated mice group. This data suggests that exon skipping may represent a promising therapeutic approach for treating severe male XLAS cases., Alport syndrome is a progressive inherited nephritis accompanied by sensorineural loss of hearing and ocular abnormalities, for which there is currently no effective therapy. Here, the authors develop an exon-skipping therapy using an antisense-oligonucleotide and show it is effective in mouse models.

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 (P(meta)=6.71E-28, OR=1.88) and TNFSF15 (P(meta)=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).

Mutations of OCRL cause Lowe syndrome, which is characterised by congenital cataracts, infantile hypotonia with mental retardation, and renal tubular dysfunction and Dent-2 disease, which only affects the kidney. While few patients with an intermediate phenotype between these diseases have been reported, the mechanism underlying variability in the phenotype is unclear. We identified an intronic mutation, c.2257-5GA, in intron 20 of OCRL in an older brother with atypical Lowe syndrome without eye involvement and a younger brother with renal phenotype alone. This mutation created a splice acceptor motif that was accompanied by a cryptic premature termination codon at the junction of exons 20 and 21. The mutation caused incomplete alternative splicing, which created a small amount of wild-type transcript and a relatively large amount of alternatively spliced transcript with a premature termination codon. In the patients' cells, the alternatively spliced transcript was degraded by nonsense-mediated decay and the wild-type transcript was significantly decreased, but not completely depleted. These findings imply that an intronic mutation creating an incomplete alternative splicing acceptor site results in a relatively low level of wild-type OCRL mRNA expression, leading to partial phenotypes of Lowe syndrome.

The dosing of intravenous immunoglobulin (IVIG) therapy for Kawasaki disease (KD) has been a matter of debate for decades, with recent studies implicating that larger doses lead to better outcomes. Despite this, few have investigated post-IVIG infusion immunoglobulin G (IgG) levels in relation to outcomes of KD such as response to IVIG and development of coronary artery abnormalities (CAAs). The present study investigated how varying levels of post-infusion IgG affected these outcomes.We collected demographic and laboratory data, including post-infusion IgG, from children with KD who were admitted to six hospitals in Japan between 2006 and 2012. We conducted multivariate analyses to examine the relationship between independent variables and non-response to IVIG and development of CAAs. We used random forest, a decision tree-based machine learning tool, to investigate the marginal effect of varying post-infusion IgG levels on non-response to IVIG and development of CAAs.Of 456 patients included in the study, 130 (28.5%) were non-responders and 38 (8.3%) developed CAAs. Sodium, post-infusion IgG, and AST were significantly associated with non-response. Post-infusion IgG and sodium were significantly associated with CAA development. The random forest plots revealed a decrease in non-response and CAA rates with increasing post-infusion IgG until post-infusion IgG was near the median (2821 mg/dL), after which the non-response and CAA rates leveled off.Greater post-infusion IgG is associated with better response to IVIG and decreased CAA development in KD patients, but this effect levels off at post-infusion IgG levels greater than the median. Key points • Though previous studies have shown that post-intravenous immunoglobulin (IVIG) infusion immunoglobulin G (IgG) is associated with non-response to IVIG therapy and coronary artery abnormality (CAA) development in Kawasaki disease (KD) patients, no study has investigated the relationship between varying levels of post-infusion IgG and these clinical outcomes. • Our study showed that non-response to IVIG therapy and CAA development in Kawasaki disease patients follow a decreasing trend with increasing post-infusion IgG at post-infusion IgG levels below the median. • At values of post-infusion IgG greater than the median, non-response and CAA development rates remain relatively constant with increasing post-infusion IgG. • Our study suggests that when post-infusion IgG is greater than the median, IgG may have fully bound to the therapeutic targets of KD, and in these patients, there may be limited benefit in administering additional IVIG.

A broad range of genetic and non-genetic factors can lead to kidney injury that manifests as focal segmental glomerulosclerosis (FSGS), which can be classified into primary (idiopathic) and secondary forms. Previous genetic approaches to familial or sporadic cases of FSGS or steroid-resistant nephrotic syndrome identified causal mutations in a subset of genes. Recently, next-generation sequencing (NGS) approaches are becoming a part of a standard assessment in medical genetics. Current knowledge of the comprehensive genomic information is changing the way we think about FSGS and draws attention not only to identification of novel causal genes, but also to potential roles for combinations of mutations in multiple genes, mutations with complex inheritance, and susceptibility genes with variable penetrance carrying relatively minor but significant effects. This review provides an update on recent advances in the genetic analysis of FSGS and highlights the potential as well as the new challenges of NGS for diagnosis and mechanism-based treatment of FSGS.

Rituximab (RTX) is a murine–human chimeric monoclonal antibody against CD20 that has been proven effective for preventing relapse in frequently-relapsing or steroid-dependent nephrotic syndrome (NS). Serum sickness, a type-3 hypersensitivity reaction resulting from injection of foreign proteins, has been reported in patients treated with RTX. Herein, we describe a case of RTX-induced serum sickness (RISS) in a 6-year-old boy with steroid-dependent NS. He presented to the hospital with fever and polyarthralgia at 10 days after his fourth dose of RTX. Although he was started on empiric intravenous antibiotics, there was no evidence of septic arthritis and his symptoms resolved over the course of 4 days. He was diagnosed with RISS based on the chronology of RTX administration and the acute-onset self-limiting course of the polyarthritis. His serum human anti-chimeric antibody (HACA) level on day 53 exceeded the limit of quantification (5000 ng/mL). The pathogenesis of RISS and the role of HACAs remain unclear. It is important for clinicians to recognize RISS, because further infusions of RTX may cause more severe reactions in patients with a history of RISS.

Cubilin (CUBN) and amnionless (AMN), expressed in kidney and intestine, form a multiligand receptor complex called CUBAM that plays a crucial role in albumin absorption. To date, the mechanism of albumin endocytosis mediated by CUBAM remains to be elucidated. Here, we established an assay to quantitatively evaluate the albumin uptake by CUBAM using cells expressing full-length CUBN, which elucidated the crucial role of C-terminal part of CUBN and endocytosis signal motifs of AMN in albumin endocytosis. We also demonstrated that nuclear valosin-containing protein-like 2 (NVL2), an interacting protein of AMN, is involved in this process. While NVL2 was mainly localized in the nucleolus in cells without AMN expression, it was translocated to the extranuclear compartment when co-expressed with AMN. NVL2 knockdown significantly impaired the internalization of the CUBN-albumin complex in cultured cells, demonstrating an involvement of NVL2 in endocytic regulation. These findings uncover a link between membrane and nucleolar proteins which is involved in endocytic processes.

Nail-patella syndrome (NPS) is a multi-system disorder characterized by hypoplastic nails, hypoplastic patella, skeletal deformities, and iliac horns, which is caused by heterozygous variants of LMX1B. Nephropathy ranging from mild urinary abnormality to end-stage renal disease occurs in some individuals with NPS. Because of the low prevalence of NPS and the lack of longitudinal studies of its kidney involvement, the clinical, pathological, and genetic features characterizing severe nephropathy remain unclear. We conducted a Japanese survey of NPS with nephropathy, and analyzed their clinical course, pathological features, and factors associated with severe renal phenotype. LMX1B gene analysis and luciferase reporter assay were also performed. Among 13 NPS nephropathy cases with genetic validation, 5 patients who had moderate-to-massive proteinuria progressed to advanced chronic kidney disease or end-stage renal disease. Pathological findings in the early phase did not necessarily correlate with renal prognosis. Variants associated with deteriorated renal function including a novel variants were confined to the N-terminal region of the LIM domain and a short sequence in the LMX1B homeodomain, which were distinct from reported variants found in isolated nephropathy without extrarenal manifestation (LMX1B-associated nephropathy). Luciferase reporter analysis demonstrated that variants in patients with severe renal phenotype caused haploinsufficiency, but no dominant-negative effects on promoter activation. A distinct proportion of NPS nephropathy patients progressed to end-stage renal disease in adolescence or young adulthood. Patients with moderate or severe proteinuria, especially those with variants in specific regions of LMX1B, should be monitored for potential deterioration of renal function.

BACKGROUND: Researchers have identified about 40 genes with mutations that result in the most common cause of CKD in children, congenital anomalies of the kidney and urinary tract (CAKUT), but approximately 85% of patients with CAKUT lack mutations in these genes. The anomalies that comprise CAKUT are clinically heterogenous, and thought to be caused by disturbances at different points in kidney development. However, identification of novel CAKUT-causing genes remains difficult because of their variable expressivity, incomplete penetrance, and heterogeneity. METHODS: We investigated two generations of a family that included two siblings with CAKUT. Although the parents and another child were healthy, the two affected siblings presented the same manifestations, unilateral renal agenesis and contralateral renal hypoplasia. To search for a novel causative gene of CAKUT, we performed whole-exome and whole-genome sequencing of DNA from the family members. We also generated two lines of genetically modified mice with a gene deletion present only in the affected siblings, and performed immunohistochemical and phenotypic analyses of these mice. RESULTS: We found that the affected siblings, but not healthy family members, had a homozygous deletion in the Cobalamin Synthetase W Domain–Containing Protein 1 (CBWD1) gene. Whole-genome sequencing uncovered genomic breakpoints, which involved exon 1 of CBWD1, harboring the initiating codon. Immunohistochemical analysis revealed high expression of Cbwd1 in the nuclei of the ureteric bud cells in the developing kidneys. Cbwd1-deficient mice showed CAKUT phenotypes, including hydronephrosis, hydroureters, and duplicated ureters. CONCLUSIONS: The identification of a deletion in CBWD1 gene in two siblings with CAKUT implies a role for CBWD1 in the etiology of some cases of CAKUT.

Nail-patella syndrome (NPS) is an autosomal-dominant disease caused by LMX1B mutations and is characterized by dysplastic nails, absent or hypoplastic patellae, elbow dysplasia, and iliac horns. Renal involvement is the major determinant of the prognosis for NPS. Patients often present with varying degrees of proteinuria or hematuria, and can occasionally progress to chronic renal failure. Recent genetic analysis has found that some mutations in the homeodomain of LMX1B cause isolated nephropathy without nail, patellar or skeletal abnormality (LMX1B-associated nephropathy). The classic term "nail-patella syndrome" would not represent disease conditions in these cases. This review provides an overview of NPS, and highlights the molecular genetics of NPS nephropathy and LMX1B-associated nephropathy. Our current understanding of LMX1B function in the pathogenesis of NPS and LMX1B-associated nephropathy is also presented, and its downstream regulatory networks discussed. This recent progress provides insights that help to define potential targeted therapeutic strategies for LMX1B-associated diseases.

Background Evidence to guide rescue therapy in refractory Kawasaki disease (KD) is lacking. The aim of this study was to determine the most important variables in predicting non-response to rescue therapy in refractory KD. Methods We retrospectively analyzed 171 patients diagnosed with refractory KD resistant to initial i.v. immunoglobulin (IVIG). Participants received rescue therapy consisting of IVIG monotherapy or IVIG plus prednisolone. Characteristics and laboratory variables were compared between rescue therapy non-responders and responders. Multivariate logistic regression analysis was performed to determine the independent predictors of non-response to rescue therapy. Results Among the 171 participants, 54 (31.6%) were non-responders to rescue therapy. On univariate analysis, fever pattern after initial IVIG, day of illness at rescue therapy, rescue therapy regimen and six laboratory variables (pre-IVIG sodium, C-reactive protein [CRP]; post-IVIG white blood cell count, platelet count, sodium, CRP) were useful in discriminating between non-responders and responders. These nine variables were included in multivariate logistic regression analysis. Persistent fever after initial IVIG (aOR, 2.39; 95%CI: 1.07–5.37) and post-IVIG CRP (aOR, 1.09; 95%CI: 1.02–1.17, per 1 mg/dL increase) were identified as independent predictors of non-response to rescue therapy. IVIG rescue monotherapy (aOR, 3.05; 95%CI: 1.05–8.84) also predicted non-response after adjusting for fever pattern and post-IVIG CRP. Conclusions Persistent fever and elevated CRP after initial IVIG are predictive of non-response to rescue therapy for refractory KD. For patients at high risk of non-response, IVIG plus prednisolone, or even further intensified rescue therapy regimens may be preferable.

We report the case of a 9-year-old girl who presented with mixed-type fulminant autoimmune hemolytic anemia (AIHA) at the onset of systemic lupus erythematosus (SLE). On admission, laboratory investigations indicated very severe anemia (Hb, 2.7 g/dL) with reticulocytosis and positive direct/indirect Coombs tests. In addition, agglutinative reaction was clinically observed. Based on further examinations, the patient was diagnosed with AIHA complicated with SLE, and mixed-type AIHA was clinically identified. With oral prednisolone and methylprednisolone pulse therapy, the patient entered remission.

Mutations in either cubilin (CUBN) or amnionless (AMN) genes cause Imerslund–Gräsbeck syndrome (IGS), a hereditary disease characterised by anaemia attributed to selective intestinal malabsorption of cobalamin and low-molecular weight proteinuria. Although cubilin protein does not have a transmembrane segment, it functions as a multi-ligand receptor by binding to the transmembrane protein, amnionless. We established a system to quantitatively analyse membrane targeting of the protein complex in cultured renal and intestinal cells and analysed the pathogenic mechanisms of mutations found in IGS patients. A novel CUBN mutation, several previously reported CUBN missense mutations and all previously reported AMN missense mutations resulted in endoplasmic reticulum (ER) retention and completely inhibited amnionless-dependent plasma membrane expression of cubilin. The ER retention of cubilin and amnionless was confirmed in renal proximal tubular cells of a patient with IGS. Notably, the interaction between cubilin and amnionless was not sufficient, but amnionless-mediated glycosylation of cubilin was necessary for their surface expression. Quantitative mass spectrometry and mutagenesis demonstrated that N-linked glycosylation of at least 4 residues of cubilin protein was required for its surface targeting. These results delineated the molecular mechanisms of membrane trafficking of cubilin in renal and intestinal cells.

In kidney glomeruli, mesangial cells provide structural support to counteract for expansile forces caused by pressure gradients and to regulate the blood flow. Glomerular injury results in proliferation and aberrant migration of mesangial cells, which is the pathological characteristic of mesangial proliferative glomerulonephritis. To date, molecular changes that occur in mesangial cells during glomerular injury and their association with the pathogenesis of glomerulonephritis remain largely unclear. During the search for proteins regulating the morphology of mesangial cells, we found that afadin, a multi-domain F-actin-binding protein, and β-catenin are expressed in cell-cell contact sites of cultured mesangial cells and mesangial cells in vivo. Afadin forms a protein complex with β-catenin in glomeruli and in cultured mesangial cells. Protein expression of afadin at mesangial intercellular junctions was dramatically decreased in mesangial proliferative nephritis in rats and in patients with glomerulonephritis. RNA interference-mediated depletion of afadin in cultured mesangial cells did not affect proliferation rate but resulted in delayed directional cell migration. Furthermore, reorientation of the Golgi complex at the leading edges of migrating cells in wound-healing assay was disturbed in afadin-depleted cells, suggesting the role of aberrant migratory polarity in the pathogenesis of proliferative glomerulonephritis. These data shed light on glomerulonephritis-associated changes in cell-cell adhesion between mesangial cells, which might be related to migratory polarity.

The function of kidney podocytes is closely associated with actin cytoskeleton. Rho family small GTPase RhoA promotes stress fiber assembly through Rho-associated protein kinase (ROCK)-dependent myosin II phosphorylation and plays an important role in maintenance of actin stress fibers of podocytes. However, little is known how stress fiber assembly is regulated in podocytes. Here, we found that afadin, an actin filament-binding protein, is required for RhoA/ROCK-dependent formation of actin stress fibers in rat podocyte C7 cells. We show that depletion of afadin in C7 cells induced loss of actin stress fibers. Conversely, forced expression of afadin increased the formation of actin stress fibers. Depletion of afadin inactivated RhoA and reduced the phosphorylation of myosin II. Moreover, the DIL domain of afadin appears to be responsible for actin stress fiber formation. Thus, afadin mediates RhoA/ROCK signaling and contributes to the formation of actin stress fibers in podocyte cells. © 2015 Wiley Periodicals, Inc.

Background Resistance to intravenous immunoglobulin (IVIG) therapy is a risk factor for coronary lesions in patients with Kawasaki disease (KD). Risk-adjusted initial therapy may improve coronary outcome in KD, but identification of high risk patients remains a challenge. This study aimed to develop a new risk assessment tool for IVIG resistance using advanced statistical techniques. Methods Data were retrospectively collected from KD patients receiving IVIG therapy, including demographic characteristics, signs and symptoms of KD and laboratory results. A random forest (RF) classifier, a tree-based machine learning technique, was applied to these data. The correlation between each variable and risk of IVIG resistance was estimated. Results Data were obtained from 767 patients with KD, including 170 (22.1%) who were refractory to initial IVIG therapy. The predictive tool based on the RF algorithm had an area under the receiver operating characteristic curve of 0.916, a sensitivity of 79.7% and a specificity of 87.3%. Its misclassification rate in the general patient population was estimated to be 15.5%. RF also identified markers related to IVIG resistance such as abnormal liver markers and percentage neutrophils, displaying relationships between these markers and predicted risk. Conclusions The RF classifier reliably identified KD patients at high risk for IVIG resistance, presenting clinical markers relevant to treatment failure. Evaluation in other patient populations is required to determine whether this risk assessment tool relying on RF has clinical value.

Background Nail-patella syndrome (NPS) is an autosomal dominant disorder caused by mutations in the LMX1B gene and is characterized by nail dysplasia, skeletal abnormalities, and nephropathy. We herein report a case of steroid-resistant nephrotic syndrome (SRNS) prior to overt orthopedic symptoms in a patient with NPS. Case presentation A 24-year-old woman presented to our hospital with knee pain. She had poorly developed nails, hypoplastic patellas, dislocation of the elbows, and iliac horns in the pelvis. At the age of 7, she developed nephrotic syndrome and was diagnosed with primary focal segmental glomerulosclerosis by renal biopsy. She received long-term corticosteroid therapy with no obvious response. Her clinical course and orthopedic manifestations indicated NPS, and a genetic analysis showed a de novo mutation in the LMX1B gene (c.819 + 1G > A). Nephropathy in this case was considered to be associated with NPS. Therefore, we discontinued corticosteroids without the exacerbation of nephrotic syndrome. Conclusions Patients with NPS may develop nephrotic syndrome prior to overt orthopedic symptoms and only show non-specific findings in renal biopsy at an early stage of NPS nephropathy. Hereditary nephrotic syndrome, often presenting as childhood-onset SRNS, may also be difficult to diagnose in patients with the following conditions: renal symptoms prior to overt extrarenal symptoms, de novo mutations, and non-specific findings in renal biopsy. Therefore, in the management of SRNS in children, we need to reconsider the possibility of hereditary diseases such as NPS even without a family history.

Background Although early treatment of Kawasaki disease (KD) with i.v. immunoglobulin (IVIG) is expected to prevent coronary artery abnormalities, the effectiveness of IVIG by day 4 of illness remains to be determined. Methods This was a multi-institutional, retrospective cohort study. Patients diagnosed with KD at ≤4 days of illness were divided into two groups: those who received initial IVIG before and on day 5 of illness. Baseline characteristics were adjusted using propensity scores. The primary endpoint was the need for additional treatment. Results Of 339 patients diagnosed with KD by day 4, 181 and 158 received IVIG before and on day 5 of illness, respectively. Patients in the early treatment group had more adverse prognostic factors: infancy, early onset of the principal symptoms, and abnormal laboratory data. We thus adjusted baseline characteristics before treatment decisions using propensity scores. Propensity score matching of the two groups yielded 100 observations. More patients required additional treatment in the matched early treatment group: 37% vs 24% (adjusted OR, 1.7; 95%CI: 1.06-2.8; P = 0.047). The difference was more pronounced for risk of relapse after initial resolution of fever: 14% vs 5.0% (adjusted OR, 3.2; 95%CI: 1.3-7.7; P = 0.02). The risk of coronary artery lesion did not differ significantly. Conclusions IVIG treatment by day 4 of illness is associated with the requirement for additional treatment even after adjustment of baseline characteristics. Increased resistance to IVIG when given by day 4 should be considered in order to improve the treatment regimen for early-diagnosed KD.

Mesangial cell migration, regulated by several growth factors, is crucial after glomerulopathy and during glomerular development. Directional migration requires the establishment of a polarized cytoskeletal arrangement, a process regulated by coordinated actin dynamics and focal adhesion turnover at the peripheral ruffles in migrating cells. Here we found high expression of the actin cross-linking protein EPLIN (epithelial protein lost in neoplasm) in mesangial cells. EPLIN was localized in mesangial angles, which consist of actin-containing microfilaments extending underneath the capillary endothelium, where they attach to the glomerular basement membrane. In cultured mesangial cells, EPLIN was localized in peripheral actin bundles at focal adhesions and formed a protein complex with paxillin. The MEK–ERK (extracellular signal-regulated kinase) cascade regulated EPLIN–paxillin interaction and induced translocalization of EPLIN from focal adhesion sites to peripheral ruffles. Knockdown of EPLIN in mesangial cells enhanced platelet-derived growth factor–induced focal adhesion disassembly and cell migration. Furthermore, EPLIN expression was decreased in mesangial proliferative nephritis in rodents and humans in vivo . These results shed light on the coordinated actin remodeling in mesangial cells during restorative remodeling. Thus, changes in expression and localization of cytoskeletal regulators underlie phenotypic changes in mesangial cells in glomerulonephritis.

We report the first case of a girl who presented with Papillon-Lefèvre syndrome (PLS) and subsequently developed systemic lupus erythematosus and liver cirrhosis. This indicates that autoimmune diseases can be a complication in patients with PLS. Cathepsin C gene mutations were not found in our patient or her mother. Thus, other genetic factors may have been involved in this patient.

Circulating factor(s) has been thought to be the underlying cause of focal segmental glomerulosclerosis (FSGS), and recent studies foster this idea by demonstrating increased soluble urokinase receptor (suPAR) levels in the serum of FSGS patients.To explore the possible contribution of suPAR in FSGS pathogenesis, we analyzed serum suPAR levels in 17 patients with FSGS and compared them with those in patients with steroid-sensitive nephrotic syndrome, chronic glomerulonephritis, or non-glomerular kidney diseases.Serum suPAR levels in patients with FSGS were higher than those in patients with steroid-sensitive nephrotic syndrome or chronic glomerulonephritis, but not higher than those in patients with non-glomerular kidney diseases. suPAR levels negatively correlate with estimated glomerular filtration rate and were decreased after renal transplantation in patients with FSGS as well as in those with non-glomerular kidney diseases. Furthermore, 6 FSGS patients with post-transplant recurrence demonstrated that suPAR levels were not high during the recurrence.Based on our results, elevated suPAR levels in FSGS patients were attributed mainly to decreased glomerular filtration. These data warrant further analysis for involvement of possible circulating factor(s) in FSGS pathogenesis.

Background Nail-patella syndrome (NPS) is a rare autosomal-dominant disorder caused by LMX1B mutation. In patients with the renal lesions typical of NPS without skeletal or nail findings, it is described as nail-patella-like renal disease (NPLRD). However, the pathogenesis of NPLRD is largely unknown. Methods A 6-year-old girl with microscopic haematuria and mild proteinuria was diagnosed with NPLRD because of an aberrantly thickened glomerular basement membrane (GBM) and deposition of Type III collagen in the GBM observed by electron microscopy. Immunohistological analyses of podocyte protein expression were performed on biopsy tissues. Sequence analysis of LMX1B was performed, and the functional consequences of the detected mutation were analysed by luciferase reporter assay. Results When analysing molecules that are important for podocyte development, maintenance and maturation, CD2AP expression was found to be altered in the podocytes. A novel LMX1B missense mutation (R246Q) was identified. Functional analyses revealed partial but significant impairment of R246Q transcriptional activity. However, no dominant-negative effect of R246Q was detected, which suggests that NPLRD is caused by LMX1B haploinsufficiency. Conclusions This is the first report on LMX1B mutation identified in a patient with NPLRD. Residual transcriptional activity would account for normality of the nails and patella in this case. Genetic and pathological analyses of additional cases would clarify the role of LMX1B in glomerulopathy without systemic symptoms, which, together with nephropathy in NPS, can be designated as 'LMX1B nephropathy'.

BACKGROUND Previous studies have identified significant associations between the development of idiopathic focal segmental glomerulosclerosis (FSGS) and MYH9 encoding nonmuscle myosin heavy chain-IIA (NMMHC-IIA). However, these studies focused only on the linkage of MYH9 polymorphisms and development of FSGS. There have been no reports on pathological changes of NMMHC-IIA in human glomerular diseases. Here we report on the precise localization of NMMHC-IIA in podocytes and changes in NMMHC-IIA expression in pathological states in rats and humans. METHODS Immunocytochemical (immunofluorescence and immunoelectron microscopy) studies were performed to determine the precise localization of NMMHC-IIA. Expression levels of NMMHC-IIA were investigated in puromycin aminonucleoside (PAN)-treated rats; and expression levels of NMMHC-IIA and other podocyte-related proteins were investigated in glomeruli of patients with idiopathic FSGS and other heavy proteinuric glomerular diseases. RESULTS NMMHC-IIA was located primarily at the cell body and primary processes of podocytes; this localization is distinct from other podocyte-related molecules causing hereditary FSGS. In PAN-treated rat kidneys, expression levels of NMMHC-IIA in podocytes decreased. Immunohistochemical analysis revealed that expression levels of NMMHC-IIA markedly decreased in idiopathic nephrotic syndrome, especially FSGS, whereas it did not change in other chronic glomerulonephritis showing apparent proteinuria. Changes in NMMHC-IIA expression were observed in glomeruli where expression of nephrin and synaptopodin was maintained. CONCLUSIONS Considering previous genome-wide association studies and development of FSGS in patients with MYH9 mutations, the characteristic localization of NMMHC-IIA and the specific decrease in NMMHC-IIA expression in idiopathic nephrotic syndrome, especially FSGS, suggest the important role of NMMHC-IIA in the development of FSGS.

Knowledge about age-related differences in the course of the acute phase symptoms is helpful to make an accurate and timely diagnosis of Kawasaki disease (KD).We performed a retrospective study involving 100 consecutive patients with KD. Time to the first detection of the principal symptoms was examined. The first day of fever was defined as day 1.Median age was 24 months. In patients24 months, cervical lymphadenopathy was the earliest symptom other than fever and appeared earlier than in younger patients (2.6 ± 2.2 versus 3.8 ± 1.9 days of illness; P0.0001). Of the total, 67% of the older patients initially presented with cervical lymphadenopathy alone, which remained the only symptom for 2.8 days on an average. In younger patients, polymorphous rash was the most common initial symptom and appeared earlier than in older patients (2.8 ± 1.6 versus 4.2 ± 1.8 days of illness; P0.0001). Time to diagnosis since the initial symptoms was shorter in younger patients (2.1 ± 1.5 versus 3.2 ± 1.6 days; P = 0.006).A high index of suspicion for KD is required in febrile patients ≤24 months presenting with rash and in those24 months with cervical lymphadenopathy. Younger patients need close observation because their acute phase symptoms progress rapidly. On the contrary, in older patients, cervical lymphadenopathy often remains the only manifestation for more than a few days and complicates the diagnosis. Recognizing age-specific patterns is useful for accurate and timely diagnosis of KD.