Akemi Shono

Monitoring and robust induction of nephrogenic intermediate mesoderm from human pluripotent stem cells

A method for stimulating the differentiation of human pluripotent stem cells into kidney lineages remains to be developed. Most cells in kidney are derived from an embryonic germ layer known as intermediate mesoderm. Here we show the establishment of an efficient system of homologous recombination in human pluripotent stem cells by means of bacterial artificial chromosome-based vectors and single-nucleotide polymorphism array-based detection. This system allowed us to generate human-induced pluripotent stem cell lines containing green fluorescence protein knocked into OSR1, a specific intermediate mesoderm marker. We have also established a robust induction protocol for intermediate mesoderm, which produces up to 90% OSR1(+) cells. These human intermediate mesoderm cells can differentiate into multiple cell types of intermediate mesoderm-derived organs in vitro and in vivo, thereby supplying a useful system to elucidate the mechanisms of intermediate mesoderm development and potentially providing a cell source for regenerative therapies of the kidney.

Phosphorylation of Nephrin Triggers Its Internalization by Raft-Mediated Endocytosis

Proper localization of nephrin determines integrity of the glomerular slit diaphragm. Slit diaphragm proteins assemble into functional signaling complexes on a raft-based platform, but how the trafficking of these proteins coordinates with their signaling function is unknown. Here, we demonstrate that a raft-mediated endocytic (RME) pathway internalizes nephrin. Nephrin internalization was slower with raft-mediated endocytosis than with classic clathrin-mediated endocytosis. Ultrastructurally, the RME pathway consisted of noncoated invaginations and was dependent on cholesterol and dynamin. Nephrin constituted a stable, signaling-competent microdomain through interaction with Fyn, a Src kinase, and podocin, a scaffold protein. Tyrosine phosphorylation of nephrin triggered its own RME-mediated internalization. Protamine-induced hyperphosphorylation of nephrin led to noncoated invaginations predominating over coated pits. These results demonstrate that an RME pathway couples nephrin internalization to its own signaling, suggesting that RME promotes proper spatiotemporal assembly of slit diaphragms during podocyte development or injury.

Podocin Participates in the Assembly of Tight Junctions between Foot Processes in Nephrotic Podocytes

The predominant type of cellular junction between normal podocyte foot processes is the slit diaphragm. Under nephrotic conditions,however, foot process effacement leads to the loss of slit diaphragms and the new formationof tight junctions composed of the proteins coxsackievirus and adenovirus receptor (CAR) and zonula occludens 1 (ZO-1). Podocin, a protein that plays a key role in maintaining the integrity of the slit diaphragm, has also been localized to these tight junctions, but its function at this site is unknown. In this study, we confirmed that podocin colocalizes with CAR and ZO-1 at the tight junction between foot processes in nephrotic rats. Using primary cultures of rat podocytes, as well as cell lines that co-expressed podocin and CAR, we observed that podocin was recruited to sites of cell-cell contact and that it co-localized with CAR and ZO-1. Immunoprecipitation suggested that these three junctional proteins from a multi-protein complex. Consistent with this, we found that podociin facilitated the coalescence of preassembled lipid rafts containing CAR and restricted their lateral mobility, the latter likely a result of dynamic actin reorganization and subsequent tethering of CAR-podocin complexes to the cytoskeleton. In conclusion, in addition to serving as a structural protein of the slit diaphragm of normal podocytes, our data suggest that podocin may also serve as a scaffold that links tight junction proteins to the actin cytoskeleton in nephrotic foot processes.

Steroid-resistant nephrotic syndrome

Akiko Kitamura, Hiroyasu Tsukaguchi, Kenichi Maruyama, Akemi Shono, Kazumoto Iijima, Shoji Kagami and Toshio Doi Department of Pediatrics, University of Tokushima Graduate School, Tokushima, Japan; Department of Clinical Biology and Medicine, University of Tokushima Graduate School, Tokushima, Japan; Department of Pediatrics, Gunma Children’s Medical Center, Gunma, Japan and Department of Nephrology, National Center for Child Health and Development, Tokyo, Japan

Differential diagnosis of Bartter syndrome, Gitelman syndrome, and pseudo–Bartter/Gitelman syndrome based on clinical characteristics

Purpose:Phenotypic overlap exists among type III Bartter syndrome (BS), Gitelman syndrome (GS), and pseudo-BS/GS (p-BS/GS), which are clinically difficult to distinguish. We aimed to clarify the differences between these diseases, allowing accurate diagnosis based on their clinical features.Methods:A total of 163 patients with genetically defined type III BS (n = 30), GS (n = 90), and p-BS/GS (n = 43) were included. Age at diagnosis, sex, body mass index, estimated glomerular filtration rate, and serum and urine electrolyte concentrations were determined.Results:Patients with p-BS/GS were significantly older at diagnosis than those with type III BS and GS. Patients with p-BS/GS included a significantly higher percentage of women and had a lower body mass index and estimated glomerular filtration rate than did patients with GS. Although hypomagnesemia and hypocalciuria were predominant biochemical findings in patients with GS, 17 and 23% of patients with type III BS and p-BS/GS, respectively, also showed these abnormalities. Of patients with type III BS, GS, and p-BS/GS, 40, 12, and 63%, respectively, presented with chronic kidney disease.Conclusions:This study clarified the clinical differences between BS, GS, and p-BS/GS for the first time, which will help clinicians establish differential diagnoses for these three conditions.Genet Med 18 2, 180–188.

Genetic, Clinical, and Pathologic Backgrounds of Patients with Autosomal Dominant Alport Syndrome

BACKGROUND AND OBJECTIVES Alport syndrome comprises a group of inherited heterogeneous disorders involving CKD, hearing loss, and ocular abnormalities. Autosomal dominant Alport syndrome caused by heterozygous mutations in collagen 4A3 and/or collagen 4A4 accounts for <5% of patients. However, the clinical, genetic, and pathologic backgrounds of patients with autosomal dominant Alport syndrome remain unclear. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS We conducted a retrospective analysis of 25 patients with genetically proven autosomal dominant Alport syndrome and their family members (a total of 72 patients) from 16 unrelated families. Patients with suspected Alport syndrome after pathologic examination who were referred from anywhere in Japan for genetic analysis from 2006 to 2015 were included in this study. Clinical, laboratory, and pathologic data were collected from medical records at the point of registration for genetic diagnosis. Genetic analysis was performed by targeted resequencing of 27 podocyte-related genes, including Alport-related collagen genes, to make a diagnosis of autosomal dominant Alport syndrome and identify modifier genes or double mutations. Clinical data were obtained from medical records. RESULTS The median renal survival time was 70 years, and the median age at first detection of proteinuria was 17 years old. There was one patient with hearing loss and one patient with ocular lesion. Among 16 patients who underwent kidney biopsy, three showed FSGS, and seven showed thinning without lamellation of the glomerular basement membrane. Five of 13 detected mutations were reported to be causative mutations for autosomal recessive Alport syndrome in previous studies. Two families possessed double mutations in both collagen 4A3 and collagen 4A4, but no modifier genes were detected among the other podocyte-related genes. CONCLUSIONS The renal phenotype of autosomal dominant Alport syndrome was much milder than that of autosomal recessive Alport syndrome or X-linked Alport syndrome in men. It may, thus, be difficult to make an accurate diagnosis of autosomal dominant Alport syndrome on the basis of clinical or pathologic findings. No modifier genes were identified among the known podocyte-related genes.

Predisposition to relapsing nephrotic syndrome by a nephrin mutation that interferes with assembly of functioning microdomains

Minimal-change disease (MCD) is the most common cause of nephrotic syndrome (NS) and is characterized only by minor morphological alterations in podocytes. A subtype of MCD arises from mutations in nephrin, a major component of the slit diaphragm (SD). Idiopathic MCD is a complex trait where interactions of genetic and immunological factors are implicated. However, the pathogenic mechanisms remain unclear. Here we studied the molecular basis for familial NS characterized by frequent relapses and minimal-change histology. Our previous mutational analysis revealed that the two affected children were compound heterozygotes for nephrin variants C265R and V822M (Kidney Int., 2008). When heterologously expressed, these variants exhibited normal metabolic half-life and raft binding. C265R exhibited substantial ER retention, reflecting an intracellular trafficking defect. In contrast, V822M was able to reach the plasma membrane, but was restricted in lateral diffusion as well as trafficking at the cell surface. Clustering of V822M failed to evoke a maximum tyrosine-phosphorylation and actin reorganization, suggesting the inability to assemble into functioning membrane microdomains. Our results suggest that C265R and V822M compose a dysfunctional SD complex due to their mixed defects comprising reduced cell surface targeting and ineffective assembly of signaling microdomains. The defective SD likely confers a susceptibility to immunogenic stimuli and predisposes to a relapsing phenotype.

Identification of mutations in FN1 leading to glomerulopathy with fibronectin deposits

BackgroundGlomerulopathy with fibronectin deposits (GFND) is a rare autosomal dominant disease characterized by massive fibronectin deposits, leading to end-stage renal failure. Although mutations within the heparin-binding domains of the fibronectin 1 gene (FN1) have been associated with GFND, no mutations have been reported within the integrin-binding domains.MethodsIn this study, FN1 mutational analysis was conducted in 12 families with GFND. Biochemical and functional features of mutated proteins were examined using recombinant fibronectin fragments encompassing both the integrin- and heparin-binding domains.ResultsWe report six FN1 mutations from 12 families with GFND, including five that are novel (p.Pro969Leu, p.Pro1472del, p.Trp1925Cys, p.Lys1953_Ile1961del, and p.Leu1974Pro). p.Pro1472del is localized in the integrin-binding domain of fibronectin, while the others are in heparin-binding domains. We detected p.Tyr973Cys, p.Pro1472del, and p.Leu1974Pro mutations in multiple families, and haplotype analysis implied that p.Pro1472del and p.Leu1974Pro are founder mutations. The protein encoded by the novel integrin-binding domain mutation p.Pro1472del showed decreased cell binding ability via the integrin-binding site. Most affected patients developed urine abnormalities during the first or second decade of life, and some mutation carriers were completely asymptomatic.ConclusionsThis is the second large-scale analysis of GFND families and the first report of an integrin-binding domain mutation. These findings may help determine the pathogenesis of GFND.

Natural History and Genotype-Phenotype Correlation in Female X-Linked Alport Syndrome

Introduction X-linked Alport syndrome (XLAS) is a hereditary disease characterized by progressive nephritis, hearing loss, and ocular abnormalities. Affected male patients usually progress to end-stage renal disease in early or middle adulthood, and disease severity is strongly correlated with genotype. However, the clinical course in female patients has rarely been reported. Methods We conducted a retrospective analysis of females with genetically proven XLAS (n = 275) and their affected female family members (n = 61) from 179 Japanese families. Patients suspected to have Alport syndrome from pathologic findings or a family history who were referred from anywhere in Japan for genetic diagnosis between 2006–2015 were included in this study. Clinical and laboratory data were collected from medical records at the time of registration for genetic analysis. Results Proteinuria was detected in 175 genetically proven patients (72.6%), and the median age for developing proteinuria was 7.0 years. Fifty-two of 336 patients developed end-stage renal disease with a median renal survival age of 65.0 years. No obvious genotype–phenotype correlation was observed. Additionally, targeted sequencing for podocyte-related genes in patients with severe phenotypes revealed no obvious variants considered to be modifier genes except for 1 patient with a COL4A3 gene variant. Discussion This study revealed that phenotypes in female XLAS patients may be severe, but genotype does not help to predict the disease severity. Clinicians must therefore pay careful attention to the clinical course and appropriate treatment in females with XLAS.

X-linked Alport syndrome associated with a synonymous p.Gly292Gly mutation alters the splicing donor site of the type IV collagen alpha chain 5 gene

BackgroundX-linked Alport syndrome (XLAS) is a progressive hereditary nephropathy caused by mutations in the type IV collagen alpha chain 5 gene (COL4A5). Although many COL4A5 mutations have previously been identified, pathogenic synonymous mutations have not yet been described.MethodsA family with XLAS underwent mutational analyses of COL4A5 by PCR and direct sequencing, as well as transcript analysis of potential splice site mutations. In silico analysis was also conducted to predict the disruption of splicing factor binding sites. Immunohistochemistry (IHC) of kidney biopsies was used to detect α2 and α5 chain expression.ResultsWe identified a hemizygous point mutation, c.876A>T, in exon 15 of COL4A5 in the proband and his brother, which is predicted to result in a synonymous amino acid change, p.(Gly292Gly). Transcript analysis showed that this mutation potentially altered splicing because it disrupted the splicing factor binding site. The kidney biopsy of the proband showed lamellation of the glomerular basement membrane (GBM), while IHC revealed negative α5(IV) staining in the GBM and Bowman’s capsule, which is typical of XLAS.ConclusionsThis is the first report of a synonymous COL4A5 substitution being responsible for XLAS. Our findings suggest that transcript analysis should be conducted for the future correct assessment of silent mutations.