OBJECTIVE

We performed association studies between 118 single-nucleotide polymorphisms (SNPs) of 22 candidate genes (or gene family) and hypertension in a Japanese population.

METHODS

The study population consisted of 1880 subjects representing the general population in Japan, recruited from the Suita study. The candidate genes were selected based on their functions, including insulin resistance (APM1, CD36, HSD11B1), oxidative stress (CYBA, GPX1, GSTMs), steroid hormone (ESR1, ESR2, HSD11B2), renal functions (PTGS2, KLK1, NPHS1, NPHS2, SGK, SLC12A1, PTGES), and others related to cardiovascular physiology (GJA4, NOS1, NTRK3, P2RX4, SPP1, ALDH2).

RESULTS

Multiple logistic analyses, with age and body mass index as covariates, indicated that 13 SNPs (eight genes), six SNPs (four genes) and 11 SNPs (four genes) were associated with hypertension (P < 0.05) in the total, male, and female populations, respectively. PTGS2 seems to be a promising candidate gene for hypertension in men. GSTM3 and SLC12A1 seem to be promising candidate genes for hypertension in women. Especially, a polymorphism in SLC12A1 was significantly associated with hypertension in women even after correction by the Bonferroni method (corrected P = 0.0236). Multiple logistic analyses, with age and body mass index as covariates, indicated that the prevalence of hypertension in females was significantly higher in subjects with the CC genotype than in those with the TT + TC genotypes (P < 0.0001, odds ratio = 1.967, 95% confidence interval = 1.430-2.712).

CONCLUSIONS

Although the present results should be replicated in other study populations for confirmation, the present results suggest that SLC12A1 may contribute to hypertension in Japanese women.

BACKGROUND

Minimal change nephrotic syndrome (MCNS) is a major problem in pediatric nephrology. While the pathogenesis of MCNS is not known, the latest discoveries in the genetic diseases indicate that glomerular epithelial cells (podocytes) and the slit diaphragm play a primary role in development of proteinuria. Because nephrin is known to be a major component of the slit diaphragm, we analyzed the structure of nephrin gene (NPHS1) in patients with MCNS of different severity.

METHODS

Clinical data and DNA samples were collected from 25 adults who had biopsy-proven MCNS in childhood. A direct sequencing was performed to all 29 exons of the NPHS1 gene. The significance of the findings was evaluated by similar analysis of DNA samples from 25 healthy control patients.

RESULTS

The analysis of NPHS1 revealed no specific MCNS-associated mutation. However, 5 of the 25 MCNS patients had heterozygous allelic variants leading to nonconservative amino acid substitutions not previously reported (G879R; R800C; T294I; A916S). One of the five patients also had the Fin-major mutation, and two had new, conservative amino acid substitutions (S786N; A342G). Three of the five patients were classified as steroid sensitive, one was an early nonresponder, and one patient showed clear resistance to steroid treatment. Six known polymorphic changes in NPHS1 were also found, three of them leading to amino acid changes. The number of allelic variants was high both in MCNS patients and control patients (mean 3.0 and 2.6).

CONCLUSIONS

The results suggest that genetic changes in nephrin may have a pathogenetic role in some patients with MCNS.

APOL1 risk variants are associated with kidney disease in blacks, but the mechanisms of renal injury associated with APOL1 risk variants are unknown. Because APOL1 is unique to humans and some primates, we created transgenic (Tg) mice using the promoter of nephrin-encoding Nphs1 to express the APOL1 reference sequence (G0) or the G2 risk variant in podocytes, establishing Tg lines with a spectrum of APOL1 expression levels. Podocytes from Tg-G0 and Tg-G2 mice did not undergo necrosis, apoptosis, or autophagic cell death in vivo, even in lines with highly expressed transgenes. Further, Tg-G0 and Tg-G2 mice did not develop kidney pathology, proteinuria, or azotemia as of 300 days of age. However, by 200 days of age, Tg-G2 mice had significantly lower podocyte density than age-matched WT and Tg-G0 mice had, a difference that was not evident at weaning. Notably, a pregnancy-associated phenotype that encompassed eclampsia, preeclampsia, fetal/neonatal deaths, and small litter sizes occurred in some Tg-G0 mice and more severely in Tg-G2 mice. Similar to human placenta, placentas of Tg mice expressed APOL1. Overall, these results suggest podocyte depletion could predispose individuals with APOL1 risk genotypes to kidney disease in response to a second stressor, and add to other published evidence associating APOL1 expression with preeclampsia.

The central role of the multifunctional protein nephrin within the macromolecular complex forming the glomerular slit diaphragm is well established, but the mechanisms linking the slit diaphragm to the cytoskeleton and to the signaling pathways involved in maintaining the integrity of the glomerular filter remain incompletely understood. Here, we report that nephrin interacts with the bicarbonate/chloride transporter kidney anion exchanger 1 (kAE1), detected by yeast two-hybrid assay and confirmed by immunoprecipitation and co-localization studies. We confirmed low-level glomerular expression of kAE1 in human and mouse kidneys by immunoblotting and immunofluorescence microscopy. We observed less kAE1 in human glomeruli homozygous for the NPHS1(FinMaj) nephrin mutation, whereas kAE1 expression remained unchanged in the collecting duct. We could not detect endogenous kAE1 expression in NPHS1(FinMaj) podocytes in primary culture, but heterologous re-introduction of wild-type nephrin into these podocytes rescued kAE1 expression. In kidneys of Ae1(-/-) mice, nephrin abundance was normal but its distribution was altered along with the reported kAE1-binding protein integrin-linked kinase (ILK). Ae1(-/-) mice had increased albuminuria with glomerular enlargement, mesangial expansion, mesangiosclerosis, and expansion of the glomerular basement membrane. Glomeruli with ILK-deficient podocytes also demonstrated altered AE1 and nephrin expression, further supporting the functional interdependence of these proteins. These data suggest that the podocyte protein kAE1 interacts with nephrin and ILK to maintain the structure and function of the glomerular basement membrane.

Indoxyl sulfate is a uremic toxin and a ligand of the aryl-hydrocarbon receptor (AhR), a transcriptional regulator. Elevated serum indoxyl sulfate levels may contribute to progressive kidney disease and associated vascular disease. We asked whether indoxyl sulfate injures podocytes in vivo and in vitro. Mice exposed to indoxyl sulfate for 8 w exhibited prominent tubulointerstitial lesions with vascular damage. Indoxyl sulfate-exposed mice with microalbuminuria showed ischemic changes, while more severely affected mice showed increased mesangial matrix, segmental solidification, and mesangiolysis. In normal mouse kidneys, AhR was predominantly localized to the podocyte nuclei. In mice exposed to indoxyl sulfate for 2 h, isolated glomeruli manifested increased Cyp1a1 expression, indicating AhR activation. After 8 w of indoxyl sulfate, podocytes showed foot process effacement, cytoplasmic vacuoles, and a focal granular and wrinkled pattern of podocin and synaptopodin expression. Furthermore, vimentin and AhR expression in the glomerulus was increased in the indoxyl sulfate-exposed glomeruli compared to controls. Glomerular expression of characteristic podocyte mRNAs was decreased, including Actn4, Cd2ap, Myh9, Nphs1, Nphs2, Podxl, Synpo, and Wt1. In vitro, immortalized-mouse podocytes exhibited AhR nuclear translocation beginning 30 min after 1 mM indoxyl sulfate exposure, and there was increased phospho-Rac1/Cdc42 at 2 h. After exposure to indoxyl sulfate for 24 h, mouse podocytes exhibited a pro-inflammatory phenotype, perturbed actin cytoskeleton, decreased expression of podocyte-specific genes, and decreased cell viability. In immortalized human podocytes, indoxyl sulfate treatment caused cell injury, decreased mRNA expression of podocyte-specific proteins, as well as integrins, collagens, cytoskeletal proteins, and bone morphogenetic proteins, and increased cytokine and chemokine expression. We propose that basal levels of AhR activity regulate podocyte function under normal conditions, and that increased activation of podocyte AhR by indoxyl sulfate contributes to progressive glomerular injury.

OBJECTIVE

Several genes have been identified to be causative for the disease in a subset of patients with focal segmental glomerulosclerosis (FSGS) and nephrotic syndrome (NS). Mutations in genes with autosomal dominant inheritance mostly affect adolescent or adult patients. In rare cases recessive mutations in NPHS2 are associated with late-onset FSGS. Hereditary FSGS is associated with poor renal survival and low rates of disease recurrence after renal transplantation. Aim of the study was to evaluate the incidence of gene mutations within a cohort of adult patients with primary FSGS and/or NS and progression to end-stage renal disease (ESRD).

METHODS

Genotyping for TRPC6, ACTN4, CD2AP, WT1, INF2, NPHS2 and NPHS1 was performed in all patients with primary FSGS and ESRD registered on the waiting list for kidney transplantation of a large German transplant center (n = 26 out of 478 registered patients). Mean age at onset was 31.7 years; a positive family history for renal disease was documented in 11 (42%) patients, of these one with familiar history of FSGS.

RESULTS

A missense mutation (p.R360H) was identified in TRPC6, 2 missense mutations in compound heterozygous state in NPHS1 (p.P368L; p.G412C), a sequence variation of unknown significance (p.R310Q) in ACTN4 and the non-neutral NPHS2 polymorphism p.R229Q in two additional patients. No mutations were detected in INF2, CD2AP and WT1.

CONCLUSIONS

The observed mutation rate was 8% in this single-center cohort of adult patients with primary FSGS. Mutations in podocyte genes seem to be a rare cause of FSGS and renal failure in adult patients. However, they should be considered as the underlying cause in a subset of patient as the impact on family counseling and patients' life perspectives are significant.

BACKGROUND

Identification of genetic markers of antihypertensive drug responses could assist in individualization of hypertension treatment.

RESULTS

We conducted a genome-wide association study to identify gene loci influencing the responsiveness of 228 male patients to 4 classes of antihypertensive drugs. The Genetics of Drug Responsiveness in Essential Hypertension (GENRES) study is a double-blind, placebo-controlled cross-over study where each subject received amlodipine, bisoprolol,hydrochlorothiazide, and losartan, each as a monotherapy, in a randomized order. Replication analyses were performed in 4 studies with patients of European ancestry (PEAR Study, N=386; GERA I and II Studies, N=196 and N=198; SOPHIA Study, N=372). We identified 3 single-nucleotide polymorphisms within the ACY3 gene that showed associations with bisoprolol response reaching genome-wide significance (P<5x10(-8))however, this could not be replicated in the PEAR Study using atenolol. In addition, 39 single-nucleotide polymorphisms showed P values of 10(-5) to 10(-7). The 20 top-associated single-nucleotide polymorphisms were different for each antihypertensive drug. None of these top single-nucleotide polymorphisms co-localized with the panel of >40 genes identified in genome-wide association studies of hypertension. Replication analyses of GENRES results provided suggestive evidence for a missense variant (rs3814995) in the NPHS1 (nephrin) gene influencing losartan response, and for 2 variants influencing hydrochlorothiazide response, located within or close to the ALDH1A3 (rs3825926) and CLIC5 (rs321329) genes.

CONCLUSIONS

These data provide some evidence for a link between biology of the glomerular protein nephrin and antihypertensive action of angiotensin receptor antagonists and encourage additional studies on aldehyde dehydrogenase–mediated reactions in antihypertensive drug action.

Congenital nephrotic syndrome of the Finnish type (NPHS1) is a rare genetic disease caused by mutations in the NPHS1 gene encoding a major podocyte slit-diaphragm protein, nephrin. Patients with NPHS1 have severe nephrotic syndrome from birth and develop renal fibrosis in early childhood. In this work, we studied the development of glomerular sclerosis in kidneys removed from 4- to 44-month-old NPHS1 patients. The pathological lesions and expression of glomerular cell markers were studied in nephrectomized NPHS1 and control kidneys using light and electron microscopy and immunohistochemistry. An analysis of 1528 glomeruli from 20 patients revealed progressive mesangial sclerosis and capillary obliteration. Although few inflammatory cells were detected in the mesangial area, paraglomerular inflammation and fibrosis was common. The podocytes showed severe ultrastructural changes and hypertrophy with the upregulation of cyclins A and D1. Podocyte proliferation, however, was rare. Apoptosis was hardly detected and the expression of antiapoptotic B-cell lymphoma-2 and proapoptotic p53 were comparable to controls. Moderate amounts of podocytes were secreted into the urine of NPHS1 patients. Shrinkage of the glomerular tuft was common, whereas occlusion of tubular opening or protrusion of the glomerular tuft into subepithelial space or through the Bowman's capsule were not detected. The results indicate that, in NPHS1 kidneys, the damaged podocytes induce progressive mesangial expansion and capillary obliteration. Podocyte depletion, glomerular tuft adhesion, and misdirected filtration, however, seem to play a minor role in the nephron destruction.

BACKGROUND

Congenital nephrotic syndrome of the Finnish type (CNF) is an autosomal recessive disorder mainly caused by mutations in the nephrin gene (NPHS1). The frequency of this gene is highest in Finland but the condition occurs in all populations, with and without Finnish ancestry. The NPHS1 gene is located in the chromosomal region 19q13.1 and consists of 29 exons.

METHODS

Polymerase chain reaction (PCR), restriction and sequence analyses were used to screen 15 CNF Italian patients for mutations in this gene.

RESULTS

No Italian patients had the typical Finnish mutations, a 2bp deletion in exon 2 (Fin-major) and a nonsense mutation in exon 26 (Fin-minor). We found 13 mutations including deletions, insertions, nonsense and missense mutations. Seven of these have never been described before. We also found one nucleotide change in the promoter region and one common polymorphism. NPHS1 missense mutations were confirmed by analysis of a healthy control population.

CONCLUSIONS

Our study provides further evidence that loss of function of the nephrin gene is the main cause of congenital nephrotic syndrome of the Finnish type in Italian patients.

Nephrin is a 180 KD trans-membrane protein expressed in glomerular podocytes. It was first identified in children with congenital nephrotic syndrome of the Finnish type (NPHS1). Nephrin forms an integral part of podocytes, which-together with endothelial cells and the basement-form the glomerular filtration barrier. Podocytopathies result in the detection of nephrin in the urine. We reviewed the literature to determine if urine nephrin measurements could become useful as a biomarker to detect early podocyte injury. Our search identified a total of 19 studies that have been published to date. The most common clinical conditions for which urine nephrin analyses were carried out included diabetic nephropathy, glomerulonephritis and pre-eclampsia. Nephrin measurement was carried out using commercially available ELISA kits, the messenger ribonucleic acid real-time polymerase chain Reaction, or electrophoresis. Nephrinuria showed positive correlation with proteinuria and severity of podocyte injury. In two studies, the level of nephrinuria declined in conjunction with clinical improvement in the patient following immunosuppressive treatment. Currently, there is no published data on the value of measuring urinary nephrin in pediatric patients.

High concentrations of alpha-fetoprotein (AFP) are used for prenatal diagnosis of the Finnish type of congenital nephrotic syndrome (NPHS1). We investigated the validity of this test. We retrospectively established fetal NPHS1 genotype and assessed renal pathology in 21 pregnancies that had been terminated because of raised concentrations of AFP in amniotic fluid. 12 fetuses were homozygous and nine were heterozygous (carriers) for NPHS1 mutations. Raised concentrations of AFP and similar proteinuric features in fetal kidneys were seen in both groups, indicating that these signs are unreliable for prenatal diagnosis of congenital nephrosis. We strongly recommend the use of mutation analysis of the NPHS1 gene to confirm the AFP results in prenatal diagnosis of NPHS1.

The aim of the study was to present our experience in treating children with genetic forms of nephrotic syndrome and diagnosing these diseases. We retrospectively reviewed the clinical data, mutational analyses, histopathological features, treatment modalities, and outcome of 26 consecutive children (20 families) suffering from congenital and/or steroid-resistant nephrotic syndrome who were assessed by genetic analysis. Ten out of 26 children (38%) had congenital nephrotic syndrome, 4/26 (15%) had infantile nephrotic syndrome, 10/26 (38%) had late-onset nephrotic syndrome, and 2/26 (9%) had asymptomatic proteinuria. We detected a mutation in 21/26 (81%) patients and in 15/20 (75%) families. NPHS1 mutation analyses were positive in 4/20 (20%), NPHS2 mutations in 4/20 (20%), WT1 mutations in 4/20 (20%), and PLCE1 mutations in 3/20 (15%) families. NPHS1 and PLCE1 mutations were solely found in patients with the earliest onset. The majority of patients, especially those with early onset of nephrotic syndrome, had serious adverse events related to the nephrotic status, and 19/26 (73%) reached end-stage renal failure at a median age of 27 months. Genetic forms of nephrotic syndrome comprise a heterogeneous group of genetic mutations. The progression toward end-stage renal failure is the rule but is highly variable between patients.

OBJECTIVE

Steroid-resistant nephrotic syndrome overwhelmingly progresses to ESRD. More than 30 monogenic genes have been identified to cause steroid-resistant nephrotic syndrome. We previously detected causative mutations using targeted panel sequencing in 30% of patients with steroid-resistant nephrotic syndrome. Panel sequencing has a number of limitations when compared with whole exome sequencing. We employed whole exome sequencing to detect monogenic causes of steroid-resistant nephrotic syndrome in an international cohort of 300 families.

METHODS

Three hundred thirty-five individuals with steroid-resistant nephrotic syndrome from 300 families were recruited from April of 1998 to June of 2016. Age of onset was restricted to <25 years of age. Exome data were evaluated for 33 known monogenic steroid-resistant nephrotic syndrome genes.

RESULTS

In 74 of 300 families (25%), we identified a causative mutation in one of 20 genes known to cause steroid-resistant nephrotic syndrome. In 11 families (3.7%), we detected a mutation in a gene that causes a phenocopy of steroid-resistant nephrotic syndrome. This is consistent with our previously published identification of mutations using a panel approach. We detected a causative mutation in a known steroid-resistant nephrotic syndrome gene in 38% of consanguineous families and in 13% of nonconsanguineous families, and 48% of children with congenital nephrotic syndrome. A total of 68 different mutations were detected in 20 of 33 steroid-resistant nephrotic syndrome genes. Fifteen of these mutations were novel. NPHS1, PLCE1, NPHS2, and SMARCAL1 were the most common genes in which we detected a mutation. In another 28% of families, we detected mutations in one or more candidate genes for steroid-resistant nephrotic syndrome.

CONCLUSIONS

Whole exome sequencing is a sensitive approach toward diagnosis of monogenic causes of steroid-resistant nephrotic syndrome. A molecular genetic diagnosis of steroid-resistant nephrotic syndrome may have important consequences for the management of treatment and kidney transplantation in steroid-resistant nephrotic syndrome.

Mutations in podocyte genes have been identified in patients with steroid-resistant nephrotic syndrome (SRNS). Point mutations in the ACTN4 gene cause an autosomal dominant form of human focal segmental glomerular sclerosis (FSGS); however, reports of CD2AP mutations remain scarce. Based on the phenotype of Actn4 and Cd2ap null mice, we aimed to define the role of recessive CD2AP and ACTN4 mutations in a cohort of children with SRNS for which NPHS1, NPHS2, and PLCE1 mutations had been previously excluded. CD2AP and ACTN4 mutational analysis was performed in 42 children from 35 unrelated families. The median age of disease onset was 20 (range 0-102) months. Sixteen patients reached end-stage kidney disease at a median age of 84 (range 4-161) months. Renal histology showed FSGS lesions and minimal glomerular changes in 49% and 20% of patients, respectively. Microsatellite marker analysis excluded linkage to the CD2AP locus in 26 families and to the ACTN4 locus in 31 families. No disease-causing mutations were identified in the remaining families. Recessive CD2AP and ACTN4 mutations are rare in children with SRNS. The absence of mutations in this study suggests that there are other genetic causes of SRNS that still need to be identified.

Recent discoveries indicate that the molecules in glomerular podocytes and slit diaphragms may play an important role in the development of proteinuria and nephrotic syndrome. Mutational analyses of NPHS1 and NPHS2 were performed to verify this hypothesis in sporadic nephrotic syndrome (NS) patients. Clinical characteristics and DNA samples were collected from 38 Chinese children with sporadic steroid-sensitive NS, 22 with steroid-resistant NS and 30 controls. Direct sequencing was performed after PCR amplification of all 29 and 8 exons of the NPHS1 and NPHS2 genes, respectively. In NPHS1, 4 patients had heterozygous missense mutations leading to amino acid substitutions (R800C, Q453R). Furthermore, 3 known single nucleotide polymorphism (SNP) were found (T741T, V763V, S1105S). In NPHS2, 3 patients had novel heterozygous allelic variants leading to amino acid substitutions (S206I, E188D), while 1 patient was found to carry a novel nonsense mutation leading to a truncated protein product (Glu237STOP). Two known polymorphisms were also found (A318A, L346L). The results demonstrate that NPHS1 and NPHS2 mutations are also present in Chinese sporadic NS patients, suggesting that genetic changes of nephrin and podocin may play pathogenetic roles in some patients with sporadic steroid resistant NS.

BACKGROUND

Congenital nephrotic syndrome (CNS) is defined as nephrotic syndrome that manifests within the first 3 months of life. Mutations in the NPHS1 gene encoding nephrin, are a major cause for CNS. Currently, more than 173 different mutations of NPHS1 have been published as causing CNS, affecting most exons.

METHODS

We performed mutation analysis of NPHS1 in a worldwide cohort of 20 families (23 children) with CNS. All 29 exons of the NPHS1 gene were examined using direct sequencing. New mutations were confirmed by demonstrating their absence in 96 healthy control individuals.

RESULTS

We detected disease-causing mutations in 9 of 20 families (45%). Seven of the families showed a homozygous mutation, while two were compound heterozygous. In another 2 families, single heterozygous NPHS1 mutations were detected. Out of 10 different mutations discovered, 3 were novel, consisting of 1 splice site mutation and 2 missense mutations.

CONCLUSIONS

Our data demonstrate that the spectrum of NPHS1 mutations is still expanding, involving new exons, in patients from a diverse ethnic background.

Nephrin (NPHS1) has been described as an important structural protein of kidney podocytes. Mutations in this gene lead to the Finnish-type congenital nephrotic syndrome. More recently, a role of nephrin as a signalling molecule in kidney podocytes has been identified. Here, we show that nephrin not only has a function in kidney podocytes, but is also required for cardiovascular development. Nephrin is expressed in the epicardium and coronary vessels during human and mouse embryonic development. Nephrin knockout embryos showed abnormal epicardial cell morphology and, at later stages of development, a reduced number of coronary vessels due to increased apoptosis, and in addition, cardiac fibrosis. Connexin 43, which is required for coronary vessel formation, was downregulated in nephrin knockout embryos. Expression of the p75NTR neurotrophin receptor, a known mediator of apoptosis, was increased in mutants. Furthermore, co-immunoprecipitation studies demonstrated a direct interaction of nephrin with p75NTR. Primary nephrin-deficient cardiac cells showed a 5-fold higher rate of apoptosis in response to progenitor of nerve growth factor compared with wild-type cells, which could be rescued by RNAi against p75NTR. Taken together, our data demonstrate that nephrin directly interacts with p75NTR and reveal an important role for nephrin in murine cardiac development by permitting survival of cardiovascular progenitor cells.

Recent advances show that human focal segmental glomerulosclerosis (FSGS) is a primary podocytopathy caused by podocyte-specific gene mutations including NPHS1, NPHS2, WT-1, LAMB2, CD2AP, TRPC6, ACTN4 and INF2. This review focuses on genes discovered in the investigation of complex FSGS pathomechanisms that may have implications for the current FSGS classification scheme. It also recounts recent recommendations for clinical management of FSGS based on translational studies and clinical trials. The advent of next-generation sequencing promises to provide nephrologists with rapid and novel approaches for the diagnosis and treatment of FSGS. A stratified and targeted approach based on the underlying molecular defects is evolving.

OBJECTIVE

Mutations in nephrin (NPHS1) and podocin (NPHS2) genes represent a major cause of idiopathic nephrotic syndrome (NS) in children. It is not yet clear whether the presence of a single mutation acts as a modifier of the clinical course of NS.

METHODS

We reviewed the clinical features of 40 patients with NS associated with heterozygous mutations or variants in NPHS1 (n = 7) or NPHS2 (n = 33). Long-term renal survival probabilities were compared with those of a concurrent cohort with idiopathic NS.

RESULTS

Patients with a single mutation in NPHS1 received a diagnosis before those with potentially nongenetic NS and had a good response to therapies. Renal function was normal in all cases. For NPHS2, six patients had single heterozygous mutations, six had a p.P20L variant, and 21 had a p.R229Q variant. Age at diagnosis and the response to drugs were comparable in all NS subgroups. Overall, they had similar renal survival probabilities as non-NPHS1/NPHS2 cases (log-rank chi(2) 0.84, P = 0.656) that decreased in presence of resistance to therapy (P < 0.001) and in cases with renal lesions of glomerulosclerosis and IgM deposition (P < 0.001). Cox regression confirmed that the only significant predictor of dialysis was resistance to therapy.

CONCLUSIONS

Our data indicate that single mutation or variant in NPHS1 and NPHS2 does not modify the outcome of primary NS. These patients should be treated following consolidated schemes and have good chances for a good long-term outcome.

BACKGROUND

The aim of this study was to elucidate whether genetic screening test results of pediatric patients with steroid-resistant nephrotic syndrome (SRNS) vary with ethnicity.

METHODS

Using high-throughput DNA sequencing, 28 nephrotic syndrome-related genes were analyzed in 110 chil-dren affected by SRNS and 10 children with isolated proteinuria enrolled by 5 centers in China (67 boys, 53 girls). Their age at disease onset ranged from 1 day to 208 months (median, 48.8 months). Patients were excluded if their age at onset of disease was over 18 years or if they were diagnosed as having Alport syndrome.

RESULTS

A genetic etiology was identified in 28.3% of our cohort and the likelihood of establishing a genetic diagnosis decreased as the age at onset of nephrotic syndrome increased. The most common mutated genes were ADCK4 (6.67%), NPHS1 (5.83%), WT1 (5.83%), and NPHS2 (3.33%), and the difference in the frequencies of ADCK4 and NPHS2 mutations between this study and a study on monogenic causes of SRNS in the largest international cohort of 1,783 different families was significant. A case of congenital nephrotic syndrome was attributed to a homozygous missense mutation in ADCK4, and a de novo missense mutation in TRPC6 was detected in a case of infantile nephrotic syndrome.

CONCLUSIONS

Our results showed that, in the first and the largest multicenter cohort of Chinese pediatric SRNS reported to date, ADCK4 is the most common causative gene, whereas there is a low prevalence of NPHS2 mutations. Our data indicated that the genetic testing results for pediatric SRNS patients vary with different ethnicities, and this information will help to improve management of the disease in clinical practice.

Mutations in the NPHS1 gene, which encodes NEPHRIN, cause congenital nephrotic syndrome, resulting from impaired slit diaphragm (SD) formation in glomerular podocytes. However, methods for SD reconstitution have been unavailable, thereby limiting studies in the field. In the present study, we established human induced pluripotent stem cells (iPSCs) from a patient with an NPHS1 missense mutation, and reproduced the SD formation process using iPSC-derived kidney organoids. The mutant NEPHRIN failed to become localized on the cell surface for pre-SD domain formation in the induced podocytes. Upon transplantation, the mutant podocytes developed foot processes, but exhibited impaired SD formation. Genetic correction of the single amino acid mutation restored NEPHRIN localization and phosphorylation, colocalization of other SD-associated proteins, and SD formation. Thus, these kidney organoids from patient-derived iPSCs identified SD abnormalities in the podocytes at the initial phase of congenital nephrotic disease.

Molecular and genetic studies in the last 2 decades have shed new light on the understanding of congenital and infantile nephrotic syndrome (NS). Glomerular pathology may appear as minimal change disease, focal segmental glomerulosclerosis, or diffuse mesangial sclerosis, glomerular diseases now recognized as podocyte injuries and in part caused by altered podocyte genes. Even though genetic mutations are not implicated in all infants with NS, the study of familial disease and congenital NS reveals that proteinuria is in many patients due to specific gene mutations. The most common mutations are in 4 genes, 3 of which are podocyte genes: NPHS1 (Finnish nephropathy), NPHS2 (podocin-induced focal segmental glomerulosclerosis), WT1 (diffuse mesangial sclerosis), and LAMB2 (Pierson syndrome). Furthermore, these studies have improved our understanding of steroid-resistant NS in older children, particularly girls, in whom proteinuria may be due to WT1 mutations. Availability of molecular genetic testing and antibodies to specific gene products are closing the gap between histopathology of pediatric glomerular disease and molecular genetic diagnosis. Recognition of NS variants, which may be reversible (eg, mitochondrial mutations, viral disease), is important. This review discusses the most common entities and the differential diagnosis of pediatric NS from the pathologist's point of view, with an emphasis on congenital (<3 months) and infantile (3 months to 1 year) NS in light of molecular and genetic studies.

NPHS1 encodes nephrin, the core protein of the interpodocyte slit diaphragm of the kidney glomerulus. NPHS1 is the causative gene for congenital nephrotic syndrome of the Finnish type (CNF) with massive, treatment resistant proteinuria. We report here the establishment of a novel nephrin-like gene, NLG1 encoding filtrin, a protein with substantial homology to human nephrin. Filtrin is a type I transmembrane protein consisting of 708 amino acids. Together with the recently cloned NEPH1, NLG1 establishes a new nephrin-like subgroup of genes belonging to the immunoglobulin superfamily of cell adhesion molecules. The RNA dot blot experiment revealed that the NLG1 mRNA expression is widely distributed but most prominently observed in the pancreas and lymph nodes. The expression of NLG1 mRNA in kidney glomeruli was verified with RT-PCR. Further immunoblotting studies with antifiltrin antibody showed a specific band at 107kDa in the human and rat glomeruli. In immunofluorescence microscopy specific staining of glomeruli but also proximal and distal parts of the nephron was seen in human kidney cortex. Due to its structural similarity and sequence homology as well as partially consistent expression pattern with nephrin we propose that filtrin belongs to a functionally important complex of proteins of the glomerular filtration barrier.

A novel immunoglobulin superfamily (Igsf) protein gene was discovered by computational analysis of human draft genomic DNA, and multiple cDNA clones were obtained. The protein encoded by this gene contains five Ig domains, one transmembrane domain, and an intracellular domain. It has significant similarity with several known Igsf proteins, including Drosophila RST (irregular chiasm C-roughest) protein and mammalian KIRREL (kin of irregular chiasm C-roughest), NEPH1, and NPHS1 (nephrin) proteins. All these proteins have multiple Ig domains, possess properties of cell adhesion molecules, and play important roles in organ development. RT-PCR and Northern blot results indicate this gene is predominantly expressed in pancreas, and public sequence databases indicate there is also expression in the nervous system. We have named this gene Kirrel2 (kin of irregular chiasm-like 2), to reflect its similarity to irregular chiasm C-roughest and Kirrel. Four splice forms of Kirrel2 were observed, including two that we cloned from pancreas mRNA as well as two GenBank entries, one from the brain and one from a retinoblastoma cell line. A partial cDNA clone of the mouse orthologue was obtained by RT-PCR from mouse brain, and the inferred protein sequence has 85% sequence identity to the human protein. Immunohistochemical staining results indicate that the KIRREL2 protein is conserved from rodents to primates, and it is highly expressed in pancreatic islets. RT-PCR results on mouse pancreatic cell lines indicate that expression in the pancreas is restricted to beta cells. Thus, KIRREL2 protein is a beta-cell-expressed Ig domain protein and may be involved in pancreas development or beta cell function.