The objective of this paper was to explore the role and molecular mechanism of miR-193a in membranous nephropathy (MN). Experimental rats and podocytes were randomly divided into four groups: control, MN, miR-NC, and miR-193a inhibitor groups. The relative mRNA level of miR-193a was determined. The mRNA level and protein expression of PODXL, NPHS1, and Notch1 were determined by real-time polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. The mRNA level and protein expression of WT1 in podocytes were also determined by RT-PCR and Western blot analysis. The relative mRNA level of miR-193a in the MN group was significantly higher than that in the control group, and inhibition of miR-193a inhibited the increase successfully. Inhibition of miR-193a inhibited renal injury, podocyte injury, and tissue cell apoptosis resulting from MN. The expression of PODXL, NPHS1, and Notch1 was decreased in the MN group, while the expression was increased in the miR-193a inhibitor group. WT1 was verified as a target gene of miR-193a and the expression of WT1 increased after inhibition of miR-193a. Inhibition of miR-193a by targeting WT1 could inhibit renal function injury, renal tissue cell apoptosis, and podocytosis.

BACKGROUND

In 2010, INF2 mutations were associated with autosomal-dominant focal segmental glomerulosclerosis (FSGS), clinically presenting with moderate proteinuria in adolescence. However, in the meantime, cases with more severe clinical courses have been described, including progression to end-stage renal disease (ESRD) during childhood. INF2 mutations in patients with isolated FSGS are clustered in exons 2 to 4, encoding the diaphanous inhibitory domain, involved in the regulation of the podocyte actin cytoskeleton.

METHODS

We report a family with 14 affected individuals (autosomal-dominant mode of inheritance), most of whom presented with nephrotic-range proteinuria, hypertension, and progressive renal failure. Four members received a kidney transplant without disease recurrence. Two patients underwent renal biopsy with the result of minimal-change glomerulopathy and IgA nephropathy respectively. We performed mutational analysis of ACTN4, CD2AP, COQ6, INF2, LAMB2, NPHS1, NPHS2, PLCE1, TRPC6, and WT1 in the index patient by next-generation sequencing. Additionally, in 6 affected and 2 unaffected family members target diagnostics were performed.

RESULTS

We identified a novel heterozygous mutation c.490G>C (p.(Ala164Pro) in exon 3 of the INF2 gene in the index patient and 6 additionally examined affected family members. In silico analysis predicted it as "probably damaging". Additionally, three patients and 2 unaffected relatives harbored a novel heterozygous variant in ACTN4 (c.1149C>G, p.(Ile383Met)) with uncertain pathogenicity.

CONCLUSIONS

Mutations in INF2 are associated with familial proteinuric diseases - irrespective of the presence of FSGS and in the case of rapid disease progression. Therefore, mutational analysis should be considered in patients with renal histology other than FSGS and severe renal phenotype.

Background: Diabetes is a chronic metabolic disease characterized by disorders of glucose and lipid metabolism. Its most serious microvascular complication is diabetic nephropathy (DN), which is characterized by varying degrees of proteinuria and progressive glomerulosclerosis, eventually progressing to end-stage renal failure.

Objective: The aim of this research is to identify hub genes which might serve as genetic markers to enhance the diagnosis, treatment, and prognosis of DN.

Method: The procedures of the study include access to public data, identification of differentially expressed genes (DEGs) by GEO2R, and functional annotation of DEGs using enrichment analysis. Subsequently, construction of the protein-protein interaction (PPI) network and identification of significant modules were performed. Finally, the hub genes were identified and analyzed, including clustering analysis, Pearson's correlation coefficient analysis, and multivariable linear regression analysis.

Results: Between the GSE30122 and GSE1009 datasets a total of 142 DEGs were identified, which were mainly enriched in cell migration, platelet activation, glomerulus development, glomerular basement membrane development, focal adhesion, regulation of actin cytoskeleton, and the PI3K-AKT signaling pathway. The PPI network was composed of 205 edges and 142 nodes. A total of 10 hub genes (VEGFA, NPHS1, WT1, PODXL, TJP1, FYN, SULF1, ITGA3, COL4A3, and FGF1) were identified from the PPI network.

Conclusion: The DEGs between DN and control glomeruli samples may be involved in the occurrence and development of DN. We speculated that hub genes may be important inhibitory genes in the pathogenesis of diabetic nephropathy, so they are expected to become the new gene targets for the treatment of DN.

Keywords: Diabetic nephropathy; bioinformatics technology; differentially expressed genes; hub genes; glomeruli; public data..

Podocytes are crucial cells of the glomerular filtration unit and plays a vital role at the interface of the blood-urine barrier. Podocyte slit-diaphragm is a modified tight junction that facilitates size and charge-dependent permselectivity. Several proteins including podocin, nephrin, CD2AP, and TRPC6 form a macromolecular assembly and constitute the slit-diaphragm. Podocin is an integral membrane protein attached to the inner membrane of the podocyte via a short transmembrane region (101-125). The cytosolic N- and C-terminus help podocin to attain a hook-like structure. Podocin shares 44% homology with stomatin family proteins and similar to the stomatin proteins, podocin was shown to associate into higher-order oligomers at the site of slit-diaphragm. However, the stoichiometry of the homo-oligomers and how it partakes in the macromolecular assemblies with other slit-diaphragm proteins remains elusive. Here we investigated the oligomeric propensity of a truncated podocin construct (residues:126-350). We show that the podocin domain majorly homo-oligomerizes into a 16-mer. Circular dichroism and fluorescence spectroscopy suggest that the 16-mer oligomer has considerable secondary structure and moderate tertiary packing.

Keywords: CD, Circular dichroism; CD2AP, CD-2 associated protein; GFB, Glomerular filtration barrier; IDRs, Intrinsically disordered regions; MALS, multi-angle light scattering; NEPH, Nephrin-like protein; NPHS1 & 2, Nephrotic syndrome-type I and type II; NS, Nephrotic syndrome; Nephrotic syndrome; Podocin; Podocyte; Proteinuria; SD, slit-diaphragm; SEC, Size-exclusion chromatography; SRNS, steroid-resistant NS; Slit-diaphragm; TRPC6, Transient receptor potential cation channel subfamily C member 6; ZO-1, Zonula occludens-1.

OBJECTIVE

To report the first case of congenital nephrotic syndrome of the Finnish type (SNCF) in Togo associated with a new mutation of NPHS1.

METHODS

Our study focused on a female infant of 10months, born premature at 34weeks 6days, followed from birth to pure SNC discovered the 10th day of life. Monitoring and pregnancy outcome unremarkable. It is the third in a family of three children, the first two are killed in a similar table but not explored before 1year of age. The diagnosis is confirmed by the SNCF genetic study NPHS1 gene encoding nephrin performed in our patient and her parents showed a double mutation of which c.[106delG]+[2728T>C] and p. at the nucleotide level. [Ala36fs*6]+[Ser91OPro] at the protein level inherited from each parent. The change was made to the 10th month of death in life after sepsis in a third of cortico-resistance.

CONCLUSIONS

The SNCF, autosomal recessive disease early, which remains a serious diagnosis, is genetic. This new mutation could she explained the severity of the SNCF in this family?

Background and Objectives: Congenital nephrotic syndrome (CNS), a genetic disease caused by mutations in genes on autosomes, usually occurs in the first three months after birth. A number of genetic mutations in genes, which encode for the components of the glomerular filtration barrier have been identified. We investigated mutations in NPHS1, NPHS2, PLCE1 (NPHS3), and WT1 genes that relate to the disease in Vietnamese patients. Materials and Methods: We performed genetic analysis of two unrelated patients, who were diagnosed with CNS in the Vietnam National Children's Hospital with different disease status. The entire coding region and adjacent splice sites of these genes were amplified and sequenced using the Sanger method. The sequencing data were analyzed and compared with the NPHS1, NPHS2, PLCE1, and WT1 gene sequences published in Ensembl (ENSG00000161270, ENSG00000116218, ENSG00000138193, and ENSG00000184937, respectively) using BioEdit software to detect mutations. Results: We detected a new variant p.Ser607Arg and two other (p.Glu117Lys and p.Ser1105Ser) in the NPHS1 gene, as well as two variants (p.Arg548Leu, p.Pro1575Arg) in the PLCE1 gene. No mutations were detected in the NPHS2 and WT1 genes. Patient 1, who presented a heterozygous genotype of p.Ser1105Ser and p.Arg548Leu had a mild disease status but patient 2, who presented a homozygous genotype of these alleles, had a severe phenotype. Conclusions: These results suggest that variants p.Ser1105Ser (in NPHS1 gene) and p.Arg548Leu (in PLCE1 gene) in the homozygous form might play a role in the development of the disease in patients.

BACKGROUND

NPHS1, which encodes nephrin, recently has been identified as the gene in which mutations cause congenital nephrotic syndrome of the Finnish type (CNF). We previously reported novel missense mutations of NPHS1 in a Japanese patient with CNF. However, the mechanism by which these missense mutations cause the disorder remains to be clarified.

METHODS

Wild-type nephrin and mutated nephrin complementary DNA were each tagged by the green fluorescence protein (GFP) gene; the expressing vectors of the fusion protein were each transfected to human embryonic kidney 293 cells. We compared intracellular localization of mutated nephrin with that of wild-type nephrin by using GFP and immunostaining examination.

RESULTS

In both wild-type and mutated nephrin (Glu(447)Lys), GFP and immunostaining resulted in a colocalized microgranular pattern along the cell membrane that indicated these recombinant proteins were located at the cell surface. Conversely, in mutated nephrin (Asp(819)Val), GFP aggregation was observed in the cytoplasm, and no fluorescence was observed at the cell membrane, indicating that recombinant mutated nephrin (Asp(819)Val) could not be distributed at the cell membrane and instead was retained in cytoplasm.

CONCLUSIONS

We confirmed that the missense mutation GAC-to-GTC transversion leading to an Asp(819)Val caused the disorder. The present study analyzes in vitro distribution of nephrin with a missense point mutation. The analysis uses a new convenient method, construction of a nephrin-GFP fusion protein.

Renal transplantation is the optimal renal replacement therapy (RRT) in children, but some primary diseases can recur after transplantation, and recurrence accounts for a significant proportion of graft losses, being second only to acute rejection. The risk of disease recurrence is highest among patients with idiopathic focal segmental glomerulosclerosis (FSGS), presumably due to a circulating permeability factor. Less is clear about the genetic forms of FSGS, where the data regarding the frequency of recurrence are rather conflicting. We present a 12-year-old girl with rapidly progressive FSGS and end-stage renal disease in her native kidneys associated with heterozygous mutations in NPHS1 and in NPHS2, suffering from early post-transplant recurrence. On the basis of reviewed literature, and until further and more conclusive evidence considering pathogenicity is provided, we propose that FSGS patients with heterozygous mutations in NPHS1 or NPHS2 should be considered as having idiopathic FSGS, and post-transplant recurrence should be anticipated.

Although congenital nephrotic syndromes (CNS) form a heterogenous group of disease characterized by proteinuria shortly after birth, the most common type of CNS is the congenital NS of the Finnish type (CNF). CNF is an autosomal recessive disease, and caused by mutations in the gene (NPHS1) for nephrin which is a key component of the podocyte slit diaphragm. In this review, some special issues concerning clinical and molecular diagnosis for CNS and optimal management of CNF patients were briefly summarized.

OBJECTIVE

To investigate the association of the polymorphism of NPHS1, coding gene of nephrin, with the degree of proteinuria, renal function, and prognosis of IgA nephropathy (IgAN) in patients in north China.

METHODS

Peripheral blood samples were collected from 532 patients with IgAN confirmed by biopsy, 285 males and 230 females, aged (31+/-11). Genomic DNA was isolated from the peripheral blood leucocytes. Polymorphism of the exon G349A of NPHS1 was detected by polymerase chain reaction combined with restriction fragment length polymorphism (PCR-RFLP). 138 patients were followed up for 4-99 months. The correlation between the NPHS1 polymorphism and renal function at the time of renal biopsy, and that between NPHS1 polymorphism and the prognosis were analyzed.

RESULTS

The frequency of the genotype with the allele G (AG/GG) in the patients with the estimated glomerular filtration rate (eGFR)<60 mlxmin(-1)x(1.73 m2)(-1) was significantly higher than that of the patients with the eGFR>60 ml.min(-1)x(1.73 m2)(-1) (P=0.008). Even after adjusting for the effects of proteinuria, hypertension, and age, AG/GG genotype was an independent risk factor of the exacerbation of renal damage at the time of diagnosis (P=0.011), and GG genotype was an independent risk factor of the prognosis (P<0.001).

CONCLUSIONS

G allele and AG/GG genotype are associated with the severity of renal function at the time of diagnosis the GG genotype is associated with the prognosis of IgAN patients.

The aim of the review is to discuss recent investigations on the glomerular filtration barrier. The barrier consists of three layers: the vascular endothelium, the glomerular basement membrane and the slit diaphragm located between podocyte foot processes. The main components of the slit diaphragm are nephrin, the product of NPHS1 gene and podocin, the product of NPHS2 gene. Mutations in NPHS1 lead to congenital nephrotic syndrome of the Finnish type (CNF), whereas NPHS2 mutations result in focal segmental glomerulosclerosis (FSGS). In both cases massive proteinuria is accompanied by the effacement of podocyte foot processess. Reduced expression and redistribution of nephrin and podocin are also seen in podocytes of patients with acquired glomerulopathies. The results suggest that those proteins play a pivotal role in the processes responsible for glomerular filtration. Together with podocin and CD2AP (CD2-associated protein), nephrin forms a complex determining the integrity of the slit diaphragm. Its function has not yet been fully understood and the pathways of signal transduction need to be elucidated.

Objective: Information on etiology of congenital nephrotic syndrome in non-Caucasian populations is limited. This study aimed to determine the genetic basis of congenital nephrotic syndrome in Indian patients.

Methods: In this observational, cross-sectional study, whole exome sequencing was performed on samples from all children diagnosed with congenital nephrotic syndrome, presenting at centers collaborating in a nationwide registry and biorepository. Analysis was targeted to focus on reported or novel, pathogenic or likely pathogenic variants in 89 genes implicated in etiology of nephrotic syndrome. Sanger sequencing was used to confirm disease-causing variants in patients and allelic segregation of compound heterozygous variants in samples from parents. Inheritance of a shared haplotype was analyzed among ten individuals carrying the most common variant.

Results: During 2017-2019, 34 patients with congenital nephrotic syndrome were screened. Consanguinity and similar illness in siblings were reported in eleven patients each. Homozygous or compound heterozygous, pathogenic or likely pathogenic variants were found in NPHS1 in 24 cases, including two novel variants. One patient each had homozygous pathogenic or likely pathogenic known or novel variant in NPHS2, PLCE1, OSGEP and LAMB2 genes. Patients with OSGEP and LAMB2 mutations had phenotype typical of Galloway Mowat and Pierson syndromes, respectively. Three variants in NPHS1 were common to 16 individuals. One reported variant in exon 19 (c.2600G>A; p.Gly867Asp) appears to share a common founder.

Conclusions: A genetic cause was determined for 82.4% patients with congenital nephrotic syndrome. Variants in NPHS1 are most common in Indian patients and founder mutations might be present.

The research of novel markers in urinary samples, for the description of renal damage, is of high interest, and several works demonstrated the value of urinary mRNA quantification for the search of events related to renal disease or affecting the outcome of transplant kidneys. In the present pilot study, a comparison of the urine mRNA expression of specific podocyte markers among patients who had undergone clinical indication to renal transplanted (RTx, n = 20) and native (N, n = 18) renal biopsy was performed. The aim of this work was to identify genes involved in podocytes signaling and cytoskeletal regulation (NPHS1, NPHS2, SYNPO, WT1, TRPC6, GRM1, and NEUROD) in respect to glomerular pathology. We considered some genes relevant for podocytes signaling and for the function of the glomerular filter applying an alternative normalization approach. Our results demonstrate the WT1 urinary mRNA increases in both groups and it is helpful for podocyte normalization. Furthermore, an increase in the expression of TRPC6 after all kinds of normalizations was observed. According to our data, WT1 normalization might be considered an alternative approach to correct the expression of urinary mRNA. In addition, our study underlines the importance of slit diaphragm proteins involved in calcium disequilibrium, such as TRPC6.

Keywords: glomerular disease; podocyte mRNA; renal transplantation; signaling molecules; urine biomarkers.

The most common immunological causes of delayed renal function failure in kidney grafts are recurrent glomerular disease, de novo glomerulonephritis, and chronic cellular or antibody-mediated rejection. Glomerulonephritis can recur any time in the natural history of renal allografts, with the same morphological features of the disease occurring in the native kidney. It has a frequency varying from 100% to 1% and a generally favorable prognosis with the exception of FSGS, SHU and diabetic glomerulosclerosis. The most frequent glomerular diseases to occur de novo in the kidney graft are membranous glomerulopathy, antiglomerular basement membrane disease in patients with Alport's syndrome, and nephrotic syndrome of the Finnish type with antinephrin antibodies in patients with NPHS1 gene mutations. Chronic rejection, including chronic transplant arteriopathy and chronic transplant glomerulopathy, is the cause of renal failure in up to 20% of kidney grafts and may occur as early as a few months after transplant.

Genetic screening paradigms for the nephrotic syndrome (NS) in the developed world are well established; however, screening in developing countries has received only minor attention. We retrospectively analyzed a cohort of all children who underwent genetic testing for challenging NS from our registry in the 10-year interval from 2000 to 2010 and based on 58 patients aged 0-12 years with at least one of the following clinical diagnosis: Nonsyndromic steroid-resistant nephrotic syndrome (SRNS), familial NS, and congenital NS. Of these, 23 patients (~40%) had a history of familial disease occurrence. All cases were screened for NPHS2 and WT1 mutations by direct sequencing of all exons of the genes. In addition, all patients who were diagnosed during the first three months of life were screened for NPHS1 mutations too. A genetic disease cause was identified in 12 patients (20.7%); of these, five novel mutations, all in NPHS2 accounting for 42% of all mutations and 9% of the cohort. Nine patients were found to have NPHS2 mutations. Only one case with SRNS had a mutation in WT1. Of the five congenital NS, two cases were found to have NPHS1 mutations and one case with NPHS2 mutation. Therefore, mutations in NPHS2 were the most commonly identified and explained in 15.5% of the screened patients and WT1 mutation in 1.7% of cases, whereas NPHS1 mutations were found in 40% of congenital NS cases. A genetic disease cause was identified in 20.7% of the screened patients. Among 12 identified mutations, abnormalities in NPHS2 (n = 9) were most commonly identified.

Podocyte loss and changes to the complex morphology are major causes of chronic kidney disease (CKD). As the incidence is continuously increasing over the last decades without sufficient treatment, it is important to find predicting biomarkers. Therefore, we measured urinary mRNA levels of podocyte genes NPHS1, NPHS2, PODXL and BDNF, KIM-1, CTSL by qRT-PCR of 120 CKD patients. We showed a strong correlation between BDNF and the kidney injury marker KIM-1, which were also correlated with NPHS1, suggesting podocytes as a contributing source. In human biopsies, BDNF was localized in the cell body and major processes of podocytes. In glomeruli of diabetic nephropathy patients, we found a strong BDNF signal in the remaining podocytes. An inhibition of the BDNF receptor TrkB resulted in enhanced podocyte dedifferentiation. The knockdown of the orthologue resulted in pericardial oedema formation and lowered viability of zebrafish larvae. We found an enlarged Bowman's space, dilated glomerular capillaries, podocyte loss and an impaired glomerular filtration. We demonstrated that BDNF is essential for glomerular development, morphology and function and the expression of BDNF and KIM-1 is highly correlated in urine cells of CKD patients. Therefore, BDNF mRNA in urine cells could serve as a potential CKD biomarker.

Oligonucleotide ligation assay combined with polymerase chain reaction (PCR-OLA) is a technique which can be used for the detection of characterized sequence variations. In the present study, new PCR-OLA methods were developed for the detection of the major mutations causing infantile neuronal ceroid lipofuscinosis (INCLFin), congenital nephrotic syndrome of Finnish type (NPHS1 FinMajor and FinMinor) and medium chain acyl-CoA dehydrogenase deficiency (MCAD A985G). The prevalence of these mutations in the Finnish population was studied by analyzing blood samples collected in eastern Finland. The throughput of PCR-OLA was further enhanced by optimizing the direct use of dried blood spot (DBS) specimens for PCR. This study demonstrated that PCR-OLA is an accurate method for the detection of gene defects causing inherited disorders. With automation, PCR-OLA can be applied for routine diagnosis and for carrier screening from a large number of specimens.

The congenital nephrotic syndrome is a rare and severe pathology, and its management represents a real challenge for pediatric nephrologists. We report the case of a congenital nephrotic syndrome secondary to a homozygous mutation of the NPHS1. The young patient has a severe clinical course, and benefits of a management by anti-proteinuric treatment and a unilateral nephrectomy. This clinical case illustrates the difficulties of the management of a severe congenital nephrotic syndrome. To date, it is difficult to identify these patients beforehand because there is a poor correlation between the genotype and the phenotype of the NPHS1 mutation. There are two managements described in the literature: an early bilateral nephrectomy at 7 kg of weight with a renal transplant around 10 kg, versus a conservative management via an anti-proteinuric treatment and/or an unilateral nephrectomy. Current evidence is based on retrospective studies and the choice of a conservative approach versus early bilateral nephrectomy should take into account the severity of protein loss and its complications.

Le syndrome néphrotique congénital est une pathologie rare et sévère, dont la prise en charge représente un défi pour les néphrologues pédiatriques. Nous rapportons le cas d’un jeune patient présentant cette pathologie secondaire à une mutation homozygote du gène NPHS1. Il présente un tableau clinique sévère et bénéficie d’un traitement anti-protéinurique et d’une néphrectomie unilatérale. Ce cas clinique illustre les difficultés de la prise en charge des cas sévères, dont l’identification préalable est difficile à ce jour car la corrélation entre le génotype et le phénotype de la mutation NPHS1 est pauvre. Il existe deux prises en charges décrites dans la littérature : une néphrectomie bilatérale précoce vers 7 kg de poids et une greffe rénale vers 10 kg, ou bien une prise en charge conservative via un traitement anti-protéinurique et/ou une néphrectomie unilatérale permettant de postposer la greffe. Les données actuelles n’étant basées que sur des études rétrospectives, le choix entre une approche conservative et une néphrectomie bilatérale précoce doit prendre en compte la sévérité de la déperdition protéique et ses complications.

Keywords: NPHS1; Unilateral nephrectomy; Congenital nephrotic syndrome; Pediatrics.

Congenital nephrotic syndrome of the Finnish type (CNF) is a rare autosomal recessive disorder. The incidence of CNF is relatively high in Finland but considerably lower in other countries. We encountered a male newborn with CNF, associated with compound heterozygous mutations in nephrosis 1, congenital, Finnish type (NPHS1). The patient was admitted to hospital as a preterm infant. Physical and laboratory findings fulfilled the diagnostic criteria of nephrotic syndrome, and were compatible with a diagnosis of CNF, but there was no family history of the disease. On genetic analysis of NPHS1 a paternally derived heterozygous frame-shift mutation caused by an 8 bp deletion, resulting in a stop codon in exon 16 (c.2156-2163 delTGCACTGC causing p.L719DfsX4), and a novel, maternally derived nonsense mutation in exon 15 (c.1978G>T causing p.E660X) were identified. Early genetic diagnosis of CNF is important for proper clinical management and appropriate genetic counseling.