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Pediatric Research Institute, Department of Nephrology, Ministry of Education Key Laboratory of Child Development and Disorders, National Clinical Research Center for Child Health and Disorders, China International Science and Technology Cooperation base of Child Development and Critical Disorders, Chongqing Key Laboratory of Pediatrics, Children's Hospital of Chongqing Medical University, Chongqing, China
Pediatric Research Institute, Department of Nephrology, Ministry of Education Key Laboratory of Child Development and Disorders, National Clinical Research Center for Child Health and Disorders, China International Science and Technology Cooperation base of Child Development and Critical Disorders, Chongqing Key Laboratory of Pediatrics, Children's Hospital of Chongqing Medical University, Chongqing, China
Pediatric Research Institute, Department of Nephrology, Ministry of Education Key Laboratory of Child Development and Disorders, National Clinical Research Center for Child Health and Disorders, China International Science and Technology Cooperation base of Child Development and Critical Disorders, Chongqing Key Laboratory of Pediatrics, Children's Hospital of Chongqing Medical University, Chongqing, China
Pediatric Research Institute, Department of Nephrology, Ministry of Education Key Laboratory of Child Development and Disorders, National Clinical Research Center for Child Health and Disorders, China International Science and Technology Cooperation base of Child Development and Critical Disorders, Chongqing Key Laboratory of Pediatrics, Children's Hospital of Chongqing Medical University, Chongqing, China
Pediatric Research Institute, Department of Nephrology, Ministry of Education Key Laboratory of Child Development and Disorders, National Clinical Research Center for Child Health and Disorders, China International Science and Technology Cooperation base of Child Development and Critical Disorders, Chongqing Key Laboratory of Pediatrics, Children's Hospital of Chongqing Medical University, Chongqing, China
Pediatric Research Institute, Department of Nephrology, Ministry of Education Key Laboratory of Child Development and Disorders, National Clinical Research Center for Child Health and Disorders, China International Science and Technology Cooperation base of Child Development and Critical Disorders, Chongqing Key Laboratory of Pediatrics, Children's Hospital of Chongqing Medical University, Chongqing, China
Centre for Lipid Research and Key Laboratory of Molecular Biology for Infectious Diseases (Ministry of Education), Institute for Viral Hepatitis, Department of Infectious Diseases, The Second Affiliated Hospital, Chongqing Medical University, Chongqing, China
Pediatric Research Institute, Department of Nephrology, Ministry of Education Key Laboratory of Child Development and Disorders, National Clinical Research Center for Child Health and Disorders, China International Science and Technology Cooperation base of Child Development and Critical Disorders, Chongqing Key Laboratory of Pediatrics, Children's Hospital of Chongqing Medical University, Chongqing, China
Pediatric Research Institute, Department of Nephrology, Ministry of Education Key Laboratory of Child Development and Disorders, National Clinical Research Center for Child Health and Disorders, China International Science and Technology Cooperation base of Child Development and Critical Disorders, Chongqing Key Laboratory of Pediatrics, Children's Hospital of Chongqing Medical University, Chongqing, China
Address for correspondence: Dr Wei Jiang and Dr Qiu Li, Pediatric Research Institute, Department of Nephrology, Ministry of Education Key Laboratory of Child Development and Disorders, National Clinical Research Center for Child Health and Disorders, China International Science and Technology Cooperation Base of Child Development and Critical Disorders, Chongqing Key Laboratory of Pediatrics, Children's Hospital of Chongqing Medical University, Chongqing, China.
Pediatric Research Institute, Department of Nephrology, Ministry of Education Key Laboratory of Child Development and Disorders, National Clinical Research Center for Child Health and Disorders, China International Science and Technology Cooperation base of Child Development and Critical Disorders, Chongqing Key Laboratory of Pediatrics, Children's Hospital of Chongqing Medical University, Chongqing, China
Address for correspondence: Dr Wei Jiang and Dr Qiu Li, Pediatric Research Institute, Department of Nephrology, Ministry of Education Key Laboratory of Child Development and Disorders, National Clinical Research Center for Child Health and Disorders, China International Science and Technology Cooperation Base of Child Development and Critical Disorders, Chongqing Key Laboratory of Pediatrics, Children's Hospital of Chongqing Medical University, Chongqing, China.
Pediatric Research Institute, Department of Nephrology, Ministry of Education Key Laboratory of Child Development and Disorders, National Clinical Research Center for Child Health and Disorders, China International Science and Technology Cooperation base of Child Development and Critical Disorders, Chongqing Key Laboratory of Pediatrics, Children's Hospital of Chongqing Medical University, Chongqing, China
The glomerular basement membrane (GBM) consists of laminins, collagen IV, nidogens, and fibronectin and is essential for filtration barrier integrity in the kidney. Critically, structural and functional abnormalities in the GBM are involved in chronic kidney disease (CKD) occurrence and development. Fibronectin is encoded by FN1 and is essential for podocyte-podocyte and podocyte-matrix interactions. However, disrupted or disordered fibronectin occurs in many kidney diseases. In this study, we identified a novel mutation (c.3415G>A) in FN1 that causes glomerular fibronectin-specific deposition in a gain-of-function manner, that may be associated with thin basement membrane nephropathy (TBMN) and expand the spectrum of phenotypes seen in glomerulopathy with fibronectin deposits (GFND). Our studies confirmed this variant increased fibronectin's ability to bind to integrin, thereby maintaining podocyte adhesion. Also, we hypothesised that TBMN arose as the fibronectin variant exhibited a decreased capacity to bind COL4A3/4. Our study is the first to identify and link this novel pathogenic mutation (c.3415G>A) in FN1 to GFND as well as TBMN, which may broaden the phenotype and mutation spectrums of the FN1 gene. We believe our data will positively impact genetic counselling and prenatal diagnostics for GFND with TBMN and other associated conditions that may be commonly benign conditions in humans, and may not require proteinuria-lowering treatments or renal biopsy.
Chronic kidney disease (CKD) is a progressive disease affecting more than 10% of the global population and is associated with an increased risk of morbidity and mortality.
CKD is characterised by structural and functional changes to the kidney and is typically defined as a reduction in normal kidney function, accompanied by a breakdown in the glomerular filtration barrier (GFB).
The GFB is a highly specialised capillary wall and consists of two cell types; podocytes and endothelial cells, and also a glomerular basement membrane (GBM) which is a specialised and condensed extracellular matrix (ECM) structural region between cells. The GBM is essential for filtration barrier integrity in the kidney and any loss of functional integrity is associated with changes in both cell morphology and the ECM.
Integrin adhesion complexes have key roles in these structures; however, the regulation of protein complexes in glomerular disease is largely unknown.
The GBM is a unique basement membrane formed by secreted products from both endothelial cells and podocytes during glomerulogenesis, and is a complex gel-like structure consisting of core basement membrane components, including laminins, collagen IV, nidogens, and heparan sulfate proteoglycans.
Therefore, the GBM is critical as numerous kidney diseases arise from mutations in genes encoding GBM proteins, including mutations in COL4A3, COL4A4, and COL4A5 which are associated with Alport syndrome, while LAMA5 mutations are associated with nephrotic syndrome.
Alport syndrome: a unified classification of genetic disorders of collagen IV α345: a position paper of the Alport Syndrome Classification Working Group.
Fibronectin is encoded by FN1 and encodes a large non-collagenous glycoprotein which exists as cellular/extracellular matrix and circulating forms. The protein is involved in several key functional processes, including cell adhesion, differentiation, and migration, and is also associated with the GBM.
An adapted passive model of anti-MPO dependent crescentic glomerulonephritis reveals matrix dysregulation and is amenable to modulation by CXCR4 inhibition.
Expressed in whole basal membranes and mesangial matrix, fibronectin is required for cell-cell and cell-matrix interactions and mesenchymal cell condensation.
GFND is a rare, inherited, autosomal dominant kidney disease characterised by glomerular fibronectin deposits deriving from the plasma that may lead to kidney failure, and typically presents with mild to nephrotic-range proteinuria and haematuria.
However, the pathogenic mechanisms underpinning fibronectin deposits are largely unclear, and could be related to poorly cleared fibronectin variants, or fibronectin variants formed by the attachment of circulating factors.
Using whole exome sequencing (WES), we linked a pathogenic FN1 variant [c.3415G>A (p.V1139I)] and GFND to an autosomal dominant inheritance modality in a family. Importantly, the variant was related to glomerular fibronectin specific deposits, leading to thin basement membrane nephropathy (TBMN) in the proband. TBMN is the most common cause of persistent glomerular bleeding in children and adults and occurs in at least 1% of the population. Most affected individuals exhibit haematuria, minimal proteinuria, normal renal function, a uniformly thinned GBM, and a family history of haematuria, the clinical course of which is typically benign.
However, some individuals with TBMN display severe proteinuria. Recent in vivo and in vitro mechanistic evidence suggested that unlike the increased expression of wild-type fibronectin in focal segmental glomerulosclerosis (FSGS), massively aberrant variant fibronectin levels in the glomerulus significantly decreased fibronectin binding to collagen IV (COL4A3 and COL4A4), but increased integrin (integrin β1) expression. These mechanistic and functional insights putatively suggest fibronectin variant induces renal dysfunction with GFND with TBMN.
Methods and materials
Histological analysis and staining
The fn1 patient together with the hospitalised patients of FSGS identified as non-hereditary nephropathy and Alport syndrome with similar age to the proband underwent kidney biopsy at our institution (Children's Hospital of Chongqing Medical University, Chongqing, China). The decision to biopsy was at the discretion of the attending nephrologist. Core needle biopsy material was examined under the stereomicroscope and divided for light and electron microscopy studies. The sample for light microscopy was fixed in neutral buffered formalin and was embedded in paraffin using standard procedures. Paraffin sections were stained with haematoxylin and eosin staining (H&E), periodic acid–Schiff reaction (PAS), periodic acid–silver methenamine (PASM), Masson, immunohistochemistry (IHC) and immunofluorescence (IF). Digital images were obtained with a light microscope (Olympus, Japan).
Transmission electron microscopy (TEM)
Electron microscopic sample handling and detection were performed by the electron microscopic core lab of Chongqing Medical University and the images were analysed using Image Pro plus 6.0 (Media Cybernetics, USA), randomly selecting four glomeruli and taking 10 electron micrographs in each glomerulus.
Bioinformatics analysis
Schematic of the fibronectin protein domains was analysed by SMART (a Simple Modular Architecture Research Tool).
Kidney biopsies and HeLa cells fixed in neutral buffered formalin were embedded in paraffin or optimal cutting temperature compound using standard procedures. Paraffin sections were stained with immunofluorescence, respectively. Immunofluorescent staining and images were obtained with a Nikon A1R Meta confocal microscope (Nikon, Japan). Coverslips were observed.
HeLa cells were cultured in DMEM supplemented with 10% (v/v) FBS (10100147, Hyclone; Cytiva, Germany) and 1% (v/v) penicillin/streptomycin (C0222; Beyotime) at 37°C and 5% CO
in a humidified atmosphere and passaged every 2–3 days.
Plasmid construction and transient transfection
The plasmid pCMV-HA-N was digested by EcoR I and 7431 bp of human FN1 gene and linearised pCMV-HA-N were purified. Then, FN1 and pCMV-HA-N were linked utilising In-Fusion Cloning (ClonExpress II One Step Cloning Kit, C112; Vazyme, China) to generate shuttle recombinant plasmids pLVX-IRES-ZsGreen1-AMN and pCMV-HA-N-CUBN. The shuttle plasmids were identified by Sanger sequencing analysis. Site-directed mutagenesis of FN1 was performed using Mut Express MultiS Fast Mutagenesis Kit V2 (C215; Vazyme) and also verified by Sanger sequencing analysis.
The day prior to transfection, the HeLa cells were seeded into 6-well plates at 1∗106 cells/well. The cells were transfected using Lipofectamine 3000 (Fisher Scientific, USA) according to the manufacturer's instructions with 2 μg of respective plasmid DNA per well. After 6–7 h, the medium was exchanged with fresh medium.
Co-immunoprecipitation (Co-IP) assay and Western blot analysis
Protein extracts were prepared and incubated with anti-bodies against HA or IgG for 24 h on a rotating wheel. Then, Pierce Protein A/G Magnetic Beads (Thermo Scientific, USA) were added and incubated for another 24 h. After the beads were boiled, the precipitated proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes for further analysis. For western blotting, cell samples were extracted and quantified then boiled at 95°C, 10 min. Protein sample was separated on a 6% SDS-PAGE gel and then transferred on a PVDF membrane. Primary antibodies were incubated overnight at 4°C, with specific primary antibodies against HA (1:1000, ab236632; Abcam), Integrin β1 (1:1000, R24729; Zen-Bio, USA), β-tubulin (1:3000, AB0039; Abways, China), Col4A3 antibody (1:350, #7076; Chondrex), Col4A3 antibody (1:350, #7073; Chondrex) in Tris-Buffered Saline Tween-20 (TBST) containing 5% skim milk. After being washed three times with TBST, the membranes were incubated for 1 h at room temperature with a respective IgG-HRP labelled second antibody (1:10000) in TBST containing 5% skim milk. Antigens were revealed using a chemiluminescence assay (Pierce ECL Western Blotting Substrate, 32209; Thermo Scientific) and quantification of bands was achieved by densitometry using Image J software (NIH, USA).
Proximity ligation assay
HEK 293T cells were grown in 24-well plates containing coverslips (14 mm diameter) and cultured overnight. Then cells were treated with plasmid as described for transient transfection. Coverslips were washed with PBS twice and fixed in 4% paraformaldehyde for 15 min. Then coverslips were blocked with Duolink blocking solution for 60 min at 37°C. The primary antibodies fibronectin, COL4A3 and COL4A4, diluted in blocking solution, were added to the coverslips and incubated overnight at 4°C. Then coverslips were washed with 1×Wash Buffer A and subsequently incubated with Duolink PLA Probe (Duolink In Situ PLA Probe Anti-Rabbit PLUS, DUO92002; Duolink In Situ PLA Probe Anti-Mouse MINUS, DUO92004; Sigma, USA) for 60 min at 37°C. The subsequent steps of ligation and amplification were performed according to the manufacturer's instructions (DUO92013, Sigma). Finally, coverslips were covered with Duolink In Situ Mounting Medium with DAPI (DUO82040, Sigma). Images were obtained using a Nikon A1 confocal microscope.
Statistical analyses
For immunostaining analysis, images were randomly taken under light microscope. Three sections of view were subsequently examined using Image-Pro Plus 6.0 software. The percentage of positive staining was indicated with measurement of the average optical density [integrated optical density (IOD)/area]. The ratios of IOD/area were represented as relative expression levels of various proteins in kidney tissues. Quantitative results were used to perform statistical analysis. All data were analysed using GraphPad Prism 7 (Macintosh; GraphPad, USA). Quantitative values are presented as the mean ± standard deviation. For multiple comparison analysis, one-way ANOVA followed by Tukey's multiple comparison tests were performed.
Results
Correlations between proband clinical phenotypes and TBMN
The proband was a 13-year-old male who mainly manifested with recurrent proteinuria accompanied by microscopic haematuria. Patient clinical characteristics (Table 1) showed that serum and urine creatinine levels were increased, as were urinary microalbumin levels. Also, the urinary microalbumin/urinary creatinine ratio and total 24 h urinary protein levels (mg/24 h) were significantly elevated. These observations suggested that the proband had abnormal kidney function. Therefore, a kidney biopsy was performed and showed mild glomerular lesions, but without immune complex deposition. Similarly, no obvious thickening of the GBM or inflammatory cell infiltration or fibrosis was observed (Fig. 1A). In addition, immunofluorescence was performed to assess podocyte injury, but no damage was observed as evidenced by linear podocin expression patterns along the glomerular capillary wall (Fig. 1B). Of note, electron microscopy showed that most of the GBM was markedly thin in comparison to healthy control,
but without podocyte-shedding from the area (Fig. 1C). More specifically, no electron-dense deposits adjacent to GBM were found (Fig. 1C). Therefore, our initial suspicion was TBMN for which the proband met the diagnostic criteria, and the pathogenic factors inducing this condition required investigation for optimal clinical treatment options.
Fig. 1Correlations between proband clinical phenotypes and thin basement membrane nephropathy. (A) H&E, PAS, PASM and Masson staining of proband kidney biopsy samples showed no obvious glomerular mesangial cell infiltration, inflammatory cell infiltration, fibrosis, glomerular sclerosis, or segmental sclerosis. (B) Immunofluorescence staining showed podocin expression (a podocyte membrane marker) had normal, linear expression patterns. (C) Representative transmission electron microscope photomicrographs showed that most of the glomerular basement membrane was markedly thin, without podocyte shedding from the area. HC, healthy control. Each group was tested in triplicate, and data are presented as mean ± standard deviation. ∗∗∗p<0.001.
Identification of a novel mutation c.3415G>A (p.V1139I) in FN1
TBMN is the most common inherited renal disorder. Therefore, WES was used to screen for candidate genes underlying TBMN susceptibility loci and the schematic diagram of the workflow for screening pathogenic mutations is shown in Supplementary Fig. 1 (Appendix A). Interestingly, none of the collagen IV genes including COL4A3, COL4A4, and COL4A5 had gene mutations. However, we identified a novel missense mutation in FN1 [c.3415G>A (p.V1139I)] as verified by Sanger sequencing (Fig. 2A), and likely a pathogenic variant segregating with proteinuria (Table 2). In light of the family's genetic predisposition, Sanger sequencing of the proband's parents confirmed the mutation was asymptomatically present in the proband's mother (Fig. 2B). To assess variant effects on fibronectin function, we performed an amino acid conservation analysis on the missense variant site (p.V1139I). Using ClustalW multiple sequence alignments, V1139I was localised to an evolutionary, highly conserved region across different species (Fig. 2C). Furthermore, fibronectin functional domain analysis showed the mutation was located in the central binding domain of the protein (Fig. 2D). Also, a three-dimensional structural model of fibronectin, from 1107–1181 amino acids, predicted folding and functional issues in the protein (Fig. 2E).
Fig. 2The identification of and biological information on a novel FN1 variant. (A) Exome sequencing identified the FN1 variant (c.3415G>A) which was confirmed by Sanger sequencing in the proband's family. (B) The family pedigree showing the novel germline mutation in FN1. (C) ClustalW multiple sequence alignment of fibronectin across different species. The novel missense variant (p.V1139I) identified in the proband was located at a highly conserved position in the central binding domain of fibronectin (highlighted in a black box). Asterisks below the sequence alignment indicate evolutionary conserved residues, the colon indicates highly conserved residues, and the period represents less conserved residues. (D) Schematic showing fibronectin protein domains. The novel variant is marked in red font with black arrows. (E) A three-dimensional structural model of fibronectin, from 1107–1181 amino acids, predicted folding and functional issues in the protein.
Additionally, a clinical survey revealed the proband often suffered with ankle pain after exercise. Interestingly, FN1 variants were recently shown to be associated with a skeletal disorder in some individuals.
Therefore, when all data were combined, the variant appeared to be associated with dysfunctional fibronectin.
The FN1 variant causes aberrant fibronectin expression
To exclude background disease effects in the proband, renal biopsies from patients (similar age to the proband) with clinically diagnosed non-hereditary FSGS and Alport syndrome were used as controls involved in interstitial fibrosis and glomerulosclerosis by the increased wild-type fibronectin expression. We used immunohistochemistry (IHC) with the primary antibody specifically detecting the fibronectin deriving from plasma to investigate differing expression patterns of fibronectin in all FSGS, Alport syndrome, and proband renal biopsy samples. Surprisingly, distinct to broad spectrum fibronectin expression levels in FSGS or Alport samples, levels in the glomerulus of the proband's kidney biopsy were very high (Fig. 3A). To further verify variant associations with fibronectin expression levels in vitro, we generated recombinant plasmids expressing wild-type fibronectin and variant fibronectin (p.V1139I), and then transiently transfected plasmids into HeLa cells. Western blotting was used to assess fibronectin variant expression and showed that variant gain of function was reflected by elevated protein expression (Fig. 3B,C). Thus, the variant induced massive glomerular fibronectin deposits.
Fig. 3The novel fibronectin variant is a gain-of-function mutation associated with high fibronectin expression in the glomerulus. (A) Immunohistochemistry showed elevated fibronectin expression in the glomerulus in the proband's kidney biopsy sample when compared with FSGS and Alport syndrome samples. (B,C) Western blotting and immunofluorescence analysis of fibronectin variant expression in Hela cells transfected with transient expression plasmids. HC, healthy control. Each group was tested in triplicate, and data are presented as mean ± standard deviation. ns, not significant; ∗∗p<0.01; ∗∗∗p<0.001.
Early diagnosis and intervention of diseases are of great importance to inhibition of development of diseases. Based upon the findings and combined with the clinical manifestations, we were inclined to the diagnosis of GFND with TBMN for this child, although there were no typical mesangial or subendothelial deposits found on renal biopsy. However, long-term follow-up should be conducted for the child, and renal biopsy should be performed if necessary to further confirm the pathological diagnosis.
The fibronectin variant (p.V1139I) increases integrin β1 expression
Having demonstrated variant gain-of-function associated with high fibronectin expression in the glomerulus, we hypothesised if this was related to TBMN. Of note, the proband's podocytes had not detached from the GBM, suggesting podocyte adhesion to the GBM was intact. To further confirm our findings, we investigated the effects of the fibronectin variant on integrin expression, which mediates podocyte adhesion to the GBM and intercellular adhesion. Renal biopsies from the fn1 proband and adjacent healthy kidney tissue from a healthy control (HC) were used to assess in vivo integrin β1 expression by IHC. Interestingly, when compared with the HC, integrin β1 expression in the glomeruli of the fn1 patient was significantly increased (Fig. 4A). To verify fibronectin variant associations with integrin β1 expression and in vitro functions, recombinant plasmids expressing wild-type fibronectin and variant fibronectin (p.V1139I) were transiently transfected into HeLa cells as described, after which, western blotting and immunofluorescence were performed to assess the effects of the fibronectin variant on integrin β1 expression. Critically, the variant induced integrin β1 overexpression (Fig. 4B,C). When combined, the variant (p.V1139I) associated with basement membrane thinning but did not affect podocyte adhesion by increasing integrin β1 expression levels.
Fig. 4Fibronectin variant gain-of-function status was accompanied by integrin β1 overexpression. (A) Immunohistochemistry staining showed that integrin β1 expression was significantly increased in kidney tissue. (B) Western blotting showed integrin β1 expression after HeLa transfection with recombinant plasmids. (C) Immunofluorescence showed integrin β1 overexpression was generated by the fibronectin variant in in vitro transient transfections with plasmids expressing wild-type fibronectin and variant fibronectin. HC, healthy control. Each group was tested in triplicate, and data are presented as mean ± standard deviation. ns, not significant; ∗p<0.05; ∗∗∗p<0.001.
The variant significantly decreased fibronectin binding to COL4A3 and COL4A4
To examine the relationship between the variant and TBMN, we investigated collagen type IV, COL4A3, COL4A4 and COL4A5 expression, the abnormal expression of which affects GBM thickness.
In HC biopsies, immunofluorescence and immunohistochemistry showed COL4A3, COL4A4 and COL4A5 expression levels in the GBM in glomerular capillary loops were uniform and had a continuous linear distribution (Fig. 5A–C). However, COL4A3 and COL4A4 expression levels in the GBM of the fn1 proband had altered from a continuous linear distribution to a diffuse granular distribution pattern as well as COL4A5 (Fig. 5A–C). Also, cell culture transfection/western blotting analyses indicated the fibronectin variant significantly decreased collagen type IV expression that was further demonstrated by cellular immunofluorescence (Fig. 5D,E). Similarly, immunofluorescence confirmed the variant significantly decreased COL4A3 and COL4A4 expression (Fig. 5F,G).
Fig. 5The fibronectin variant significantly decreased fibronectin binding capacity to COL4A3, COL4A4 and COL4A5. (A–C) immunofluorescence (IF) and immunohistochemistry (IHC) showed COL4A3, COL4A4 and COL4A5 expression levels in the glomerular basement membrane in normal glomerular capillary loops were not uniform or continuously linearly distributed in proband biopsies when compared with the healthy control. (D,E) HeLa cells transfected with transient-expression plasmids (wild-type fibronectin and variant fibronectin) and subjected to western blotting and cellular immunofluorescence analyses showed the variant affected collagen IV expression. (F,G) Cellular immunofluorescence analysis of the fibronectin variant toward COL4A3 and COL4A4 expression. HC, healthy control. Each group was tested in triplicate, and data are presented as mean ± standard deviation. ns, not significant; ∗p<0.05; ∗∗p<0.01; ∗∗∗p<0.001.
As COL4A3 and COL4A4 may be trafficked outside the cell to form the ECM, we hypothesised if the fibronectin variant disturbed COL4A3 or COL4A4 localisation on the GBM. To this end, we performed co-immunoprecipitation (co-IP) studies and used Duolink PLA technology to investigate fibronectin variant interactions with COL4A3 and COL4A4. Wild-type fibronectin had an increased capacity to interact with COL4A3 and COL4A4, whereas the fibronectin variant displayed a decreased interactive capacity, potentially explaining decreased COL4A3 and COL4A4 expression (Fig. 6A–C). Therefore, the fibronectin variant displayed a decreased binding ability to COL4A3 and COL4A4 proteins, thereby affecting their location in the GBM, and potentially contributing to TBMN.
Fig. 6Analysis of protein interactions of fibronectin variant with COL4A3 and COL4A4. Duolink PLA (A,B) and co-immunoprecipitation (C) studies were used to analyse variant protein interactions with COL4A3 and CLO4A4. Each group was tested in triplicate, and data are presented as mean ± standard deviation. ∗∗∗p<0.001.
The GBM is a key component of the glomerular capillary wall, with critical roles maintaining the glomerular filtration barrier. However, an unusually thick or thin GMB arises due to defects in GBM components and may be associated with a range of hereditary human diseases such as TBMN and Alport syndrome caused by mutations in COL4A3, COL4A4, and COL4A5, and also LAMB2 variants associated with Pierson syndrome.
The reasons for renal function occasionally deteriorating, the relationship between a thinned GBM and secondary glomerular disease, and whether mutations in the COL4A3 and COL4A4 genes could account for all cases of TBMN, are still unknown. In this study, we identified a novel missense mutation in FN1 [c.3415G>A (p.V1139I)] which may be associated with TBMN and expand the spectrum of phenotypes seen in GFND.
In the previous literature, GFND was diagnosed without typical histopathological changes and the authors considered the reason to be the young age of onset and the short duration of the medical history.
For our patient, the course of the disease was also relatively short. The patient presented with microscopic haematuria at the onset of disease, which was the main clinical manifestation of not only GFND but also TBMN. Therefore, no obvious glomerular electron-dense deposition had been formed in a short time. Early diagnosis and intervention treatment is key to prevent the process of both GFND and TBMN. However, it was also believed that long-term follow-up should be conducted for the child, and renal biopsy should be performed again as necessary to confirm the pathological diagnosis. In addition, the fibronectin variant induced integrin overexpression but decreased fibronectin's ability to bind to COL4A3 and COL4A4, potentially accounting for the benign clinical course of TBMN in this proband.
Fibronectin is a high molecular weight glycoprotein consisting of repeated amino acids which form domains that facilitate molecular interactions with a variety of cells via integrin and non-integrin receptors. The protein is normally produced by renal mesangial cells.
Mutations in fibronectin are associated with fibronectin glomerulopathy which is a rare, inherited, autosomal dominant, glomerular disease characterised by proteinuria, microscopic haematuria, hypertension, glomerular fibronectin specific deposits, and a slow progression to end-stage renal failure.
The pathogenic mechanisms of fibronectin accumulation in the glomerulus remain unclear, but may involve poorly cleared fibronectin variants, or fibronectin variants formed by the attachment of circulating factors.
Here, the (c.3415G>A) FN1 mutation specifically and significantly elevated fibronectin expression in the glomerulus. Our comprehensive analyses showed the variant did not damage podocytic adhesive capacity or morphology, but instead generated a severely thinned GBM. Similarly, a recent study showed that a heterozygous missense mutation (c.1819 G>T, C518F) in FN1 decreased chondrogenic potential and affected cartilage deposition via decreased fibronectin binding to collagen type II and up-regulated integrin β1 expression.
However, the transcriptional mechanism of the fibronectin variant toward increased integrin expression remains unclear.
Our study is the first to link a novel pathogenic mutation in FN1 to GFND with TBMN. Importantly, the variant generated a gain-of-function for fibronectin as evidenced by elevated fibronectin expression and an increased ability to bind integrin; however, podocyte adhesion was maintained. Our observations on TBMN aetiology suggest the variant decreased fibronectin's capacity to bind COL4A3/4. Undoubtedly, our data will positively impact genetic counselling and prenatal diagnostics for GFND with TBMN and associated conditions that may be commonly benign in humans and may not require proteinuria-lowering treatments or renal biopsy.
Conclusion
This study is the first to identify and link the novel pathogenic mutation (c.3415G>A) in FN1 to GFND as well as TBMN, which may broaden the phenotype and mutation spectrums of the FN1 gene and give an insight into genetic findings for GFND and TBMN. The findings highlighted the pathogenesis and therapeutic approaches for GFND with TBMN and other associated conditions that may be commonly benign conditions in humans, and may not require proteinuria lowering treatments or renal biopsy.
Ethics approval and consent
Informed consent for participation was obtained from the patient and parents. Written informed consent was obtained from the parents of the proband for publication of the information contained within this article. The study was approved by the Ethics Committee of the Children's Hospital of Chongqing Medical University.
Availability of data and material
All data and materials included in this study are available upon request by contacting the corresponding author.
Acknowledgements
Special thanks to the Department of Nephrology at Children's Hospital of Chongqing Medical University for assistance in performing kidney biopsies and providing clinical data about serum autoantibodies and 24 h urine protein excretion. Thanks to urology and nephropathy center of the Second Affiliated Hospital of Chongqing Medical University for providing adjacent normal kidney tissue samples.
Conflicts of interest and sources of funding
This study was supported by grants from Multi-Center Innovation Platform for Early Development and Major Diseases of Perinatal Newborns in Different Altitude Areas (Special Funds for The Central Government to Guide Local Scientific and Technological Development), the Second Batch of Funds for Chongqing Talents and Famous Teachers (No. 020210), General Project of Basic Research of Key Laboratory of Ministry of Education for Research on Child Developmental Diseases, Children's Hospital of Chongqing Medical University, China (No. GBRP-202111) and the National Natural Science Foundation of China (No. 81970618 and No. 82200906). The authors state that there are no conflicts of interest to disclose.
Appendix A. Supplementary data
The following is the Supplementary data to this article.
Alport syndrome: a unified classification of genetic disorders of collagen IV α345: a position paper of the Alport Syndrome Classification Working Group.
An adapted passive model of anti-MPO dependent crescentic glomerulonephritis reveals matrix dysregulation and is amenable to modulation by CXCR4 inhibition.
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