Exome sequencing

Genomic DNA was obtained from whole blood using the QIAamp DNA Mini Kit (180134, Qiagen). 97 genes related to kidney disease were kept as a gene capture strategy, using the GenCap custom enrichment kit (MyGenostics Inc) following the manufacturer’s protocol. The enriched libraries were sequenced using an Illumina HiSeq 2000 sequencer (Illumina), which running for paired-end reads of 150 bp. The clean reads were aligned to the reference human genome (hg19) with Short Oligonucleotide Analysis Package (SOAP) aligner software (SOAP2.21; soap.genomics.org.cn/soapsnp. html). Afterwards, single nucleotide polymorphisms (SNPs) were annotated with the SOAPsnp program, and the deletions and insertions (InDels) were detected using Genome Analysis Toolkit software 3.7. Low-quality variations were filtered out using a quality score ≥ 20 and MAF ≤ 0.01 and the schematic of screening workflow were shown in Additional file 1: Fig. S1. All variants were verified by Sanger sequencing.

Minigene assay

Vector pSPL3, known as the exon trapping vector, was carried out for minigene assay. Briefly, exon 44 of CUBN and its adjacent intron 43 and 44, was PCR-amplified using the following primers: forward 5′-accagaattctggagctcgagATTCATCTAT CAGAAACATGATATATT-3′ and reverse 5′-accagaattctggagctcgagCAATGAGAATAGATAAATGGTCTGGCA-3′. XhoI and NheI were chosen as restriction sites. The PCR products were inserted into the vector pSPL3 following the standard process with ClonExpress II one step cloning kit (C112, Vazyme). The mutant type was constructed according to the procedure by Mut Express II Fast Mutagenesis Kit V2 (C214, Vazyme). Wild-type and mutant types were transfected into HEK293T cells with lipofectamine 3000 (Life Technologies). RNA was harvested using the Steadypure Quick RNA Extraction Kit (AG21023, Accurate biology) at 24 h after transfection. Then cDNA synthesis was performed with HiScript III 1st strand cDNA Synthesis Kit (R312, Vazyme). Subsequently, cDNA was PCR-amplified using the following pSPL3 specific primers:SD6-5′-TCTGAGTCACCTGGACAACC-3′ and SA2-5′-ATCTCAGTGGTATTTGTGAGC- 3′. The PCR fragments were identified by Sanger sequencing to evaluate the alternative splicing.

Bioinformatics analysis

Phylogenetic analysis of CUBN was used an online tool, the interactive tree of life (https://itol.embl.de). Schematic of the Cubilin protein domains was analyzed by the SMART (a Simple Modular Architecture Research Tool). ClustalW multiple sequence alignment of Cubilin protein in several species was achieved by Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/). Crystal structures of CUB domain were analyzed by the SWISS-MODEL (https://swissmodel.expasy.org).

Histological analysis and staining

The cubn patient together with the hospitalized patients of minimal change nephropathy (MCD) and focal segmental glomerulosclerosis (FSGS) identified as non-hereditary nephropathy with similar age to the proband underwent kidney biopsy at our institution (Children’s Hospital of Chongqing Medical University, Chongqing, P.R 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 was embedded in paraffin or optimal cutting temperature (OCT, 4583, SAKURA, America) compound by using standard procedures. Paraffin sections were stained with H&E, PAS, IHC and IF, respectively. Digital images were obtained with a light microscope (Olympus).

Transmission electron microscopy (TEM)

Electron microscopic sample handling and detection were performed by the electron microscopic core lab of Chongqing Medical University. TEM images were analyzed using Image Pro plus 6.0. Four glomeruli were randomly selected and ten electron micrographs were taken in each glomerulus.

Confocal and fluorescence microscopy

Kidney biopsies and 293 T cells fixed in neutral buffered formalin were embedded in paraffin or optimal cutting temperature compound by using standard procedures. Frozen and paraffin sections were stained with immunofluorescence, respectively. Immunofluorescent staining and images were obtained by a Nikon A1R Meta confocal microscope. Cover slips were observed.

The antibodies used were list below: anti-Cubilin-C-terminal antibody (1:500, ab191073, Abcam), Rat Cubilin(CUBN) polyclonal antibody (1:100, 31010, Bicell Scientific), anti-Synaptop-odin antibody (1:50, 21064-1-AP, Proteintech), anti-Wilms Tumor Protein antibody (1:50, ab89901, Abcam), anti-COL4A3 antibody (1:100, Kingmed, Guangzhou, China), anti-COL4A5 antibody (1:100, Kingmed, Guangzhou, China), anti-Amnionless antibody (1:10, sc-365384, Santa Cruz), anti-Megalin Antibody (1:30, CD7D5, Novus Biologicals), goat polyclonal secondary antibody to mouse Alexa fluor 488 (1:400, ab150113, Abcam), goat polyclonal secondary antibody to rabbit Alexa fluor 555 (1:400, ab150078, Abcam), rabbit monoclonal to HA tag (1:500, ab236632, Abcam), goat polyclonal secondary antibody to rabbit Alexa fluor 647 (1: 400, ab150079, Abcam), goat anti-mouse Alexa fluor 568 (1: 400, ab175473, Abcam), DAPI (1:1000, C1002, Beyotime).

Cell culture

293 T cells were cultured in DMEM supplemented with 10% (v/v) FBS (Hyclone, 10100147) and 1% (v/v) penicillin/streptomycin (Beyotime, C0222) at 37 ℃and 5% CO2 in a humidified atmosphere and passaged every 2-3 days.

Animals

Male BALB/c mice (20–22 g per mouse) and male C57BL6 mice (20–25 g per mouse) was kept under pathogen-free conditions at the Laboratory Animal Centre institution, Children’s Hospital of Chongqing Medical University (Chongqing, P.R China). After adaptive feeding for one week, BALB/c mice were injected by adriamycin (11 mg/kg, Meilunbio) through tail vein, and male c57 mice were administered intraperitoneally with Lipopolysaccharides (LPS, 12 mg/kg, Sigma-Aldrich). Control groups were received an equal volume of saline. BALB/c mice were anesthetized and sacrificed using isofluorane and euthanized by cervical dislocation at the fourth week after tail vein injection, while c57 mice were sacrificed in the same way at the 24th hour after intraperitoneal LPS injection. Kidney tissues were excised and fixed in 4% paraformaldehyde, embedded in paraffin. Paraffin-embedded sections were used to analyze the co-localization between Cubilin and Amn according to standard protocol. The experiment was approved by the Animal Ethics Committee of the Children’s Hospital of Chongqing Medical University (No. CHCMU-IACUC20220629011).

Plasmid construction and transient transfection

The plasmids pLVX-IRES-ZsGreen1 and pCMV-HA-N were digested by EcoR I, respectively, and a 10,869 bp of human CUBN gene, a 1359 bp of human AMN gene, linearized pLVX-IRES-ZsGreen1 and pCMV-HA-N were purified. Then, AMN and pLVX-IRES-ZsGreen1, CUBN and pCMV-HA-N were linked utilizing In-Fusion Cloning (Vazyme, ClonExpress II One Step Cloning Kit, C112) 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 CUBN were performed using Mut Express MultiS Fast Mutagenesis Kit V2(Vazyme, C215) and also identified by Sanger sequencing analysis.

The day prior to transfection, the cells were seeded into 24-well plates at 1 × 105 cells/well. The cells were transfected using Lipofectamine3000 (ThermoFisher, USA) according to the manufacturer’s instructions with 500 ng 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 (ThermoFisher, USA) were added and incubated for another 24 h. After the beads were boiled, the precipitated proteins were separated by SDS-PAGE and transferred to PVDF membranes for further analysis. For western blotting, cell samples were extracted and quantified then boiled at 95 ℃, 10 min. Protein sample was separated on a 6% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel then transferred on a polyvinylidene fluoride (PVDF) membrane. Incubating primary antibodies overnight at 4 ℃, with specific primary antibodies against HA (1:1000, ab236632, Abcam), Cubilin (1:1000, ab191073, Abcam), anti-Amnionless antibody (1:500, sc-365384, Santa Cruz) and β-tubulin (AB0039, 1:2000, Abways) in Tris-Buffered Saline Tween-20 (TBST) containing 5% skim milk. After washed for 3 times with TBST, the membranes were incubated for 1 h at room temperature with a respective IgG-HRP labeled second antibody (1:10,000) in TBST containing 5% skim milk. Antigens were revealed using a chemiluminescence assay (Pierce ECL Western Blotting Substrate, 32,209, ThermoFisher, USA) and quantification of bands was achieved by densitometry using the Image J software.

Proximity ligation assay

HEK 293 T 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 antibody HA and AMN, diluted in blocking solution, was 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) 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 Nikon A1 confocal microscope.

Data availability

All data included in this study are available upon request by contact with the corresponding author.