Clinical samples

This study was approved by the Ethics Committee of The First Affiliated Hospital of Zhejiang University School of Medicine (No2020057), Hangzhou, China. Written informed consent was obtained from the participant and their guardians prior to enrollment. Blood samples were collected on August 27th, 2015 at the Department of Nephrology, Zhejiang University Paediatric Hospital. Renal biopsies were performed as part of routine clinical diagnostic investigations to determine the expression of ocrl1 in the kidneys of both proband and control patients. Renal biopsy samples were obtained from the Department of Pathology, The Children’s Hospital of Zhejiang University School of Medicine. Control samples were collected from the healthy kidney poles of individuals who underwent tumor nephrectomies without renal disease.

Generation of podocyte-specific OCRL1 knockout mice

The mice study was approved by The Ethics Committee of the Laboratory Animal Center of Zhejiang University (No. 21377). With recent studies on Dent disease, attention has been focused on whether podocyte damage is the result of renal tubular disease or whether defects such as ocrl1 directly cause glomerulopathy. The purpose of generating mice with a specific knockout of ocrl1 in podocytes was to investigate whether a single factor of renal tubular disease could also cause primary glomerular damage. ocrl1flox/flox mice with a C57BL/6 background were crossed with Podocin-Cre mice to generate podocyte-specific ocrl1 knockout (Pod-ocrl1-KO) mice. ocrl1flox/flox/Cre(-) and ocrl1+/+/Cre( +) mice served as controls, while ocrl1flox/flox Cre( +) mice were used as conditional knockout (cKO) mice (n = 6). The sample size was calculated using the resource equation method [13, 14]. An "E" value that represents the degree of freedom of analysis of variance was calculated using the equation: E = total number of animals—total number of groups. Any sample size that keeps E between 10 and 20 is considered adequate. The total number of animals in our study was 12, and the number of groups was 2, generating an E value of 12—2 = 10. All mice were housed in the Laboratory Animal Center of Zhejiang University under specific pathogen-free conditions, with free access to food and water, and maintained at a temperature of 25 °C and 50% humidity. Control and Pod-ocrl1-KO mice were subjected to tail genotyping. Mice were euthanized with 4% chloral hydrate at 8, 12, 16, and 20 weeks. The kidneys of control (n = 6) and Pod-ocrl1-KO mice (n = 6) were harvested.

Whole exome and RNA sequencing

DNA was extracted from the blood of the proband and his parents using standard protocols. The Roche Nimble Gen Seq EZ exome enrichment kit with capturing probes V2.0 (Roche, Indianapolis, IN, USA) was used to construct the whole-exome library and enriched the DNA of total exons and their flanking introns. High-throughput sequencing was performed on an Illumina NovaSeq 6000 series sequencer (Illumina, San Diego, CA, USA), with a minimum of 99% of target sequences sequenced at a 150 × reading depth. To identify pathways involved in the cell cycle, total RNA from scramble controls and the OCRL knockdown group of MCP5 cells was extracted using TRIzol. Enriched signaling pathways were selected through RNA sequencing, and the relevant genes were validated using RT-PCR. Data were analyzed using GraphPad software.

Cell culture

HK-2 cells were purchased from the Shanghai Cell Bank, Chinese Academy of Sciences and cultured in RPMI culture medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin at pH 7.4. The cells were maintained at 37 °C in a humidified atmosphere of 5% CO2 and passaged upon reaching an 80–90% confluent monolayer following trypsin digestion. The immortalized mouse podocyte cell line (MPC5) was generously donated by Professor Peter Mundel (Goldfinch Bio.Inc., Cambridge, MA, USA). The MPC5 cell line was originally derived from the podocytes of a male mouse. Although it is not a human cell line, it was used in our research since it is an established and validated model. MPC5 cells were cultured in RPMI medium supplemented with 10% FBS and 10U/mL mouse recombinant interferon-γ (R&D Systems) at 33 ℃ for proliferation, followed by transfer to RPMI medium supplemented with 10% FBS without recombinant interferon-γ for differentiate at 37 ℃ for 10–14 days.

Knockdown of OCRL1 and overexpression of OCRL1 mutant

Small interfering RNA targeting ocrl1 (si-ocrl1) or ocrl1 R318H (si-ocrl1 H318) mutant, as well as lentiviral vectors carrying shRNA against ocrl1 and adenoviral vectors containing ocrl1 (R318H) were purchased from Shanghai Ruijing Biological Technology (Shanghai, China), which were used to knockdown ocrl1 or overexpress the ocrl1 mutant in HK-2 cells. The targeted sequence of si/shRNA was 5’-ACCGGCAAGCCAAAGTTACCATATTTCTCGAGAAATATGGTAACTTTGGCTTGTTTTTGAATTC-3’. Lentiviral vectors carrying shRNAs 848, 1816, and 2208 targeting ocrl1 (TRCnumber:0000012848, TRCnumber00000121816, TRCnumber 00000122208; JIMA) were obtained from GenePharma (Shanghai China) to knockdown ocrl1 in MCP5 cell line. Cells were seeded in 6-well plates at a density of 5 × 105 cells/well, and lentiviral/adenoviral vectors were added to HK-2 cells at MOI of 10 and to MCP5 cells at MOI of 20. To perform cotransfection, shRNA against ocrl1 was transfected into HK-2 cells. Once the cells reached 60%–70% confluence, transfection with the ocrl1 mutant was carried out. Puromycin (2 μg/mL) was added 72 h after transfection and maintained for 5–7 days to achieve > 95% transfection efficiency. Scrambled-shRNA was used as a control. MPC5 cells were transfected using the same protocol as for HK-2 cells, except the cells were incubated at 33 °C until transfection efficiency reached 95%. The cells were then transferred to 37 °C for differentiation.

Western blot analysis

After cell lysis and centrifugation, the supernatant was collected to determine protein concentration using a Pierce BCA Protein Assay kit (#23,225; Thermo Fisher). Equal amounts of protein were then separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were incubated with anti-OCRL1 (#8797; CST, dilution 1:1000), anti-CCND1 (#2922; CST, dilution 1:1000), anti-E2F1 (#3742; CST, dilution 1:1000), anti-CDKN2D (#77,184; CST, dilution 1:1000), anti-GAPDH (#60,004–1-Ig; Proteintech, dilution 1:1000), or anti-β-actin (#3700 l; CST, dilution 1:10,000) antibody overnight at 4 °C. The membranes were then incubated with HRP-conjugated secondary antibodies (#SA00001-1; #SA00001-2; Proteintech, dilution 1:10,000) at 37 °C for 1 h. Signals were detected with ECL solution (#32,209; Thermo Fisher) and visualized with an Odyssey infrared imaging system.

Immunoprecipitation

Protein-G Dynabeads (Life Technologies) were mixed with anti-OCRL1 (1:200 dilution, CST, #8797), anti-CCND1 (#2922; CST, dilution 1:1000), anti-E2F1 (#3742; CST, dilution 1:1000), or anti-CDKN2D (#77,184; CST, dilution 1:1000) antibodies diluted in phosphate-buffered saline (PBS) with 0.02% Tween. After rotating at 4 °C for 1 h, total cell lysate was added, and the mixture was rotated for 24 h. The proteins were eluted with sample buffer (5 ×) at 96 °C for 5 min, separated by electrophoresis, and detected by immunoblotting with anti-OCRL1, anti-E2F1, anti-CCND1, or anti-CDKN2D antibodies, followed by Alexa Fluor-conjugated secondary antibody (1:10,000 dilution, ProteinTech).

Flow cytometry

Reactive oxygen species (ROS) and phosphatidylserine levels as well as cell apoptosis were determined by flow cytometry. HK-2 cells were seeded at a density of 1 × 105 cells/mL in 6-well plates. After incubation, the cells were harvested by digestion and centrifugation at 1200 rpm for 5 min. The cells were then resuspended in 500 µL of PBS and stained with 2ʹ,7ʹ-dichlorofluorescein diacetate for ROS measurement. To detect phosphatidylserine eversion, cells were resuspended in 100 μL of binding buffer and incubated with 10 μL of FITC-labeled Annexin-V in the dark for 30 min at room temperature. For cell apoptosis assay, HK-2 cells were digested with 0.25% trypsin (EDTA free), suspended in binding buffer, and incubated with 5 μL Annexin V and 5μL propidium iodide antibodies (#556,547, BD Biosciences) in the dark for 15 min. Cells were analyzed using a Cyto FLEX S flow cytometer (Beckman, USA) and FlowJo software.

Scanning electron microscopy (SEM)

To observe crystal adhesion on the cell surface, cells were seeded on coverslips in 12-well plates at a density of 1 × 105 cells/mL. After incubation, the cells were washed twice with D-Hanks solution and exposed to a serum-free medium containing 200 μg/mL calcium oxalate monohydrate (COM) for 1 h. The supernatant was removed, and the cells were washed twice with PBS. Next, the cells were fixed with 2.5% glutaraldehyde for 24 h, washed thrice with PBS, and dehydrated with graded ethanol (50%, 70%, 90%, and 100%). The cells were then dried with CO2, sprayed with gold, and analyzed by SEM to visualize crystal adhesion.

Transmission electron microscopy (TEM)

To observe kidney tissue pathology, samples were fixed in 10% formalin for 72 h, embedded in paraffin, and cut into 4-µm sections. For TEM, 1 × 1 × 1 mm sections of the kidney were fixed in 2.5% glutaraldehyde overnight at 4 ℃. TEM images were obtained by the Center of Electron Microscopy, Zhejiang University School of Medicine, while sample handling and detection were performed by the Analysis Center of Agrobiology and Environmental Sciences & Institute of Agrobiology and Environmental Sciences. Results were analyzed using Image J software (NIH, Bethesda, MD, USA) by two investigators blinded to the experimental details.

Laser scanning confocal microscope

To examine endocytosis, an internalization assay using human serum transferrin Alexa Fluor conjugate (#148,026; Jackson Immune Research) was conducted with some modifications as described [15]. Surface-bound transferrin was removed using citrate buffer (pH 2.5). Images were collected at 4 h post-incubation using a Nikon A1 Ti laser scanning confocal microscope. Cells with punctate transferrin labeling were considered positive, while cells without distinct transferrin puncta were considered negative.

Immunohistochemistry

Paraffin sections were subjected to a deparaffinization process in a 60 °C oven for 30–60 min, followed by sequential immersion in three changes of xylene for 10 min each and ethanol gradients (100%, 95%, 80%, and 70%) for 2 min each. The sections were then washed with water for 5 min. To reduce endogenous peroxidase activity, the sections were treated with pre-warmed permeation solution for 30 min (protected from light), followed by incubation at 37 °C for 30 min with anti-OCRL1 antibody (#8797; CST, dilution 1:1000) or IgG (1:10000 dilution, ProteinTech). The sections were then stained with DAB-H2O2 for 10 min and observed under microscopy.

Immunofluorescence staining

MCP5, HK-2 cells, and frozen kidney sections were fixed with 4% paraformaldehyde and blocked with 5% bovine serum albumin for 1 h at room temperature. After blocking, cells and tissues were incubated overnight at 4 °C with anti-OCRL1 (#8797; CST, dilution 1:1000), anti-podocin (#ab50339; Abcam, dilution 1:1000), or anti-nephrin (#AF3159; R&D Systems, dilution 1:250) antibodies. The following day, the samples were stained with a mixture of Alexa Fluor 488 donkey anti-goat IgG (#A11055; Life Technologies, dilution 1:500) and Alexa Fluor 594 donkey anti-rabbit IgG (#A21207; Life Technologies, dilution 1:500) secondary antibodies at 37 °C for 1 h in the dark. Images were captured using a Nikon A1 Ti laser scanning confocal microscope.

Transwell assay

MPC5 cells were suspended in serum-free medium and adjusted to a cell density of 1–10 × 105/mL. A 100–200 μL of cell suspension was added to the upper chamber containing serum-free medium, and the medium containing 10% FBS was added to the lower chamber. The cells were then incubated for 12–48 h. After removing the chambers, the medium was aspirated, and the cells in the Matrigel and upper chamber were gently wiped with a cotton swab. The chambers were then fixed with 4% paraformaldehyde (600 μL) in a new 24-well plate for 20–30 min. After discarding the fixative, the cells were stained with 0.1–0.2% crystalline violet for 5–10 min and washed three times with PBS. The samples were then examined under a microscope.

Statistical analysis

Data were presented as the mean ± standard deviation. Statistical analysis was performed using GraphPad Prism version 8.0. Nonparametric tests were used for comparing two groups. Statistical significance was considered at P < 0.05.