Antibodies and reagents
The antibodies and reagents used in this study are listed with their sources in parentheses as follows: monoclonal antibody against glyceraldehyde-phosphate dehydrogenase (GAPDH) (ImmunoWay Biotechnology, Texas, USA); polyclonal antibody against ANGPTL3 (R&D Systems, Minneapolis, USA); polyclonal antibody against LPL (Santa Cruz Biotechnology, Santa Cruz, CA). LPS was purchased from Pfizer Inc, USA.
Production of Cas9 mRNA and sgRNA
T7 promoter was added to Cas9 coding sequence by PCR amplification using PX330 vector (Addgene) and the primer Cas9 (F and R) (Table S1). T7-Cas9 PCR product was gel purified by QIAquick Gel Extraction Kit (Qiagen, USA) and used as the template (500 ng) for in vitro transcription (IVT) using mMESSAGEmMACHINE T7 ULTRA Transcription Kit (Thermo Fisher Scientific, USA). Both T7 promoter and targeting sequence was added to sgRNA by PCR amplification using the primer sgAngptl3 (F and R) (Table S1). The T7-sgRNA PCR product was also purified on gels using QIAquick Gel Extraction Kit (Qiagen, USA) and then used as templates (250 ng) for IVT using MEGA short script T7 kit (Thermo Fisher Scientific, USA). Both the Cas9 mRNA and the sgRNA were purified according to the standard protocol by phenol: chloroform extraction and ethanol precipitation, and then dissolved in DNase/RNase-free water (Thermo Fisher Scientific, USA).
Generation of Angptl3 knockout mice
C57BL/6 female mice (6–8 weeks old) were used as embryo donors. C57BL/6 female mice were supervulated by intraperitoneally injecting with PMSG and hCG, and then mated to C57BL/6 male mice. Fertilized embryos (zygotes) were collected from their oviducts. Cas9 mRNA (100 ng/μL) and sgRNA (angptl3) (50 ng/μL) were mixed and injected into the cytoplasm of fertilized eggs with both pronuclei visible in CZB (Chatot–Ziomek–Bavister) medium. The injected zygotes were then cultured in Quinn’s Advantage cleavage medium (In-Vitro Fertilization, Inc.) at 37 °C under 5% CO2 in air for about 24 h, and 18–20 2-cell stage embryos were transferred into the oviduct of a pseudo-pregnant ICR female mouse at 0.5 dpc. This work was taken in Shanghai Gemple Biotech Co.Ltd.
Mouse identification and maintenance
Angptl3-/- mice were generated by CRISPR/Cas9 system. All mice had access to food and water. All experiments were performed in accordance with the Health Guide for the Care and Use of Laboratory Animals and were approved by the Biological Research Ethics Committee of Gansu province People’s hospital (No. syll20130331). Genotyping of angptl3-/- mice was performed by PCR of mice tail-tip genomic DNA using primer angptl3 (F and R) (Table S2) and then analyzed by Sanger sequencing. This work was done in the animal center of Gansu University of Traditional Chinese medicine. All mice were housed in an air-conditioned room and were provided free access to food and water (22 ± 2°C; 12:12-hour light: dark cycle). After the 10% chloral hydrate(400 mg/kg) anesthesia, the mice were euthanized by cervical dislocation, and all efforts were undertaken to minimize pain and discomfort. The mice did not exhibited signs of peritonitis after the administration of 10% chloral hydrate(400 mg/kg).
Generation of angptl3 gene knock-out mice
The double-strand breaks (DSBs) induced by the CRISPR/Cas9 system stimulate DNA repair by at least two distinct mechanisms, non-homologous end joining (NHEJ) and homology-directed repair (HDR). NHEJ is error-prone and introduces unpredictable patterns of insertions and deletions (Indel), which can lead to disruption of the protein-coding capacity of a defined locus. To generate angptl3 knockout mice, we injected into fertilized eggs with Cas9 mRNAs and sgRNA targeting the angptl3 exon 1, which contains the start codon (Fig S1a). Subsequent sub-cloning of flanking regions surrounding the sgRNA targeting site identified four founder mice with frame shift mutations (Fig S1b).
Generation of angptl3 transgenic mice
The murine angptl3 cDNA, synthesized by Shanghai Gemple Biotechnology, was cloned into the pCDNA3.1 vector (Fig S2a). This plasmid, designated pcDNA3.1- Angptl3, was linearized by MluI/DraIII, and fragment of interest was then purified for oocyte injection using C57Bl/6 mouse-derived fertilized eggs[19-20]. Transgenic mice were identified by PCR using oligonucleotide primers specific for the construct was inserted (CMV-F, 5’-CGCGTTGACATTGATTATTGA CTA -3’ and angptl3-R, 5’- CAGGAGGCCATTCG CTAAAA -3’; PCR fragment =892bp) (Fig S2b).
Generation of LPS nephrosis in mice
All animal studies were approved by the Subcommittee on Research Animal Care of the Gansu province People’s Hospital (No. syll20130331) and performed in the animal center of Gansu University of Traditional Chinese medicine. Either of thirty-six wild type and Angptl3-/- male 6-8week C57BL/6 mice were given free access to standard laboratory food and water. The both mice were injected intraperitoneally with either 200 μg LPS (1 mg/ml in sterile LPS-free PBS) in a total volume of 200 μl. As the control group, the both mice (n＝5) received an identical volume of intravenous saline. After these four group were injected at 24th hour, 48th hour, 72th hour, the 24h urinary protein excretion was measured, and kidney and liver tissues were harvested and processed for H&E. FP effacement was assessed by transmission electron microscopy according to our published protocols.After LPS injection, the mice were killed at 24h, 48h, 72h, during which no death was found. The humane endpoint is defined as weight loss of 20%, dyspnea, or difficulty in feeding after LPS injection within 72 hours. Death was confirmed by the absence of a pulse, breathing, corneal reflex, response to toe pinch as well as a lack of respiratory sounds and heartbeat.
In this study, 196 patients with PNS admitted to Gansu Province People's Hospital from Jan 2016 to Jan 2018 were collected, including 124 males and 72 females. The health control was 60 cases. The study protocol conforms to the ethical guidelines of the 2013 Declaration of Helsinki. We insured that all patients and healthy controls provided informed and a written consent for the study, and the ethical approval was obtained from the Gansu province People’s Hospital Research Ethics Committee（syll20160037）.
PNS met the inclusion criteria of nephrotic syndrome, with urinary protein > 3.5 g/d and plasma albumin < 30 g/L, accompanied by edema of varying degrees and/or hyperlipidemia[9-10]. Exclusion criteria: secondary NS; other acute and chronic kidney diseases; tumors, infectious diseases; liver dysfunction, thyroid dysfunction, systemic sclerosis and dermatomyositis, heart failure, etc.
(1) Sample collection: All fasting subjects' elbow venous blood 3 ml in the morning was collected and placed in anticoagulant test tube. Serum was separated after centrifugation for 5 minutes (800 x g) and separated into EP tube. Urine 5 ml was collected and placed in a test tube without any additives. After centrifugation for 5 minutes (800 x g), the supernatant was taken and separated into EP tubes. Serum and urine are frozen in - 80 C refrigerator for use.
(2) Detection of serum and urine biochemical indicators: Biochemical indicators such as triglyceride (TG), total cholesterol (TC), high density lipoprotein (HDL), low density lipoprotein (LDL), serum creatinine (Scr), urea nitrogen (BUN), 24-hour urea protein (24 hUP) were detected by automatic biochemical analyzer(ABBOTT ARCHITECT c1600, USA).
(3) Determination of ANGPTL3 level in serum: the concentration of ANGPTL3 in serum was detected by ELISA kit of human ANGPTL3, and the specific operation was carried out strictly according to the instructions.
Hepatocyte cell line culture and treatment
A nontumorigenic mouse hepatocyte cell line, AML12, was obtained from American type culture collection (ATCC), which was cultured in DMEM/F12 medium (Gibco) containing 5 μg/ml ITS premix (Sigma-Aldrich, USA), 40 ng/ml dexamethasone (Sigma-Aldrich), and 10% fetal bovine serum (FBS, Gibco) at 37 °C in a humidified atmosphere of 5% CO2. Lipopolysaccharide (LPS; working concentration: 25μg/ml) were purchased from Sigma-Aldrich. AML12 was collected after LPS treatment for 24h.
For production of ANGPTL3 overexpression or knockdown lentivirus, the lentivirus with mouse angptl3 coding sequence or angptl3 shRNA and blank control were purchased from Gemple Biotechnology (Shanghai, China). The target sequences of angptl3 shRNA are as follows: sh angptl3#1, 5’-GCTGGG TCATGGACTTAAAG-3’; and sh angptl3#2, 5’-GCAGCTAACCAACTTAA TTC-3’. AML12 cells were infected with recombinant lentivirus units plus 8 ug/ml polybrene (Sigma-Aldrich) with a multiplicity of infection (MOI) of 20. The stable lentivirus infection cells were selected and enriched by flow cytometry (BD).
RNA extraction and quantitative RT-PCR
Total RNA was extracted using Direct-zol RNA MiniPrep (Zymo Research, USA) according to the manufacturer's instructions. Total mRNA (1 μg) was reverse transcribed using 5X All-In-One RT MasterMix (Abm, Canada) according to the manufacturer's instructions. Real-time PCR was performed using SYBR FAST qPCR Kit Master Mix (2X) Universal (KAPA, USA) on an Applied Biosystems 7,500 Fast Real-Time PCR System (Foster City, USA). The RT-PCR system involved in cDNA 1.0 μl, 2X SYBR‑Green Mix 10 μl, Forward Primer (10 μM) 0.5 μl, Reverse Primer (10 μM) 0.5 μl, and made up to 20 μl with RNase‑free water. The reaction conditions were: 2 min of denaturation at 94 ˚C, 40 cycles of 1 min at 94 ˚C, 30 sec at 56 ˚C, 2 min at 72 ˚C, and a final extension step at 72 ˚C for 10 min. The cycle threshold (Ct) values were analyzed using the comparative Ct (ΔΔCt) method following MIQE Guidelines. The amount of target was normalized to an endogenous reference (GAPDH) and expressed relative to a control (non-treated cells). Used primers are as follows: ANGPTL3: (forward, 5’- GCGAACATACAAGTGGCGTG-3’; reverse, 5’-CTGTGAGCCATCT TTCCGGT-3’); LPL: (forward, 5’- GAAAACCCCAGC AAGGCATAC -3’; reverse, 5’- CATCTTGCTGCTTCTCTTGGC -3’).
Oil Red O Staining
Oil Red O Staining was performed with Oil Red O Stain Kit (Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions. Results were examined by a light microscope and OD560 was measured for quantification.
We performed immunoblotting experiments as described.
Quantitative data consistent with normal distribution is represented by x±s, using independent sample t test; non-normal distribution quantitative data is expressed by M(1/4, 3/4), using Mann-Whitney U test and Kruskal-Wallis test; the difference of sex ratio was tested by X2 test; relationship between indicators was determined by Spearman correlation analysis. The values from animal or cells were subjected to one-way ANOVA tests, and Pearson correlations among the groups were calculated. P-values of <0.05 were considered statistically significant. The data was statistically processed using SPSS 20.0 software.