Ethics statement
The research procedure follows the principles outlined in the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85 − 23, modified 1996). Additionally, it has received approval from the Animal Ethics Committee at Guangdong Provincial People’s Hospital in Guangzhou, China.
Animals
Eight groups of C57BL/6 mice, each with five to six animals (male and female are evenly proportioned), were assessed at different stages: embryo (18.5 days), new-born (1 day), adolescent (1 month), young (3 months), adulthood (6 months), middle-aged (10 months), old (20 months), and senescent (30 months) [12].
Blood and urine chemistry
Blood samples were obtained at each stage using the retro-orbital method under isoflurane anesthesia. Additionally, body weight (BW) and kidney weight (KW) was measured and documented. Urine sample was collected using the reflex urination strategy at 9:00–10:00 AM. Blood glucose, serum blood urea nitrogen (BUN), and serum and urine creatinine levels were measured using a Beckman Coulter AU480 Chemistry Analyzer (Beckman). The evaluation of urine albumin was conducted using a mouse-specific albumin ELISA kit (E99-134, Bethyl Laboratories, Montgomery, TX). The results were reported as the urine albumin to creatinine ratio (UACR). Every experiment was carried out three times.
Tissue collection and animal euthanasia
Pentobarbital sodium (P3761, 30 mg/kg, Sigma-Aldrich, St. Louis, MO) was injected intraperitoneally into eight groups of mice to anesthetize them. Their body weight was then measured. The left kidneys were infused with 0.1 M PBS (pH = 7.4) at 80 to 100 mmHg for two minutes through the abdominal artery to remove circulating blood. Kidney weight (KW) was measured as the left kidneys were quickly removed.
Histology analysis
Tissue slices were fixed in 10% formalin for 24 h, rinsed in buffer, dehydrated, and embedded in paraffin for light microscopy and histological examination. 2 μm thick paraffin sections were cut, stained with hematoxylin and eosin (H&E) for examination of cell structure, periodic acid-Shiff (PAS) and periodic acid-silver methenamine (PASM) to detect alterations in basement membrane architecture and glycogen deposition. Masson’s trichrome staining (MTS) and Picric acid-Sirius red stain were used to demonstrate the deposition of collagen matrix. At least 20 randomly selected kidney sections from each mouse were evaluated for structural alterations. Two masked independent investigators analyzed twenty glomeruli in each of the twenty fields per section, using a 100 objective oil immersion lens.
Transmission electron microscopy
Several 1-mm cubes were cut from the cortex of left kidneys, fixed in 2.5% glutaraldehyde for no less than four hours, rinsed with cacodylate buffer, postfixed in 1% osmium tetroxide, and block-stained in uranyl acetate before being embedded in Poly/Bed812 resin (Polysciences, Inc., Warrington, PA). Ultrathin sections were procured from a minimum of three glomeruli that were randomly chosen from each animal. These sections were then examined by staining using uranyl acetate and lead citrate. Digital micrographs of the glomeruli from each group of mice were captured using a Hitachi 7700 transmission electron microscope (Tokyo) at magnifications of 2000 and 6000.
Morphometric analysis of the kidney
On PAS-stained kidney tissue sections from 30 glomeruli from each group of mice, the degree of mesangial expansion was assessed to determine the degree of glomerular injury. Assessment of the mesangial and glomerular cross-sectional areas was performed using Image Pro Plus software (Media Cybernetics, Bethesda, MD) [13]. Evaluation of GBM thickness and podocyte foot process width were performed on five glomeruli with electron microscope [14, 15]. Using an automated analyzer in the transmission electron microscope, the thickness of the GBM was measured (Hitachi 7700, Hitachi High Technologies Corporation, Japan). By calculating the proportion of the effaced foot process per length of the glomerular basement membrane, podocyte foot process effacement was determined. In the analysis, the mean value was noted and used.
Single cell separation
Tissues were washed three times with PBS, cut into blocks of 2 mm and placed on DMEM-1640 medium (Gibco, Gaithersburg MD, USA). Using buffer containing collagenase IV (1 mg/ml; Thermo Fisher), pronase E (1 mg/ml), and DNase I (50 U/ml), the tissues were digested at 37 °C for 30 min. Cells were filtered through a 70 μm cell strainer and centrifuged at 400×g for 5 min. Take a small amount of single-cell suspension, add 0.4% trypan blue staining solution of the same volume, count the cells with Countess II Automated Cell Counter, and adjust the concentration of living cells to the ideal range (1000 to 2000 cells/µl).
Library construction and scRNA-seq
The gel beads containing barcode information combine with a mixture of cells and enzymes,then enter the reservoir to be separated by oil to form GEMs (GelBeads-In-Emulsions). After that, the gel beads dissolved and released the capture sequence containing the Barcode sequence, reverse transcribed the cDNA fragment, and labeled the sample. Break up the gel beads and oil droplets then use cDNA as a template for PCR amplification. All the products of GEMs are mixed to construct a standard sequencing library. The cDNA was cut into 200 ∼ 300 bp fragments, then the second-generation sequencing library was constructed. Finally, the DNA library was amplified by PCR. The high-throughput sequencing of the library was carried out by using the PE150 sequencing mode of the Illumina sequencing platform. Quality control of sequencing was performed using cellranger.
Bioinformatics analysis
The expression matrix was transferred to Scanpy for subsequent analysis, and a set of cells were clustered according to both cell types and cell subgroups by singleR and Seurat. Gene differential expression analysis was performed separately for different classes of cell populations using a rank sum test to screen subgroups for up-regulated expression genes. Gene Ontology (GO; http://geneontology.org/) and Reactome pathway enrichment analyses were used to perform functional enrichment analyses using Homo sapiens as the genetic model.
Immunofluorescence
Immunofluorescence microscopy was performed on snap-frozen kidney tissues to rule out immunological complex diseases involving IgG, C3, IgA, IgM, and C1q. Kidney tissues embedded in paraffin and formalin-fixed were used to detect the expression profile of specific kidney-related markers. Mouse monoclonal synaptopodin (1:200) (sc-515842, Santa Cruz Biotechnology, Dallas, TX, USA) and rabbit polyclonal Wilm’s Tumor 1 (WT1) antibody (1:100) (ab89901, Abcam, Cambridge, MA, USA) were the primary antibodies utilized. The FV1000-IX81 confocal laser scanning microscope (Olympus, Germany) was used to capture the images. Primary antibodies were replaced with PBS as the negative control.
Statistical analysis
The previously data are shown as mean ± SD. One-Way ANOVA and Two-Way ANOVA followed by the Tukey’s post hoc test was utilized to determine the statistical significance between groups. All statistical analyses were carried out using version 25.0 of SPSS and GraphPad Prism 10.0. A significance level of 0.05 was established.
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- Source: https://bmcnephrol.biomedcentral.com/articles/10.1186/s12882-024-03514-0