Experimental reagentsAnimals

Male 8-week-old Sprague–Dawley rats weighing 150 g were purchased from Jiangsu Laboratory Animal Center to prepare animal models. The rats were housed in a specific pathogen-free environment at the Animal Center of Jiangsu University at the optimal temperature with a 12 h light/12 h dark cycle. DKD model by high-fat diet combined with streptozotocin (STZ, 35 mg/kg) injected into tail vein. All animal experiments were performed in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals guidelines and were approved by the Ethics Committee of Jiangsu University (2,022,264, Jiangsu, China).

Isolation and identification of MSC-sEV

MSC-sEV were isolated and purified. Briefly, the conditioned medium was collected and centrifuged at 1,000 g for 20 min to remove cell debris, followed by centrifugation at 2,000 g for 20 min and 10,000 g for 20 min. The supernatant was collected and concentrated using 100 KDa molecular weight cut off (MWCO) (Millipore) at 1,000 g for 30 min. The concentrated supernatant was loaded 5 mL and then ultracentrifuged at 100,000 g for 60 min (optimal-90 K; Beckman Coulter). The exosome-enriched fraction was diluted with PBS, and then centrifuged thrice at 1,000 g for 30 min using 100 KDa MWCO. Finally, the purified exosomes were subjected to filtration on a 0.22-µm pore filter (Millipore) and stored at − 80 °C.

Histological analysis

Formalin-fixed, paraffin-embedded, 2 μm thick kidney sections were stained with H&E (Masson and Sirius Red), and their histological score was determined. A renal pathologist assessed the severity of tubulointerstitial fibrosis. Scoring was performed in a blinded fashion in ten consecutive fields at a magnification of 400× per section. All tests were repeated three times.

Immunohistochemistry and immunofluorescence

The kidney was fixed in 4% paraformaldehyde, embedded in paraffin and then cut into 2 μm-thick sections. Immunohistochemistry was performed to assess renal injury and fibrosis. In addition, immunofluorescence was performed to detect the colocalization of fibrotic proteins in mesangial cells and to assess renal inflammatory infiltration of macrophages observed by confocal microscopy. Immunofluorescence co-staining was performed using an immunohistochemistry kit (Boster, China). Sections were stained with the following antibodies: monoclonal rabbit anti-YAP (14,074 S, CST, USA), monoclonal mouse anti-α-SMA (19,245 S, CST, USA), monoclonal rabbit anti-F4/80 (70076T, CST, USA), monoclonal rabbit anti-CK1δ (12,417 S, CST, USA), and monoclonal rabbit anti-β-TRCP (4394 S, CST, USA). Positive cells were counted in the renal interstitial on five nonoverlapping view fields at 400× magnification.

Western blotting

Kidney tissues were harvested from DKD rats in different groups, lysed with radioimmunoprecipitation assay (RIPA) buffer (Sigma, St. Louis), and supplemented with multiple protease inhibitors (Invitrogen, USA). We separated 100-µg protein samples by 12% SDS-PAGE. After semidry transfer, nonspecific binding sites of the nitrocellulose membrane were blocked with 5% nonfat milk in Tris-buffered saline. Subsequently, the membrane was incubated with the following primary antibodies: monoclonal rabbit anti-α-SMA (19,245 S, CST, USA), monoclonal rabbit anti-TGF-β1 (3709 S, CST, USA), monoclonal rabbit anti-YAP (14,074 S, CST, USA), monoclonal rabbit anti-phospho-YAP (ser381) (13,008 S; CST, USA), monoclonal rabbit anti-CK1δ (12,417 S, CST, USA), and monoclonal rabbit anti-β-TRCP (4394 S, CST, USA). Subsequently, the conjugated antibodies incubated with secondary horseradish peroxidase (HRP) that were used were anti-mouse immunoglobulin G (IgG) and anti-rabbit IgG (Abcam) for 2 h at room temperature. Blots were analyzed with the enhanced chemiluminescence (ECL) system and captured on autoradiographic films. Glyceraldehyde 3-phosphate dehydrogenase (GADPH) and β-actin were blotted on the same membrane as the loading controls.

Macrophages and mesangial cell coculture

First, the macrophages were cultured in a high-glucose environment for 48 h, and primary mesangial (20,000 cells) were seeded into the cell coculture plate with macrophages (15,000 cells) from individual DKD rats (n = 5) in Gibco Dulbecco’s Modified Eagle Medium. All growth factor supplements were removed, and cells were cultured for 72 h in mesangial basal media. Mesangial cells were fixed in 4% paraformaldehyde for 30 min, permeabilized with 0.3% Triton phosphate-buffered saline (PBS) for 5 min, blocked with 10% serum in PBS for 30 min, and finally subject to primary antibody incubation (mouse anti-α-SMA and rabbit anti-Collagen 1) for 12 h. Next, cells were washed in 0.1% Triton PBS followed by the addition of fluorescently conjugated secondary antibodies (1:500 dilutions) for 2 h. Cells were mounted with the nuclear dye DAPI, and images were taken using a confocal microscope GE.

Preparation of single cell suspension

Euthanized rats were perfused with chilled 1x PBS via the left heart. Kidneys were harvested, minced into approximately 1 mm3 cubes and digested using Multi Tissue dissociation kit (Miltenyi, 130-110-201). Up to 0.25 g of the tissue was digested with 50 ul of Enzyme D, 35 ul of Enzyme R and 10 ul of Enzyme A in 1 ml of RPMI and incubated for 30 min at 37 degrees. Reaction was deactivated by adding 10% FBS. The solution was then passed through a 40 μm cell strainer. After centrifugation at 1,000 RPM for 5 min, cell pellet was incubated with 1 ml of RBC lysis buffer on ice for 3mins. Single cells were washed with PBS, and the cell number were analyzed using (Countess Auto Counter) and viability (Trypan).

Data quality control and preprocessing

Once the gene-cell data matrix was generated, poor quality cells were excluded, such as cells with < 200 or > 3,000 unique genes expressed genes (as they are potentially cell duplets). Only genes expressed in 10 or more cells were used for further analysis. Cells were also discarded if their mitochondrial gene percentages were over 50%. The data were natural log transformed and normalized for scaling the sequencing depth to a total of 1e4 molecules per cell, followed by regressing-out the number of UMI using Seurat package. Batch effect was corrected by using remove function of edgeR.

Dimensionality reduction and tSNE visualization

Seurat R package (version 1.4.0.5) was used for dimensionality reduction analysis. We first identified highly variable genes across the single cells, after controlling for the relationship between average expression and dispersion. Genes were placed into 20 bins based on their average expression and removed using 0.0125 low cutoff and 0.3 high cutoff. Within each bin, a z-score of log transformed dispersion measure (variance/mean) was calculated. A z-score cutoff of 0.5 was applied to identify the highly variable genes, resulting in a total of 1,140 genes. Then we performed PCA using the variable genes as input and determined significant PCs based on the jackStraw function from the Seurat package. Statistically significant 20 PCs were selected as input for t-Distributed Stochastic Neighbor Embedding (tSNE). tSNE visualized the single cells on a two-dimensional space based on expression signatures of the variable genes, and therefore similar to PC loadings.

Identification of differentially expressed genes and marker genes

cell specific marker genes were identified in two stages. The first sets of differentially expressed genes (DEGs) were identified by comparing cells in a specific cluster with cells in all other clusters (Seurat package likelihood ratio test: average expression difference > 0.5 natural log with a FDR corrected p < 0.01). Next, cells in a specific cluster were compared to cells in every other cluster in a pairwise manner to identify a second sets of DEGs (Seurat package likelihood ratio test: average expression difference > 0.25 natural log with p < 0.05). Cell specific markers were identified by overlapping first and second sets of DEGs. Since different cells in the kidney share some well-known markers (transitional cells vs. intercalated cells and proximal tubule vs. novel cells), the combination of these two approaches using the lower threshold enabled us to retain the shared markers while identifying distinct markers compared to other cells.

Cell clustering analysis

The density-based spatial clustering algorithm (DBSCAN) was used to identify cell types on the tSNE map with an initial setting of an eps value of 0.5. Clusters were removed if their number of cells was less than 10. The remaining cells were clustered again with an eps value of 1, followed by the removal of clusters if the number of cells was less than 20. After pruning, we removed 320 cells (1.1% of our data), and 27,424 cells were used for further analysis. In a post-hoc test of the final 16 clusters, every pair was found to have more than 10 differentially expressed genes (average expression difference > 1 natural log with a FDR corrected p < 0.01). We used the same procedure for subclustering with modifications. Then DBSCAN was used to identify cell types on the tSNE map with an initial eps value of 0.5. Briefly, though 6 steps (①Preparation of single renal cell suspension, ②Single cell RNA sequencing: library construction and quality control, ③Data quality control and preprocessing, ④Dimensionality reduction and tSNE visualization, ⑤Identification of differentially expressed genes and marker genes, ⑥Cell clustering analysis: dimensionality reduction analysis and tSNE map showed that renal cells were divided into 12 clustering) annotated the 12 populations in Fig. 2A.

Statistical analysis

All experiments were performed at least three times for each group, and the statistical analyses were performed using GraphPad Prism Software (version 7). The results were presented as mean values ± standard deviation. One-way analysis of variance (ANOVA) and two-way ANOVA for multiple groups and Student’s t test for two groups were applied for statistical analysis. Survival time was analyzed using the Kaplan–Meier method and log-rank test. A p value < 0.05 indicated statistical significance.