Mouse embryonic kidney transplantation identifies maturation defects in the medulla

Animals

C57BL/6J mice and ICR mice were purchased from Japan SLC, Inc., and immunodeficient mice (NOD.CB17-Prkdc^scid /J) were purchased from KBT Oriental, Inc. The mice were housed in a specific pathogen-free animal facility. All animal experiments were performed in accordance with our institutional ethical guidelines and approved by the licensing committee of Kumamoto University (approval numbers: A2021-008, A2023–009, and A2024-073). We also complied with ARRIVE guidelines. Mice were euthanized by intraperitoneal injection of 0.75 mg/kg medetomidine, 4.0 mg/kg midazolam, and 5.0 mg/kg butorphanol followed by cervical dislocation.

Transplantation of mouse embryonic kidneys

E12.5 mouse kidneys were used for all transplantation experiments. C57BL/6J embryos as the donors and C57BL/6J mice (8–10 weeks old) as the hosts were used in most of the transplantation experiments. The embryonic kidneys were transplanted into the peri-testicular fat unless indicated otherwise^12,18,34. For transplantation into the peri-ovarian fat, the embryonic kidneys were transplanted near the blood vessels in the peri-ovarian fat. In this experiment, ICR embryos as the donors and NOD.CB17-Prkdc^scid /J mice (8–10 weeks old) as the hosts were used. Host mice were anesthetized by intraperitoneal injection of 0.75 mg/kg medetomidine, 4.0 mg/kg midazolam, and 5.0 mg/kg butorphanol. After surgery, atipamezole was administered intraperitoneally as an anesthetic antagonist. The grafts were harvested at day 12 after transplantation. Fifteen grafts from eight independent transplantation experiments were analyzed for Fig. 1b and three samples were analyzed for Fig. 1c. Two grafts from two independent transplantation experiments were analyzed for Fig. 4. Two independent transplantation experiments (two grafts in each condition per experiment) were performed for Fig. 5. Two grafts from two independent transplantation experiments were analyzed for Fig S1a, S1c, S1d. Two independent transplantation experiments (two grafts in each condition per experiment) were performed for Fig. S9.

scRNA-seq analysis

scRNA-seq data of E15.5, E17.5, and P0 kidneys and the transplanted embryonic kidneys with a cloaca in the C57BL/6J background (transplanted at E12.5 and harvested at day 12 after transplantation) was described previously¹⁸. For the additional transplantation experiments, E12.5 embryonic kidneys with a cloaca from the ICR pregnant mice were transplanted into the testicular fat of the male NOD.CB17-Prkdc^scid /J mice (8- week-old), and harvested at day 8 and 12 after transplantation.

Dissociation of E15.5, E17.5, and P0 kidneys were described previously¹⁸. Grafts harvested on days 8 and 12 were dissociated using a similar method for the P0 kidney. Specifically, the grafts were digested with dissociation buffer comprising 2 mg/ml collagenase (Sigma; Cat#9407), 2.4 U/ml dispase II (Roche; Cat# 04942078001), 2 mM CaCl₂ (Wako; Cat# 031–00435), 50 µg/ml DNase I (Worthington #LS002139), and 10% fetal calf serum (FCS) (Sigma; Cat# 172012) in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma; Cat# D5796) for 20 min at 37 °C, washed with phosphate-buffered saline (PBS), and treated with 0.25% trypsin/EDTA 37 °C for 10 min. For the postnatal kidney (P7), the kidney was digested with dissociation buffer comprising 300U/ml collagenase Type I (Worthington; Cat# LS004194), 1 mg/ml Pronase E (Sigma; Cat# P8811), 50 U/ml DNase I (Worthington #LS002139) in Hanks’ balanced salt solution (HBSS) (Thermo; Cat# 14185-052) for 20 min at 37 °C, followed by treatment with 0.25% trypsin/EDTA at 37 °C for 10 min.

Aliquots containing 5000 dissociated cells from each sample were applied to a Chromium Controller (10× Genomics). A Chromium Single Cell 3′ Library & Gel Beads Kit v2 or v3 (10× Genomics) was used to generate cDNA libraries, which were then sequenced by an Illumina HiSeq X (757,548,299 reads for E15.5; 259; 180,950 reads for E17.5; 955,496,397 reads for P0; 370,196,520 reads for P7; 374,558,242 reads for the graft at day 8; 384,179,897 reads for the graft at day 12; 401,965,326 reads for the graft at day 12 (*)¹⁸. The Q30 base RNA reads (Q-scores indicating sequencing quality) of the samples were 86.2% for E15.5, 63.8% for E17.5, 93.6% for P0; 91.9% for P7, 91.9% for the graft at day 8, 92.2% for the graft at day 12, and 91.2% for the graft at day 12 (*).

The raw sequence data were processed using the cell ranger count command in Cell Ranger version 7.1.0 (10× Genomics), for E15.5, E17.5, P0, P7, for the graft at day 8, for the graft at day 12, and for the graft at day 12 (*) to generate count tables of unique molecular identifiers (UMIs) for each gene per cell. At this point, each dataset contained 32,749, 31,333, 33,951 34,048 35,486 35,115, and 30,347 genes, 3,969, 5,358 10,356 5,875 5,423 4,912, and 7,699 cells for E15.5, E17.5, P0, P7, the graft at day 8, the graft at day 12, and the graft at day 12 (*), respectively. All of these individually generated datasets were integrated using the cell ranger aggr command (10× Genomics). All subsequent analyses were performed in the R statistical programming language version 4.3.1³⁵, and the RStudio software³⁶. Seurat package (version 4.3.0 and 5.0.3) was used for analyses including quality control, data normalization, data scaling, visualization, and the differential analyses^37,38. For quality control, cells that expressed < 500 genes, > 35% of mitochondrial genes, > 1% of hemoglobin genes, were filtered out. Potential doublets were also removed by using DoubletFinger (2.0.4) before the further analysis. The final dataset contained 27,421 genes and 34,024 cells. A principal component analysis was used for dimension reduction with a dimension value of 95 determined by the JackStrawPlot function³⁹. Normalization was performed by SCTransform with the top 3000 highly variable genes selected by the SelectIntegrationFeatures function. Integrated data was generated by IntegrateData with using anchor genes create by FindIntegrationAnchors function, followed by scaling the data with regressing out cell cycle, high mitochondrial, ribosomal, and hemogrobin ratio. Cluster segmentation was performed using a resolution value of 2.0. The FindClusters command generated a total of 50 clusters that were easily distinguished with cluster-specific marker genes obtained with the FindMarkers function of the Seurat package. Uniform Manifold Approximation and Projection for Dimension Reduction (UMAP) plots were generated using the uwot package³⁹. The UMAP coordinates, Seurat cluster coordinates, and cluster-specific markers obtained were exported as csv files for confirmation analysis using Loupe Cell Browser software (10x Genomics).

Selection of maturation stage-dependent genes

To select maturation stage-dependent gene candidates in each cluster, we first used the FindMarkers function to pick up genes that were differentially expressed between P7 and E15.5 (e.g. comparison between cluster 10 at P7 and cluster 10 at E15.5 for collecting ducts). To determine the genes enriched in each cell type, we used the FindMarkers function to compare the target cluster with all other clusters and selected the genes with low p-values (p < 0.05). After selecting the overlapping genes, we verified them in UMAP plots to finalize the cell type-enriched stage-dependent genes. These gene lists were used for volcano plots using the EnhancedVolcano function⁴⁰. For GSEA analysis, the same gene lists were processed using the gseGO function⁴¹, and major pathways involved in each cell type were visualized using the dotplot function in DOSE package⁴². For CNET plots, part of the GSEA result were plotted using the cnetplot function in the DOSE package⁴².

Heatmap

We first utilized the AverageExpression function to add the average gene expression data for each cluster at each stage (log-fold change > 1 and p-value < 0.05). The top 200 genes were selected from the maturation stage-dependent gene lists described above. The RNAseqChef program⁴³ was used to obtain the heatmap, and pheatmap function⁴⁴ was also used to obtain a heatmap with unbiased hierarchal clustering analysis.

Immunohistochemistry

Paraffin sections were subjected to antigen retrieval in 10 mM citrate buffer (pH 6.0) after the deparaffinization. The sections were washed three times with PBS and blocked by incubation with 1% BSA in PBS for 1 h at room temperature. The sections were incubated with primary antibodies at 4 °C overnight, followed by incubation with secondary antibodies conjugated with Alexa Fluor 488, 568, or 633 for 90 min at room temperature. Cell nuclei were counterstained with 4,6-diamidino-2-phenylindole (Roche). The following primary antibodies were used: mouse anti-E-cadherin (CDH1) (BD; 610181; 1:100); rabbit anti-NKCC2 (SLC12A1) (StressMarq Bioscience; SPC-401D; 1:100); rat anti-CK8 (KRT8) (DSHB; 11562-1-AP; 1:20). Fluorescence images were captured with a confocal microscope (TSC SP8; Leica).

Whole-mount immunostaining

Whole-mount immunostaining was performed previously described with some minor modifications⁴⁵. The graft or P0 kidney was fixed in 25%, 50%, and 75% methanol/ H₂O for 10 min each at room temperature and then in methanol with 20% DMSO overnight on a shaker at 4℃. The samples were rehydrated in 75%, 50%, and 25% methanol/ H₂O for 60 min each at room temperature and then washed three times with 1% Triton X-100 in PBS for 5 min at room temperature. The washed samples were placed in PBS containing 0.3 M glycine, 1% TritonX-100, and 20% DMSO for 3 h at room temperature and then blocked in PBS containing 10% goat serum (Nippon Bio-Test Laboratories), 1% BSA (Sigma Life Science; A4503), 1% Triton X-100, 0.2% dry skim milk, and 10% DMSO overnight on a shaker at room temperature. After washing in PBS containing 1% TritonX-100 and 10 µg/ mL heparin (PTrH) twice for 60 min at room temperature, the tissues were incubated with primary antibodies conjugated with rabbit anti-E-cadherin (Cell Signaling; 3195; 1:50) and rat anti-CD31 (BD; 557355; 1:20) in PBS containing 10% goat serum, 1% BSA, 1% Triton X-100, 0.2% dry skim milk, and 5% DMSO overnight on a shaker at 4 °C. The samples were washed with PTrH for 1 day at 4 °C and then incubated with secondary antibodies conjugated with Alexa Fluor 488 and 568 (1:100) overnight at 4 °C. The tissues were washed with PTrH for two days at 4 °C. The samples were serially dehydrated with 50%, 70%, and 100% ethanol for 60 min each at 4 °C and cleared with ethyl cinnamate (Sigma-Aldrich) for more than 30 min at room temperature. Three-dimensional fluorescence images were captured using a confocal microscope (TSC SP8; Leica) and reconstructed using LAS X (Leica) software and Imaris (Bitplane).

In situ hybridization

RNAscope analysis²³ of 10% formalin-fixed paraffin sections was performed using an RNAscope Multiplex Fluorescent Reagent Kit v2 (Advanced Cell Diagnostics; Cat# 323100). Signal amplification was performed with TSA plus fluorophores (Thermo Fisher Scientific). The following RNAscope probes were used: Wnt7b (401131), Aqp4 (417161-C2), Aqp2 (452411-C3), Hif1a (313821), Ptgs2 (316621-C2), Gria1(426241-C2), and Cntn6 (836461).

Roxadustat treatment

E12.5 kidneys were transplanted into the peri-testicular fat. Roxadustat (FG4592) was dissolved in DMSO at 50 mg/ml and further diluted in 5% DMSO with 40% PEG300, 5% Tween 80, and 50% H₂O to 2.5 mg/ml^30,31. The host mice received daily intraperitoneal injections of 10 or 25 mg/kg/day Roxadustat from day 2 to day 11 after transplantation. The grafts at day 12 were compared histologically between those with and without Roxadustat treatment.

Blood analysis

8-week-old male mice received daily intraperitoneal injections of vehicle alone or 25 mg/kg/day Roxadustat for 10 days (n = 5 and 3, respectively). Mice were anesthetized by intraperitoneal injection of 0.75 mg/kg medetomidine, 4.0 mg/kg midazolam, and 5.0 mg/kg butorphanol. Blood samples were collected from the inferior vena cava. Blood hemoglobin levels were analyzed using an i-STAT1 analyzer with an EG6 + cartridge (Abbott, Princeton, NJ, USA). Student’s t-test was performed.

Statistical analysis

We assumed that our sample data were normally distributed for the analysis of the three groups. Then, Bartlett’s test was performed to determine the equality of variances among the three groups. If the variances were not significantly equal, Welch’s ANOVA was performed. If the ANOVA was significant, multiple comparisons were performed using Dunnett’s T3 test. Differences with values of p<0.05 were considered statistically significant. The asterisk (*) represents the p-value of the statistical test. One asterisk indicates that the p-value is less than 0.05. Four asterisks indicate that the p-value is less than 0.0001. This analysis was performed using GraphPad Prism 10 (GraphPad Software, San Diego, CA).

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• Source: https://www.nature.com/articles/s41598-024-81984-w