Animals
All procedures were performed according to the protocols approved by the Animal Care and Use Committee of Asahikawa Medical University (approval number R4-058). Experiments and methods were performed in compliance with relevant regulations and the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.
Transgenic mice were housed under temperatures of 20–26 °C and a 12-h light/dark cycle with free access to chow and water. The maximum caging density was five mice per aluminum cage (170 × 280 × 130 mm). Paper clean (Japan SLC, Shizuoka, Japan) was used as bedding. The general conditions of mice were checked daily. During the study, the health of all animals was maintained. Pdgfr-β-CreERT2, Rosa26tdTomato, and DicerFL/FL mice were purchased from Jackson laboratory (stock numbers 029684, 007909 and 006366, respectively). During inducible fate-tracing experiments, Pdgfr-β-CreERT2;tdTomato mice received tamoxifen (2 mg/20 g of body weight; Sigma-Aldrich, St. Louis, MO, USA) five times via oral administration, subjected to UUO surgery and sacrificed either 4 or 7 days after surgery or injection of FA and sacrificed either 14 or 28 days after injection.
Dicer cKO (CreERT2 + /DicerFL/FL) mice were obtained by crossing hemizygous Pdgfr-β-CreERT2 mice with homozygous Dicer-floxed (DicerFL/FL) mice. The Pdgfr-β-CreERT2 transgenic mouse lines expressed tamoxifen-activable Cre recombinase under the control of the Pdgfr-β promoter. Dicer-floxed mice (DicerFL/FL) without CreERT2 (CreERT2-/DicerFL/FL) were used as control mice. Male mice were used for all experiments. Dicer cKO and control mice were administered oral tamoxifen dissolved in neutral oil for 5 consecutive days. The experiments were initiated 7 days after the last tamoxifen dose.
Confirmation of Dicer deletion
DNA was extracted from the tails of the mice using NucleoSpin DNA RapidLyse (Takara Bio. Shiga, Japan) according to the manufacturer’s instructions and amplified with PCR using specific Dicer1 primers (forward: CCT-GAC-AGT-GAC-GGT-CCA-AAG; Dicer1 reverse: CAT-GAC-TCT-TCA-ACT-CAA-ACT). PDGFR-β-CreERT2 transgenic mice were genotyped with the PCR using specific primers (transgene forward: GAA-CTG-TCA-CCG-GGA-GGA; internal positive control forward: CAA-ATG-TTG-CTT-GTC-TGG-TG; internal positive control reverse: GTC-AGT-CGA-GTG-CAC-AGT-TT; and transgene reverse: AGG-CAA-ATT-TTG-GTG-TAC-GG). PCR products were loaded onto 1.5% agarose gels to check the Cre transgene (400 bp), its internal positive control (200 bp), the Dicer-floxed allele (420 bp), and the wild-type Dicer allele (350 bp).
UUO model
The 8–12 week-old Dicer cKO mice (n = 9) and control mice (n = 9) underwent UUO via cauterization of the ureter, as described previously20,21. Briefly, male mice were anesthetized with isoflurane and placed in an abdominal position at a controlled body temperature. The left ureter was exposed through a small suprapelvic incision and ligated at two sites within the middle portion of the ureter using a 4/0 silk thread. Mice were sacrificed by exsanguination under isoflurane anesthesia either 4 or 7 days after UUO, and the kidneys were harvested.
FA model
Eight- to twelve-week-old Dicer cKO mice (n = 11) and control mice (n = 11) were injected intraperitoneally with a single dose of FA (250 mg/kg, Sigma-Aldrich) in 0.3 M NaHCO3 or equal volume of vehicle (0.3 M NaHCO3). Mice were sacrificed by exsanguination under isoflurane anesthesia either 14 or 28 days after FA injection, and the kidneys were harvested.
Histology, immunohistochemistry, and immunofluorescence
Following whole-body perfusion with ice-cold phosphate-buffered saline (PBS), the kidneys were removed. Paraffin-embedded kidney sections fixed in 4% paraformaldehyde were used for Sirius red staining. Interstitial fibrosis was quantified as Sirius red-positive areas, as described previously20. Tissue preparation for histological staining of cryosections was performed as previously described21. Briefly, the collected kidneys were fixed with 4% paraformaldehyde for 2 h, washed in 18% sucrose solution overnight, and embedded in FSC 22 Clear Frozen Section Compound (Leica Biosystems). For immunofluorescence, compounded samples were cut into 7-µm sections on a cryostat. The frozen sections were incubated with 0.1% Triton X-100 in PBS for 15 min at room temperature (21–25 °C) and then incubated with blocking buffer (1% bovine serum albumin in 0.1% Triton X-100 in PBS) for 30 min at room temperature. This was followed by incubation with primary antibodies overnight at 4 °C.
For antigen detection by fluorescence, primary antibodies against the following proteins were used for immunolabeling: anti-PDGFR-β (ab32570; 1:200; Abcam, Cambridge, UK), anti-α-SMA-Cy3 (1:200; clone 1A4; Sigma-Aldrich), anti-F4/80 (14-4801-82; 1:200; eBioscience, San Diego, CA, USA), and anti-Dicer (20567-1-AP; 1:100, ProteinTech, Rosemont, IL, USA). Primary antibodies were detected using fluorescence-conjugated affinity-purified secondary antibody labeling (1:1000; Thermo Fisher). Nuclei were stained with Hoechst 33,342 (H3570; Life Technologies). Images were captured using confocal fluorescence microscopes (LSM-900 [Carl Zeiss, Oberkochen, Germany] and BZ-X700 [Keyence, Osaka, Japan]).
Quantitative RT-PCR
Total RNA was extracted using TRIzol (Thermo Fisher Scientific) and an RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions21. To perform quantitative PCR analysis, the extracted total RNA was reverse-transcribed using the iScript Reverse Transcription Supermix (Bio-Rad, Hercules, CA, USA). Quantitative RT-PCR was performed using the TaqMan Gene Expression Master Mix (Thermo Fisher Scientific) on a LightCycler 96 (Roche). The TaqMan probe sets used for the quantitative PCR are listed in Supplementary Table S2. All values were normalized to GAPDH and plotted as fold changes versus the control.
RNA sequencing
Libraries were sequenced using the Illumina NovaSeq 6000 platform. Sequence quality was assessed using FastQC (version 0.11.8), and low-quality sequences were removed using Trim Galore (version 0.6.4) as previously described22. Filtering was performed using default values, and each filtered read was mapped to the reference genome using STAR (version 2.7.0f). Additionally, counts were calculated using the gene symbols defined in GeneCode version 24. Genes with zero-count data from all samples were filtered. After filtering, all samples were normalized to align the total number of reads to 1 million counts per million.
The distance between the samples was determined using the count data after filtering. Spearman’s rank sum correlation coefficient was used with “1—correlation coefficient” as the definition of distance. Hierarchical clustering was performed using the group average method as the algorithm, and dendrograms were plotted graphically. A principal component analysis of the count data after counts per million normalization was performed, and scatter plots of PC1 to PC3 were created.
Using edgeR (version 3.22.3), p-values were calculated using exact probability tests for count data after Trimmed Mean of M-values normalization, and expression variation comparisons were performed. Genes with fold changes of > 2 and p-values of < 0.05 were defined as upregulated genes, whereas genes with fold changes of < 0.5 and p-values of < 0.05 were defined as downregulated genes. Map plots, volcano plots, and heatmaps were created for these genes.
GO analysis of the upregulated and downregulated genes was performed using gprofiler2 (version 0.2.1) and clusterProfiler (version 3.18.0). Using gprofiler2, Fisher’s exact probability tests were performed for terms registered in GO (BP), Molecular Function, and Cellular Component and Reactome. Then, statistical analyses were performed to determine which terms were biased toward genes with variable expression. The corrected p-value was obtained using the BH method, and a Manhattan plot was created with terms on the horizontal axis and the logarithm of the corrected p-value on the vertical axis. Next, using clusterProfiler, Fisher’s exact probability tests of terms registered in GO (BP) and WikiPathways were performed. A balloon plot was created by sorting terms in descending order of gene ratio (count data divided by the number of differentially expressed genes). GO terms with p-values of < 0.1 were considered indicative of a statistically significant difference23.
Using the TargetScan (https://www.targetscan.org/mmu_72/) and miRWalk (http://mirwalk.umm.uni-heidelberg.de/) databases, we predicted the target genes of the candidate miRNAs.
Renal primary fibroblast culture
Mouse renal fibroblasts were isolated from 8- to 12-week-old WT mice following a previously reported method20, with minor modifications. Briefly, the kidneys were harvested, and their fibrous renal capsule and medulla were removed. The renal cortex was cut into 1 mm3 sections using a scalpel blade on a Petri dish with ice-cold collagenase II solution (1 mg/mL Collagenase II in DMEM/F12 with Glutamax; Gibco, Thermo Fisher Scientific, Waltham, MO, USA). The diced sections were placed in a water bath at 37 °C for 60 min with centrifugation at 150 rpm. The digested fragments were placed in collagenase II solution with ice-cold PBS/2mM EDTA and 5% BSA. The glomeruli and other tubular fragments were removed from the cell suspension by filtering them through sterilized 100-, 70-, and 40-mm sieves. The filtered solution was collected and centrifuged at 300 G for 5 min. The pellet was incubated with the Easy-lyse Erythrocyte Lysis Solution (Dako) for 5 min in ice to remove red blood cells and centrifuged at 300 G for 5 min. The pellet was resuspended with ice-cold PBS/2mM EDTA and 5% BSA and centrifuged thrice at 300 G for 5 min. After the pellet was resuspended, the primary renal fibroblasts were placed in a 2% gelatin-coated dish and incubated with DMEM/F12 with Glutamax supplemented with 20% FBS (CORNING, Corning, NY, USA) and 1% penicillin/streptomycin. After 3–5 days, renal pieces in the supernatant were gently removed, and the medium was changed. For passage, primary cultured cells were dissociated with TrypLE™ Select (Gibco Corp.,12563-011, Grand Island, NY, USA). To examine the function of miR-9-5p in primary renal fibroblasts, cells at passage 1 were used.
Transfection of microRNA in primary cultured renal fibroblasts
Primary cultured renal fibroblasts were seeded in 6-well plates at a density of 2.0 × 105/well. At 80% confluency, cells were replaced with serum-free medium (DMEM/F12 with Glutamax (Gibco, Thermo Fisher Scientific, Waltham, MO, USA) supplemented with 0.1% FBS) and then treated with 5 ng/mL recombinant human TGF-β1 for 48 h. To examine the loss of function of miR-9-5p, the cells were transfected with mirVana hsa-miR-9-5p inhibitors (20 nM; Ambion Company, USA, Cat #4464088) using the Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. Total RNA was extracted from cultured cells using TRIzol.
Statistical analyses
Experimental data are presented as mean ± standard error. The numbers of samples are shown in the respective figure legends. Significance was determined using an analysis of variance with Tukey’s post hoc analysis. Statistical significance was set at p-values of < 0.05. Data were analyzed, and graphs were constructed using the GraphPad Prism 6 for Mac OS X software package (version 6.0h; GraphPad Software, Inc., La Jolla, CA, USA).
- The Renal Warrior Project. Join Now
- Source: https://www.nature.com/articles/s41598-024-61560-y