Animal studies
All mouse experiments were carried out in accordance with the UK Home Office regulations (licence 70/8891; protocol 2 or licence PP0604995; protocol 3) and mice were housed in a licenced, pathogen-free facility, under a 12hr light-dark cycle, at stable temperature (19-23 oC) and humidity (55±10%). The mice were bred on a mixed C57Bl6 background, were housed in conventional cages and had ad libitum access to food and water. All experiments were performed on 8-12 weeks old male and female mice according to the guidelines of the Animal Welfare and Ethical Review Body (AWERB) and were in agreement with the ARRIVE guidelines27.
Treatment with AAV was performed as described previously12. Briefly, mice were injected with 2x1011 genomic copies (GC) of either (AAV8.TBG.PI.Cre.rBG (AAV-Cre) (Addgene, 107787-AAV8) or AAV8.TBG.PI.Null.bGH (AAV-Null) (Addgene, 105536-AAV8) in 100μl sterile PBS via a single tail vein injection. Mice in this study weighed 21.9-31.9 g at the time of induction.
For the mouse model of hepatocyte-specific inactivation of Mdm2, male mice homozygous for the Mdm2tm2.1Glo allele (ID: MGI:238543917628) and the Gt(ROSA)26Sortm14(CAG-tdTomato)Hze allele (ID: MGI:380952417729) were used (Mdm2E5/E6fl; R26LSL-tdTomato/LSL-tdTomato mice). For the induction of Ras-induced liver senescence, 8-12 weeks old male and female mice heterozygous for the Krastm4Ty allele, (ID: MGI:242994817830) and the Gt(ROSA)26Sortm14(CAG-tdTomato)Hze allele (ID: MGI:3809524177) were used (KrasLSL-D12D/WT; R26LSL-tdTomato/LSL-tdTomato mice).
For treatment with TGFβR1i, the mice received either TGFβR1i or Vehicle (0.5% HPMC/0.1% Tween 80) by oral gavage twice daily. The dose of TGFβR1i was 50mg/kg in 100ml PBS.
Mice were euthanised by CO2 inhalation in a CO2 chamber, cervically dislocated and then weighed. Blood was collected immediately via cardiac puncture for whole blood analysis (EDTA-coated tubes; Sarstedt) and plasma biochemistry (Lithium-heparin coated tubes; Sarstedt). Plasma was separated by centrifugation (2350g for 10 minutes at room temperature) within 1-3 hours after culling and stored immediately at -80oC. After weighing the liver, the caudate and left median lobes lobe were snap-frozen on dry ice for protein/RNA extraction and for histology studies respectively. The remaining liver was fixed in 10% neutral buffered formalin (NBF) for 22-24 hours before transfer to 70% ethanol for further processing. The left kidney was immediately cut in half and both halves were snap frozen on dry ice for protein/RNA extraction and for histology. The right kidney, heart, brain and lungs were fixed in 10% NBF for 22-24 hours and then changed to 70% ethanol.
Assessment of cognitive function
Y-maze test
The mice were individually placed into a testing area measuring 25.66 x 17.53cm onto a Samsung Galaxy Tab 2 10.1 for 5 minutes. Steps and distance travelled were recorded using MouseTrapp software. The Y maze arena had three arms of 40cm identified as A1 A2 A3, each with a differentiating marker at the end of the arm. Mice were assigned different start arms in a rotating allocation, and tested prior to the start of the experiment and on day 4, with differing arm allocations each time. In the T1 phase mice were placed into the maze with only two arms open for 5 minutes to explore the arena. The mice were then removed to a clean cage for 1 minute (fresh cage used per cage of mice). Then in the T2 phase mice were returned to the maze in the starting position with the novel arm opened for 2 minutes, and allowed to explore. A camera was set up above the maze to record movements and the video files analysed via Ethovision XT13 software.
Brain slice electrophysiology
Animals were killed humanely by anaesthetic overdose with inhaled isoflurane and intramuscular injection ketamine (≥100 mg/kg) and xylazine (≥10 mg/kg) as previously described31. The mice were then transcardially perfused with at least 25ml of sucrose rich artificial cerebral-spinal fluid (SaCSF) – composed of (mM) 252 sucrose, 3.0 KCl, 1.25 NaH2PO4, 24 NaHCO3, 2.0 MgSO4, 2.0 CaCl2 and 10 glucose. The brain was removed and sliced at 450mm horizontal slices with a Leica VT1000S vibratome in ice cold SaCSF. Slices were trimmed to the hippocampal region and maintained at 32-34 oC at an air liquid interface between normal aCSF (sucrose replaced with 126 mM NaCl) and humidified 95% O2/5% CO2. Oscillations were evoked with 10μM cholinergic agonist carbachol, to activate transmission through acetylcholine receptors. Extracellular recording electrodes were filled with normal aCSF (resistance 2–5 MΩ), and field recordings taken from the border between stratum radiatum and stratum lacunosum moleculare in CA3. Recordings were taken with Axoclamp-2B amplifier (Axon Instruments Inc., Union City, CA, USA) and extracellular data filtered at 0.001–0.4 kHz low-using Bessel filters. Mains noise was deducted with a Humbug (Digitimer, Welwyn Garden City, Herts, UK), and data redigitized at 10 kHz using an ITC-16 interface (Digitimer, Welwyn Garden City, UK). Axograph 4.6 software (Axon Instruments Inc., Union City, CA, USA) was used for data acquisition and analysis.
To generate power spectra Axograph used Fourier analysis using 60 seconds per 10 minute recording. This was used to calculate peak frequency and area power (area under the peak). Mouse gamma frequency oscillation was measured at frequencies between 15 and 49 Hz. Oscillations were categorized as stable when area power measured within 10% for three consecutive 10 min recording intervals.
Histology
FFPE sections, 4μm thick, were used for simple immunohistochemistry (IHC) and for multiplex immunofluorescence (IF). Sections were subjected to heat-induced antigen retrieval, followed by protein blocking to reduce non-specific staining. Incubation with primary antibody overnight at 4 oC or for 1 hour at room temperature was followed by secondary antibody incubation and signal detection. Details on the antibodies can be found in Supplemental Table 1.
Photos were taken with a Zeiss Axiovert 200 microscope using a Zeiss Axiocam MRc camera. Stained slides were scanned using a Leica Aperio AT2 slide scanner (Leica Microsystems, UK) at 20x magnification. Automated quantification of positively stained cells or area was performed using the HALO image analysis software (V3.1.1076.363, Indica Labs). Manual quantification of p21+ and BrdU+ kidney cells was performed on 20 random fields at 20x magnification. For p21 IHC quantification on brain sections, total brain area was calculated using the HALO software and the p21+ cells were manually counted in the whole brain tissue area.
For multiplex IF, 4μm tissue sections underwent heat-induced antigen retrieval (HIER) by boiling (in a waterbath) in citrate buffer (10mM Na Citrate (Sigma, W302600), 0.05% Tween 20 (Sigma, P1379), pH 6) for 25 minutes, and were subsequently cooled down for 20 minutes in the retrieval solution. Peroxidase quenching with 3% H2O2 (Sigma, 95321) was followed by biotin (Invitrogen, 4303) and protein blocking (Abcam, ab64226). Sections were incubated with the primary antibodies either for 1 hour at room temperature, or overnight at 4 oC, followed by 45 minutes with the secondary antibodies (conjugated to fluorophor) together with DAPI (1mg/ml). Sudan black B was then used to quench autofluorescence. An aqueous mounting solution (DAKO, S1964 was used for mounting.
The anti-p21 antibody required additional signal amplification which was achieved by using the tyramide signal amplification (TSA) system. Briefly, after incubation with the anti-p21 primary antibody, the sections were incubated with a secondary anti-rat biotinylated antibody for 30 minutes, followed by 30 minute incubation with an avidin-HRP complex (Vectastain ABC, Vector, PK-7100). Then, the sections were incubated with TSA FITC (Perkin Elmer, NEL741B001KT) for 6 minutes (in the dark). After that, the sections were subjected to a 5 minute HIER in order to remove the anti-p21 antibody complex, underwent protein blocking and then were incubated with the other primary antibodies as described in the previous paragraph. Images were taken using a Zeiss 710 upright confocal Z6008 microscope. The Opera Phenix scanner (Perkin Elmer) was used to scan the stained sections at 20x magnification. For the analysis of scanned sections, the Columbus software (Perkin Elmer) was used to create a custom algorithm to quantify 20 random fields of view.
In situ hybridization (ISH) was performed in an autostainer (Leica Bond Rx) using the 2.5 LSx RNAScope kit (Bio-Techne, 322700) according to the manufacturer’s instructions. Probes against Smad7 mRNA (Bio-Techne, 429418), TGFβ1 (Bio-Techne, 407758), TGFβ2 (Bio-Techne, 406188) and TGFβR1 (Bio-Techne, 431048) were used for the detection of the respective mRNA, and PPIB (Bio-Techne, 313918) as a positive control of gene expression.
Senescence-associated β-Galactosidase staining
Staining for senescence-associated β-Galactosidase (SA β-Gal) was performed as described previously32. Briefly, 10mm thick cryosections or cultured cells were fixed in 2% paraformaldehyde/0.25% glutaraldehyde in PBS for 15 or 5 minutes respectively. This was followed by three washes with PBS 1mM MgCl2 (pH 5.5 or 6 for mouse or human cells/tissues respectively) and incubation with staining solution (1mM MgCl2, 0.5mM K3Fe(CN)6 0.5mM C6FeK4N6*3H2O and 1mg/ml X-Gal in PBS, pH 5.5 or 6 for mouse or human cells/tissues respectively) overnight (liver sections, cells) or for 2.5 hours (kidney sections). After three washes with PBS, cryosections were counterstained with eosin and mounted, while cells (on coverslips) were mounted immediately. For quantification, SA β-Gal+ and SA β-Gal- cultured cells were counted in 20 random fields of view.
RNA extraction
Whole tissue RNA was extracted using the Qiagen RNeasy kit (Qiagen, 74104), including the optional DNase step, as described previously12. Briefly, 20-30mg of snap frozen tissue were homogenized in 600ml buffer RLT/1% β-mercaptoethanol using the Precellys Evolution homogenizer (Bertin Technologies) and the RNA was eluted in 30μl RNase-free water. RNA concentration was estimated with the Nanodrop 2000 and only samples with a 260/280 ratio≥2 were used for further analysis.
cDNA generation and qPCR
cDNA was generated using the QuantiTect Reverse Transcription Kit (Qiagen, 205311) according to the manufacturer’s instructions from 1μg RNA. A PTC-200 Gradient cycler (MJ Research) was used to perform the gDNA wipeout and reverse transcription steps. A sample-free reaction and a reaction without the reverse transcriptase served as negative controls. Quantitative real time PCR was performed with the SYBR Green system (Qiagen, 204145)using a QuantStudio 5 Real time PCR system in a 384 well plate setting (final reaction volume 10μl per well). All primers used were purchased from Qiagen, as shown in Supplemental Table 2. Each biological replicate (mouse) was run in triplicate and the 18S ribosomal RNA (Rn18S) was used as a house keeping gene for normalization. Relative expression was calculated using the ΔΔCt method.
Whole tissue (bulk) RNA sequencing
For bulk RNA-sequencing (RNA-seq), RNA was extracted as described above. Briefly, the RNA was tested on an Agilent 2200 TapeStation (D1000 screentape) using RNA screentape and only samples with a RIN>7 were further processed for library preparation. 20ng/ml of RNA were used to prepare libraries using the TruSeq Stranded mRNA Kit. Agilent 2200 Tapestation was used to assess library quality and Qubit (Thermo Fisher Scientific) was used to check concentration. The libraries were then run on the Illumina NextSeq 500 using the High Output 75 cycles kit (paired end, 2x36 bp cycle, dual index (I5 and I7 Illumina)).
For the bioinformatics analysis, raw data quality checks and trimming were performed using FastQC version 0.11.7, FastP and FastQ Screen version 0.12.0. The reads were aligned to the mouse genome and annotation (GRCm38.92 version) using HiSat2 version 2.1.0183. Determination and statistical analysis of expression levels was done by a combination of HTSeq version 0.9.1184, the R environment version 3.4, utilizing packages from Bioconductor and differential gene expression analysis based on the negative binomial distribution using the DESeq2 package version 1.18.1186. Pathway Analysis was preformed using MetaCore from Clarivate Analytics (https://portal.genego.com/).
Single cell RNA sequencing on kidneys
3 ΔMdm2Hep and 3 control mice were culled by CO2 inhalation, blood samples were collected by cardiac puncture and 30-40ml cold PBS were used to perfuse the circulatory system via the left heart. The 6 mice were culled, and their kidneys processed, on 2 different days. 2 mice (1 ΔMdm2Hep and 1 control) were culled together on one day and the others (2 ΔMdm2Hep and 2 control) were culled 2 months later. On each occasion, the left kidney was used for dissociation and generation of single-cell suspension, while the right kidney was fixed in 10% NBF. The renal capsule of the left kidney was removed, the kidney was cut into equally-sized pieces and was dissociated using the Multi-tissue dissociation kit 1 (Miltenyi, 130-110-201) as per the manufacturer’s instructions. 0.25g of kidney tissue were placed in a GentleMACS C tube with dissociation buffer (2.35ml serum-free RPMI culture medium (Gibco), 100μl enzyme D, 50μl enzyme R and 12.5μl enzyme A). Kidney dissociation was performed in a GentleMACS dissociator using the “heart_01_01” programme (15s). The samples were placed in a waterbath (37 oC) for 30 minutes and then placed back in the dissociator (“heart_01_02” programme, 30s).
After the second round of dissociation, 8ml of sterile PBS/10% FBS were added in the C tubes to stop the reaction. The samples were passed through a 40μm cell strainer into a 50ml falcon tube. All subsequent steps were performed on wet ice or at 4 oC. The samples were spun at 300g for 5 mins and re-suspended in 5 ml cold PBS. They were spun again at 300g for 5 mins, re-suspended in 1ml red blood cell lysis buffer (8.29g NH4Cl, 1g KHCO3 and 37.2g Na2EDTA in PBS) and incubated for 30sec on wet ice. The samples were topped with 9ml of cold PBS and washed twice, as described previously (spin at 300g for 5 mins and re-suspended in PBS). The samples were re-suspended in cold PBS, were subjected to debris removal using the debris removal solution (Miltenyi) according to the manufacturer’s instructions. Finally, the samples were re-suspend in 10ml cold PBS/10% FBS/2.5mM EDTA.
Cell viability and concentration were determined using the Trypan Blue assay and flow cytometry (viability≥90%). 20,000- 40,000 cells were loaded on a 10x Chromium chip (1 sample per lane). Cleanup, reverse transcription, cDNA amplification and library preparation were performed using the Chromium Single cell 3’ Reagent Kits (v3) as per the manufacturer’s instructions.
The samples were sequenced in the Illumina NextSeq 500 using the 2x150bp kit with the following read length parameters: 26bp Read1 - cell barcode and UMI, 8bp I7 index - sample index, 98bp Read2 - transcript read. CellRanger v.4.0 (with default parameters) was used to demultiplex Illumina BCL output files and align reads to the ensemble GRCm38.99 reference genome with the addition of the AAV8-TBG-Cre and AAV8-TBG-Null sequences.
All other steps were performed using R v.4.0 and packages from Bioconductor v.3.12. Raw matrices generated by CellRanger were transformed to SingleCellExperiment (SCE) objects and they were filtered to exclude empty droplets using the DropletUtils v.1.10 package. SCE objects were merged together to perform further downstream analysis. Following similar parameters used in a previous scRNA-seq analysis murine kidney33, poor quality cells (<75 or >3000 expressed genes or >50% mitochondrial gene expression) were filtered out and only genes expressed in more than 10 cells with at least one UMI were kept for further analysis.
The normalisation by deconvolution method designed by Lun et al.34 was used to normalise and log transform the counts with functions from Scater package v.1.18. Highly variable genes were computed with default parameters and the top 10% were used to perform principal component analysis (PCA) and Uniform Manifold Approximation and Projection (UMAP) using the top 20 PCs. After examining UMAP plots coloured by batch run, it was determined that batch correction was not required.
Initial clustering was performed with functions from the Seurat package v.4.0 with default parameters and with eps value of 0.5 and resolution sequence of 0.1 to 1 by 0.1. Markers of each cluster were identified by performing a pairwise differential analysis between each pair of clusters with a minimum difference of 20% of cells expressing the gene and log2 fold change threshold of >0.25 and only keeping the differentially expressed markers in all the comparisons. Re-clustering of control cells with a new PCA and UMAP was performed and the main markers were used to manually classify clusters into different cell types. ΔMdm2Hep cells were projected onto the reference UMAP and assigned to the identified clusters. The same methodology was followed for the identified control Proximal Tubular Cells (PTCs), Distal Tubular Cells (DTCs) and the initial cluster "Mesenchymal cells" for the projection and assignment of the ΔMdm2Hep cells. Scores for senescence, p21, proliferation, TGFβ, JAK-STAT and regeneration signatures were computed using the AddModuleScore Seurat function. The genes included in each list used to create the signatures can be found on Supplemental Table 3. All transcriptomic data will be made publicly available on GEO (GSE189726) at the time of publication.
DGE analysis was performed as described by Giustacchini et al. 201735. Briefly, log2 fold change was calculated between groups and non-parametric wilcoxon test was used to compare the expression values. Fisher’s exact test was used to compare expressing cell frequency (percentage of cells per group with at least one UMI count). P-values from both tests were combined using Fisher’s method and adjusted using Benjamini-Hochberg. Genes were considered to be differentially expressed if the p-adjusted values were < 0.05. Heatmaps comparing relative gene expression by cell groups were computed using the “DoHeatmap” function from Seurat R package or “plotGroupedHeatmap” function from the scater R package. In both cases, the scaled value from each group was calculated by substracting the average logcount gene expression from the total mean gene expression and divided by the standard deviation. A two colour range scale was used to convert scale values into colour intensity.
Protein extraction and western blotting
Snap frozen tissue was homogenized in 300μl of protein extraction buffer (50mM HEPES, 100mM NaF, 150mM NaCl, 10mM Na4P207, 10mM EDTA, 1% Triton X100, 0.1% SDS, 0.5% Na Deoxycholate in ddH2O), 1:100 Protease inhibitor (Thermo Fisher, 1862209) and 1:100 Phosphatase inhibitor (Thermo Fisher, 1862495) in ddH2O) using the Precellys Evolution homogenizer (Bertin technologies). The lysate was passed through a 25G needle 5-8 times and was then spun twice at 20800g for 10 minutes at 4 oC. The Bradford assay was used to measure protein concentration using the Coomasie Plus reagent (Thermo Fisher, 23236) on a 96-well plate setting. Absorbance was measured immediately at 596nm and a standard curve was automatically created using a Spectramax reader.
Western blotting was performed on precast gels using the XCell SureLoc Mini-Cell and XCell I Blot Module (Invitrogen, 10572913) protein samples were mixed with loading buffer (4X NuPAGE loading buffer (Invitrogen, 11549166) and 5% β-mercaptoethanol) at a concentration of 20μg/μl. The samples were heated for 5 minutes at 95 oC and then were spun for 2 minutes at 20800g. 20μl sample and 5μl of protein ladder (Biorad, 1610373) were loaded on a 4 to 12%, Bis-Tris, 1.0 mm, 10-well precast gel (Invitrogen, NP0321PK2) and the gel was run at 120V for 1 hour and 50 minutes in MOPS running buffer (Invitrogen, NP-0001). Transfer onto PVDF membranes was performed by wet transfer, using the NuPAGE transfer buffer (Invitrogen, NP-0006) for 1 hour at 30V. Transfer efficiency was assessed by Ponceau S staining. The membranes were blocked for 1 hour with 5% BSA and then incubated with the primary antibody (diluted in 5% BSA) overnight at 4 oC. This was followed by 45 minute incubation with the HRP-conjugated secondary antibody and ECL incubation for the appropriate amount of time (2 minutes for pSmad2, pSmad3, Smad2 and Smad3 and 5 seconds for the b-actin). Visualisation of the bands was performed using the Chemidoc imaging system (Biorad).
ELISA and cytokine arrays
For the detection of Cystatin C in murine plasma, the "Mouse/Rat Cystatin C" Immunoassay kit (R&D Technologies, MSCTC0) was used, according to the manufacturer’s instructions. The plate was scanned in a plate reader at 450nm (wavelength correction was set to 570nm) within 10 minutes after assay completion. Standard curve concentration calculations were performed on the "Myassays" website (https://www.myassays.com/).
Cytokine arrays on murine plasma and tissue samples were performed by Eve Technologies, using the TGFβ1, 2, 3 Magnetic bead kit for the measurement of the TGFβ ligands and the Milliplex MAP mouse cytokine magnetic bead panel (Discovery assay "Mouse Cytokine Array / Chemokine Array 31-Plex (MD31)").
Metabolomics on mouse urine
For the detection of urine amino acids, urine was collected from the mice by free urination 2 and 3 days before AAV-Cre injection, on injection day as well as 3 and 4 days post AAV-Cre injection. Urine collected by free urination after scruffing the mice was diluted 1:50 in cold metabolite extraction buffer (50% methanol, 30% acetonitrile, 20% water) and were vortexed for 30 seconds. The samples were then centrifuged at 20800g for 10 minutes at 4 oC. LC-MS was performed as described previously36.
Cell culture
Murine embryonic fibroblasts
WT Murine embryonic fibroblasts (MEFs) were isolated from one E13-E14 WT C57Bl/6 embryo. The MEFs were cultured on 10cm petri dishes (Corning) in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% FBS, 1% penicillin/streptomycin, 1% L-glutamine (cDMEM) at low O2 pressure (3%). The MEFs cultures were confirmed to be free of mycoplasma contamination. For the plasma treatments, passage (P) 3-P5 MEFs were trypsinised and cell density was determined by using the CASY cell counter (Cambridge Bioscience, 5651808). 30000 cells (in 1ml medium) were plated on 24-well plate wells (with a round coverslip in each well) and were incubated with cDMEM with 1% plasma from either ΔMdm2Hep or control mice for 24 hours. The cDMEM with plasma was changed with fresh cDMEM every 2 days and 6 days after the first media change the cells were stained for SA β-Gal as described above. The coverslips were mounted on slides and the SA β-Gal+ cells were quantified by manual counting of 20 random fields of view of using a Zeiss Axiovert 200 microscope at 20x magnification.
Induced Pluripotent Stem cell-derived neuronal cells
Cells were derived from human fetal neural crest progenitors by Peter Hanson and Chris Morris at the University of Newcastle. The cells were passaged using trypsin-EDTA solution and were seeded into a geltrex coated 48 well cell culture plate at a density of 5,000 cells per well with 250μl of proliferation media. They were allowed to proliferate for 2 days before the proliferation media was withdrawn and replaced with differentiation medium. Growth medium was replaced with differentiation medium for 16 days before beginning experimental procedure. The composition of proliferation and differentiation medium has been described previously37.
For the plasma treatment experiments, plasma samples were thawed on ice, vortexed and heat inactivated for 30 minutes at 60 oC. They were diluted in media to the desired concentration (1:100) and added to the plate for 24 hours. For the additional treatment with TGFβR1i, either TGFβR1i (0.2mM) or Vehicle (DMSO) were mixed with the plasma-containing medium and stayed on the cells for 24 hours. 2 days later the cells were stained for SA β-Gal and the positive cells were quantified as described above.
Statistical analyses and graphs
The Prism 9 Software (GraphPad Software, Inc.) was used for statistical analyses. The Shapiro-Wilk test was used to assess data normality. For normally distributed data, the One-way ANOVA, 2-way ANOVA, the Brown-Forsythe and Welch ANOVA test, unpaired t-test or the Welch’s t-test were used to test for statistical significance between data groups. The Kruskal-Wallis test or the Mann-Whitney test were performed for non-parametric data. All statistical tests comparing 2 groups were two-tailed. All figures were created using the Scribus Software (v1.4.7, G.N.U. general public licence). Unless otherwise stated, all data points on line graphs represent mean ± Standard Error of Mean (S.E.M.). In bar graphs, bars represent mean ± S.E.M and each dot represents a single mouse.