Data and code availability
Raw sequencing data from all samples has been uploaded to the Sequence Read Archive (SRA) of the National Center for Biotechnology Information (NCBI, https://submit.ncbi.nlm.nih.gov/subs/sra/). It can be accessed using the accession number PRJNA648141.
All raw NMR data has been uploaded to the Metabolights online repository (https://ebi.ac.uk/metabolights/) , using the study identifier MTBLS1833.
All other raw data, including animal data for the various rodent cohorts and bacteriological data from the culture experiment and in vitro microbiological work is available in the supplementary files.
Microbiological analysis was carried out in R using packages which are publicly available via CRAN or Github.
Analysis of NMR data analysis can be replicated using Matlab scripts in the IMPACT Toolkit developed in house at Imperial College available from https://github.com/csmsoftware/IMPaCTS.
Animal experiments were conducted in accordance with the UK Home Office Animals (Scientific Procedures) Act 1986, with local ethical committee approval. All animal work was carried out in the Biological Services Unit of Queen Mary University of London at Charterhouse Square, and complied fully with all relevant animal welfare guidance and legislation (UK Home Office Project License number PPL 70/8350).
Source of animals
All rats used in the experiment were male, outbred Wistar IGS rats obtained from Charles Rivers (Kent, UK) at 7 weeks of age. All were housed in individually ventilated cages under 12h light/dark cycles and were allowed unlimited access to water and feed (RM1 diet, or RM1 with 0.75% adenine, from Special Diet Services, Essex, UK).
All mice used in the experiment were male, wild-type C57BL/6 mice. Those used in the adenine feeding experiment were obtained from Charles Rivers at 7 weeks of age, whilst germ-free mice of the same species were obtained from a colony maintained by one of the authors (MC) at the Biological Research Facility, St George’s University of London, at 8 weeks of age. All were housed in individually ventilated cages under 12 h light/dark cycles and were allowed unlimited access to water and diet (RM1 diet or RM1 + 0.15% adenine, from Special Diet Services, Essex, UK).
Male animals were used throughout because of the potential for female reproductive hormones to influence the uremic phenotype or introduce phenotypic heterogeneity into the study population.
Chemically-induced uremia in rats
The total cohort size was 18 rats. After a week-long period of acclimatization, nine rats were started on the adenine-containing intervention diet whilst another nine were maintained on standard control diet. This diet was continued for four weeks, followed by a washout period of four weeks when all animals received the control diet, after which the animals were sacrificed. Oral swabs were taken from all animals at the point of maximal uremia for those receiving the intervention diet (at the end of the 4-week period of adenine administration). Serum samples were obtained by thoracotomy at the point of sacrifice, and other tissues were obtained as outlined below.
Surgically-induced uremia in rats
The total cohort size was 24 rats. After a week-long period of acclimatization, fourteen underwent SNx, involving exteriorisation of the left kidney with decapsulation and removal of the upper and lower poles and subsequent replacement of the middle pole only, followed by total right nephrectomy two weeks later. Ten underwent sham procedures, involving exteriorisation, decapsulation and replacement of the left kidney, followed by the same procedure on the right kidney two weeks later. Oral swabs to assess the microbiota were taken four weeks after the second stage of the surgical procedure, to parallel those taken in the chemically-induced uremia protocol. A 24-hour urine collection was performed in the week prior to sacrifice (results from this have been published elsewhere ).
Additional rats for histological and salivary analysis
Thirteen additional rats were used in order to obtain saliva samples for subsequent analysis, and to undertake bone staining to assess the bone formation rate. These rats underwent SNx or sham procedures as outlined above (n=6 sham surgery, n=7 SNx), and were otherwise looked after identically to those in the ‘Surgically induced uremia in rats’ protocol above. In their final week of life, 500µg calcein green (approximately 1mg/kg) was injected intravenously three times at exact 48h intervals. The following week induced saliva collection was carried out under terminal anesthesia with ketamine/xylocaine. After full induction of anesthesia, 1mg pilocarpine was injected into the peritoneum, with a further 1mg administered 5 minutes later if there was no salivary response. Saliva was then collected over the following 8 minutes using a 100mL pipette and 1.5ml Eppendorf tubes. Salivary volume was directly assessed by weighing the filled tubes and subtracting the weight of the tube itself. Salivary pH was directly measured using a pH meter and narrow gauge probe (Mettler Toledo, Leicester, UK), before saliva was snap frozen in liquid nitrogen and transferred to a -80o freezer until the time of analysis.
Chemically-induced uremia in mice
The total cohort size was 20 mice. After a week-long period of acclimatization, ten animals were placed on an intervention diet (RM1 with 0.15% adenine), whilst ten remained on standard RM1 diet. Oral swabs to assess changes in microbiota were obtained at age 7 weeks, 10 weeks, 14 weeks, 18 w weeks, 22 weeks and 26 weeks of age – ie, prior to starting the experimental protocol, and at 2 weeks, 6 weeks, 10 weeks, 14 weeks and 18 weeks after starting it. All mice were sacrificed at age 28 weeks after a 24-hour urine collection. Additional orals swabs were obtained prior to the time of sacrifice from four ‘donor’ animals in each group, for using in the ‘oral microbial transfer’ experiment described below. Additionally, soiled cage contents including bedding and droppings from the cages in which these donor animals were housed, were frozen for further use as described below.
Oral microbial transfer in mice
Fifteen germ-free C57BL/6 mice were transferred direct from their sterile isolator at the Biological Research Facility, St George’s University of London, to the Biological Services Unit at Charterhouse Square using a clean but non-sterile specialist animal transfer company (Impex, UK) in three separate batches (one batch of seven for receipt of microbiota from control donors, two batches of four each for receipt of microbiota from uremic donors).
On arrival, each mouse received oral microbial transfer (OMT) by oral gavage of swabs taken from donor animals as described above. Each donor swab was used to transfer into two (or in one case, one) recipient(s); seven were designated control recipients and eight, uremic recipients. Gavage was carried out by using a sterile swab thoroughly immersed in transport medium that had been frozen since the time of sampling, and agitating the swab in the mouth of the recipient mouth for 15 seconds and encouraging them to suck on it. After receiving the OMT, the mice were placed in cages containing cage contents from the cage occupied by the particular donor animals, which had been frozen at -80o until the time of use, to permit ongoing microbial transfer by coprophagy.
Animals were then maintained in ordinary individually-ventilated cages in an open area of the Biological Services Unit, with standard 12h light/dark cycles. They had unlimited access to standard RM-1 diet and tap water. Oral swabs were taken to assess the efficacy and durability of bacterial transfer at 3-weeks and 9-weeks after transfer in all animals, and all animals were then culled, after a 24-hour urine collection, at 18 weeks of age (10 weeks after transfer).
Processing of blood samples:
Animals in all experimental groups were sacrificed by lethal injection of sodium thiopentone (LINK Pharmaceuticals, Horsham, UK). Blood samples were obtained by thoracotomy and cardiac puncture, and spun down directly to isolate serum which was frozen at -80o until the time of analysis. Quantification of serum urea, creatinine, calcium and phosphate concentrations was done by IDEXX Bioresearch, Ludwigsberg, Germany. Serum parathyroid hormone concentrations were assessed using a PTH ELISA kit suitable for rat serum (RayBiotech), used according to the manufacturer’s instructions.
Measurement of alveolar bone height:
Heads were removed and jaw specimens obtained from all animals using a guillotine and sharp dissection with scissors. Alveolar bone height was measured using a morphometric method previously demonstrated to have equal reliability to radiological  and histological techniques . After any samples (typically mandibles) required for conventional histology, micro-CT or scanning electron microscopy were removed, skulls were chemically defleshed by incubation in the protease-based detergent Terg-a-zyme ® (Sigma-Aldrich, UK ), for 48 hours at 55oC, with remaining soft tissue being removed mechanically after this. One of the authors (AA) who has significant expertise in the procedure obtained photographs using a dissecting microscope and measured the distance between the cemento-enamel junction and the alveolar bone crest using ImageJ software , as outlined by Baker and colleagues , although without the use of blue dye. Bone height was measured over the lingual and buccal surfaces of molar roots, and a composite measurement for each animal was calculated. These figures are expressed relative to the average bone height in control animals, with significance assessed using Student’s t-test with Welch’s correction for unequal variances.
Tissues removed prior to defleshing were fixed in formalin and processed according to standard histological procedures, and embedded in paraffin. Each jaw was sectioned in frontal buccolingual orientation using a microtome (5 mm) and mounted on charged glass slides. Every tenth section was stained by haematoxylin and eosin using an automated slide processor, and then photographed using a Nikon Eclipse 80i Stereology microscope using 4/0.13 and 10/0.45 objective lenses.
Neutrophils and IL-17 were detected using primary antibodies (Abcam, Cambridge, UK) and anti-rabbit (PK-6101) secondary antibody (Maravai LifeSciences, San Diego, US). Sections were then viewed and photographed using the same microscope and lens as used for light microscopy of the H&E stained slides.
Scanning electron microscopy:
Samples were transported in 70% ethanol to the Dental Physical Sciences unit at the Mile End Campus, QMUL. Some samples were rendered anorganic by treatment with 7% available chlorine sodium hypochlorite bleach for 3 weeks to remove all soft tissue. This treatment completely removes the periodontal ligament so that the teeth could by removed manually to expose the surface of the alveolar bone. All SEM imaging was done using 20kV accelerating voltage and a solid state backscattered electron (BSE) detector, using a chamber pressure of 50Pa.
Confocal scanning light microscopy:
Samples in 70% ethanol from calcein-injected animals were then embedded in polymethyl methacrylate (PMMA), and blocks were cut and polished to produce flat surfaces before being used for confocal scanning light microscopy (CSLM). This was carried out at the Rockefeller Building, Division of Biosciences, University College London using a Leica SPE confocal system with an inverted microscope. The PMMA blocks were cover-slipped with glycerol. Objectives used were 10/0.45, 20/0.75 and 63/1.3 oil. Images were analysed using ImageJ software and a measure of the daily rate of dentine and bone formation calculated at the incisor root and lower mandibular border, respectively. The bone formation rate (BFR) was calculated using the formula BFR = MAR * (MS/BS) as suggested by the ASBMR Histomorphometry Nomenclature Committee , where the Mineral Apposition Rate (MAR) was calculated by dividing the distance between the innermost and outermost calcein bands (given 96h apart) by 4, and the Mineralizing Surface (MS) and Bone Surface (BS) were measured directly using ImageJ.
This was carried out on samples embedded in PMMA. Samples were scanned on the MuCAT2 micro-CT system designed and operating in the Dental Physical Sciences unit at the Mile End Campus, QMUL. The samples were scanned at 90kV & 180uA at 20 or 22um voxel size. Reconstruction was performed with GPU accelerated filtered Feldkamp backprojection algorithm and the grey-level data was calibrated to linear attenuation coefficient at 40 keV using a multi-material calibration carousel and X-ray modelling software . Quantification of bone mineral density was carried out by assessing the mean linear attenuation coefficient of 20 tagged regions with a radius of three pixels in three dimensions at each tagged location and a calibration voltage of 27.5keV.
Collection of samples for analysis of oral microbiota:
Oral swabs were taken from animals at the timepoints described above by agitating sterile cotton swabs against the molars of rats or mice being held in the scruff position for a period of 30 seconds. Swabs were then placed into 100µl transport medium and transferred directly to the laboratory, where they were vortexed for 30 seconds to mobilize cells and 30µl was removed for culture. The remaining transport medium and swab was frozen at -80oC for subsequent DNA extraction.
Transport medium withdrawn for culture was serially diluted and spread onto blood agar plates containing 5% defibrinated horse blood (TCS Biosciences, UK) before being incubated under both aerobic and anaerobic conditions (80% N2, 10% H2 and 10% CO2) for 48 hours at 37oC. After this, colonies were counted according to morphology and grown to purity on new blood agar plates. DNA was extracted using the GenElute Bacterial Genomic DNA extraction kit (Sigma Aldrich, UK). PCR products were cleaned up using the NucleoSpin® Gel and PCR clean-up kit (Machery-Nagel, Germany), and then identified using Sanger sequencing of the whole 16S rRNA gene (Eurofins Scientific, Luxembourg), using the widely-used 27F-1492R primer pair, which have been used previously by our group to identify cultured oral microbes [31, 48]. Consensus sequences of forward and reverse reads were assembled using the BioEdit Sequence Alignment Editor , and full length 16S rRNA gene sequences were assembled from forward and reverse reads using the CAP3 Contig Assembly Programme  available online via the Pôle Rhône-Alpes de Bioinformatique Site (http://doua.prabi.fr/software/cap3). All consensus sequences were >1400 base pairs in length and the mean length was 1456bp.
Additional in vitro bacterial work
In vitro assessment of urease activity and tolerance of variable urea concentrations were assessed for all bacterial isolates after they were grown to purity on 5% blood agar plates under standard aerobic or anaerobic conditions.
Urease activity was assessed in all isolates by culturing under either aerobic or anaerobic conditions on Christensen’s urea agar (Sigma-Aldrich) at 37oC. A positive urease result was recorded if there was a color change to purple, and the sample was re-grown if there was no discernable growth on the top of the agar.
Two broths were used in order to assess bacterial growth at different concentrations of urea: Iso-sensitest broth (ThermoFisher Scientific) and Brain-Heart Infusion (BHI) broth (SigmaAldrich). The BHI broth was used for some samples after they could not be grown after several attempts in Iso-sensitest broth. One isolate (eventually identified as Haemophilus parainfluenzae) did not grow in either broth and after researching its specific growth requirements in the published literature, eventually grew well after filter-sterilized hemin and nicotinamide adenine dinucleotide were added to the growth medium.
Preparations of both broths were prepared at variably stronger concentrations than the manufacturer’s instructions would suggest so that when diluted with different concentrations of filter-sterilized 60% urea solution, broths with eventual concentrations of 0%, 4%, 8%, 12%, 18% and 24% urea ensued.
Bacteria grown to purity on blood agar were then transferred into 2ml sterile phosphate buffered saline (PBS). A 1ml aliquot was assessed using a spectrophotometer at 600nm and the remainder of the bacterial solution further diluted with sterile PBS to achieve a standard turbidity of 0.5 McFarland units, equating to a concentration of bacteria of 1.5x108 colony forming units/ml (cfu/ml). These solutions were further diluted 50-fold to achieve an approximate concentration of 3x106 cfu/ml, and then 34µl of this bacterial preparation were added to 200µl of varying concentrations of urea broth in a 96-well plate, to achieve 234µl incubations each containing approximately 5x105cfu bacteria in eventual urea concentrations of 0%, 3.3%, 6.6%, 10%, 15% and 20%.
These plates were then incubated at 37o in either aerobic or anaerobic conditions for 24 hours before being read on a plate reader at 620nm. The mean inhibitory concentration was defined for each organism as the urea concentration at which the optical density of the solution was decreased to less than 10% of the difference between 0% urea and control (non-inoculated) wells. One isolate did not achieve sufficient growth to allow calculation of MIC.
DNA extraction and PCR for next-generation sequencing:
The remaining transport solution not used for culture analysis, along with the swab, was transferred to bead beating tubes from the DNeasy PowerSoil kit from QIAGEN, used according to the manufacturer’s instructions including an 8-minute bead-beating step using the FastPrep-24™ homogenizer (MP Biomedicals). All samples were processed using the same kit, and negative ‘kitome’ control samples were included for each extraction kit used (Salter et al., 2014). PCR was carried out using barcoded 27F/338R primer pairs, targeting the V1/V2 hypervariable region of the 16S rRNA gene. PCR was carried out in a sterile 96-well plate using Phusion Green Hot Start II High Fidelity PCR Master Mix (ThermoFisher Scientific), using an initial denaturation step for 5 mins at 98°C followed by 25 cycles of 98°C for 10s, 53°C for 30s, 72°C for 45s and a final extension of 72°C for 10 min.
Next generation sequencing:
Normalisation of DNA concentrations was carried out using SequalPrep™ Normalisation Plates (ThermoFisher) and DNA was quantified using either the Quant-iT® PicoGreen™ dsDNA Quantitation Kit (ThermoFisher Scientific) or a Qubit® 4 Fluorometer (also ThermoFisher). The samples were pooled and sequenced in two runs; one at the Barts & the London Genome Centre, QMUL; and one in the DNA Sequencing Facility, in the Department of Biochemistry at the University of Cambridge; each using an Illumina MiSeq 2 x 250 flow cell for paired-end sequencing.
Quantification of salivary urea:
A colorimetric detection kit for urea nitrogen (ThermoFisher Scientific) was used according to the manufacturer’s instructions. Samples of saliva were processed at 1:2 and 1:20 dilutions and the mean concentration using both dilutions in duplicate was accepted. Corresponding serum samples were analysed using the same kit but at 1:20 and 1:40 dilutions to allow comparison.
NMR spectroscopy of saliva:
Saliva samples were diluted with buffer containing trimethylsilylpropanoic acid (TSP) and analysed on an NMR spectrometer (Bruker) operating at 600.22 MHz 1H frequency at Imperial College London.
Statistical analysis of bone height data:
All data for loss of periodontal bone height was found to be normally distributed when assessed by the Shapiro-Wilk test. All testing for significance of difference between two groups was carried out using Student’s t-test with Welch’s correction for unequal variances, in GraphPad Prism or Microsoft Excel.
Analysis of effect of housing on microbiology and bone height:
Two-way ANOVA was carried out in GraphPad Prism to define the significance of the different levels of bone loss (dependent variable), according to both housing and treatment class (independent variables) in the surgically-induced uremia experiment. No comparable analysis was carried out for the chemically-induced uremia protocol because it was impossible to vary the housing since all animals in a single cage received the same diet.
Analysis of urea tolerance:
Linear regression was used to draw a line of best fit between the mean inhibitory concentration of urea and the relative competitiveness of different isolates in control vs uremic animals presented in Fig 4B; standard settings in GraphPad Prism were used to accomplish this and Prism software was used to calculate the slope and the significance of its gradient.
Identification of cultured bacterial isolates:
Isolates were identified by comparing their 16S rRNA gene sequences with reference datasets using both the NCBI Nucleotide BLAST database (https://blast.ncbi.nlm.nih.gov/Blast.cgi), and the Ribosomal Database Project (RDP)  online search tool (https://rdp.cme.msu.edu/index.jsp). In many cases these tools agreed on a species level identification for the isolate, but in some cases agreement between the two was only at higher taxonomic levels (such as in the case of different species of Streptococcus or Enterobacteriaceae). Thus, for all isolates, full-length reference 16S rRNA gene sequences for all species within the genus identified by BLAST and RDP search were downloaded from the RDP Hierarchy Browser. These reference sequences were aligned with the sequences from our research isolates, trimmed to a uniform length and used to construct a maximum likelihood tree, using MEGA  version 7. Pairwise distances between all isolates within a particular genus and all references sequences within that genus were calculated and used to generate a distance matrix.
Species level identification was determined when possible at >98.5% sequence identity. Isolates that failed to obtain any match at this level were treated as potential novel species. One was a Streptococcus species (4 isolates) with a closest proximity to S. danieliae at 97.33%, and another was a Pasteurella species (closes match being P. pneumotropica at 94.7%).
Analysis of cultured microbiota data:
Once assigned a species identity, the abundance of each isolate (log10 of colony forming units/ml) was carried out using Microsoft Excel and GraphPad Prism, using the Student t-test with Welch’s correction to assess difference between growth in uremic animals and growth in controls. Comparisons were made at species level and then aggregated to allow comparisons at higher taxonomic levels.
Handling of isolates with regard to statistical analysis of in vitro culture data
For the purposes of in vitro microbiological work (urease testing and calculation of the mean inhibitory concentration of urea), where more than one isolate was assigned to a particular species identity, differences in in vitro characteristics were resolved for subsequent analysis by treating all isolates assigned to one species as urease positive if one isolate of that species was urease positive, and all isolates within a single species identification as possessing the highest urea tolerance of any isolate in that species.
Processing of 16S sequencing data:
Sequence data were processed using the DADA2 pipeline  in R, according to the author’s recommended protocol (available from https://benjjneb.github.io/dada2/tutorial_1_6.html), adjusting filter parameters to achieve maximum quality scores whilst achieving sufficient overlap between forward and reverse read. Sequences were aligned against Silva v128  in order to assign taxonomy.
Raw abundance data of sequence variants were used with taxonomic assignments and sample metadata to create phyloseq  objects. Phylogenetic trees were generated using MEGA v7.0, and rooted to a random node using the R package phytools . A pseudocount of 0.001 was added to all OTU abundances to avoid calculating log-ratios involving zeros, and then data was then made compositional through isometric log-ratio transformation using the R package philr . Ordination was carried out using the ‘ordinate’ function in Phyloseq, based on Euclidean distances in philr space. Permutational analysis of variance (PERMANOVA) and the PERMDISP test for homogeneity of variance (as proposed by Anderson) , were carried out using the R package vegan . Alpha diversity was assessed using Phyloseq. Compositional analysis of the microbiota at six taxonomic levels was based on isometric log-ratio transformation of raw sequence abundances and adjusted for multiple testing using the Benjamini-Hochberg method, carried out using the ANCOM statistical framework  in R, with code obtained from the author’s webpage: https://sites.google.com/site/siddharthamandal1985/research.
Analysis of NMR spectral data:
NMR spectral profiles were digitized and imported into Matlab (Mathworks). The peaks for water, urea and TSP were excised from the raw NMR spectra which were then aligned to adjust for variation in peak shift due to pH differences. Further normalization was carried out using the Probabilistic Quotient method between samples in order to ensure comparable baselines between samples.
Positive identification of eight metabolites achieved by identifying their spectral profiles and confirming this using Chenomx NMR Suite 8.3 evaluation version (Chenomx, Edmonton, Canada), and peak integrals were calculated from metabolite peaks. Comparisons between these integrals were used to calculate differences in relative abundance using Microsoft Excel, with the Student’s t-test and Welch’s correction used to assess significance.
Preparation of figures:
In order to achieve uniformity, all figures were generated using GraphPad Prism 7 (GraphPad Software Inc, San Diego, California).