DOI: 10.5061/dryad.zcrjdfnps
title: Data from: Cardiac dysfunction in C57Bl/6J mice with chronic kidney disease shows sex-specific effects: Comparison of dietary adenine and 5/6 nephrectomy models

description: Background Cardiovascular disease (CVD) is the leading cause of death
 among patients with chronic kidney disease (CKD). Rodent models are widely
 used to study uremic CVD pathophysiology. We compared cardiac function
 parameters in male and female animals from 2 established mouse CKD models
 and examined associations between gut-derived uremic toxins and
 echocardiogram findings. Methods Male and female adult C57Bl/6J mice were
 randomly assigned to control, adenine-induced CKD and 5/6 nephrectomy CKD
 groups. Echocardiography was performed on all mice at age 17 weeks (5
 weeks after CKD induction). Serum creatinine, cystatin C and gut-derived
 uremic toxins were analyzed at study termination, and RNA sequencing of
 left ventricle tissue was performed and analyzed. Results Markers of
 kidney dysfunction were elevated in both CKD models. The gut-derived
 uremic toxin indoxyl sulfate (IS) was increased in both CKD models, while
 trimethylamine N-oxide (TMAO) was increased in adenine CKD mice and
 p-cresyl sulfate (pCS) in nephrectomy animals. Left ventricular volume was
 increased in nephrectomy animals. Cardiac output (CO) was decreased in
 male CKD animals from both models compared to controls, and ejection
 fraction (EF) was decreased in male 5/6 nephrectomy mice. Female controls
 had lower stroke volume (SV) and CO than male counterparts. No significant
 changes in CO or EF were observed in female CKD groups. Higher serum pCS
 and IS positively correlated with increased left ventricular volume;
 higher pCS inversely correlated with EF in females but not males.
 Transcriptomics of cardiac tissue revealed sex-based variations in matrix
 metalloproteinase and mitochondrial pathways associated with cardiac
 dysfunction. Conclusions Our work highlights sex differences in cardiac
 function and serum chemistries in two established preclinical CKD models.
 Gut-derived uremic toxins may significantly impact cardiorenal
 pathophysiology in female but not male CKD animals.
description: <strong>Animal models</strong> All
 animal studies were approved by the Animal Research Committee, University
 of California, Irvine (UCI) under protocol AUP-21-157. Animals were
 maintained at the UCI vivarium in accordance with the policies instituted
 by the American Association for Accreditation of Laboratory Animal Care.
 Male and female C57Bl/6J mice aged between 8-10 weeks were purchased from
 the Jackson Laboratory (Bar Harbor, ME) and were randomly assigned to
 control vs adenine-induced CKD. Mice in adenine groups were fed a 0.2%
 adenine diet for 18 days, followed by 2 weeks on regular chow and then a
 second 1-week period on adenine diet to maintain CKD . Age-matched male
 and female 5/6 nephrectomized mice were purchased from the Jackson
 Laboratory. The initiation timepoint of uremia was considered to be after
 18 days of adenine diet (adenine-CKD model) or 14 days post-surgery (5/6
 nephrectomy model). Echocardiography was performed at 5 weeks after uremia
 induction at age 18 weeks (n=8 per group). Tail blood pressure was
 measured prior to study termination via tail-cuff plethysmography which
 included 10 habituation cycles followed by 10 measurements, with automated
 exclusion of outlier values (CODA-S2 multi-channel, Kent Scientific). Body
 weight was monitored weekly to assess the overall health of the mice and
 to track the progression of kidney disease, and body weight at termination
 is presented in Table 1. At study termination, blood was collected via
 cardiac puncture and processed for serum collection. Heart tissue was
 collected for RNA analysis.
 <strong>Echocardiography</strong>
 Echocardiography was performed at 5 weeks after uremia induction,
 at age 18 weeks . After anesthesia induction with isoflurane, animals were
 maintained under inhaled 1.5% isoflurane and 95% O<sub>2</sub>
 to allow for spontaneous breathing. Animals were imaged in a left lateral
 decubitus position and warming pads were used to maintain normothermia.
 Images of the horizontal long-axis view and short-axis view of the heart
 were collected in both M-mode and B-mode [19] using a Vevo 3100 ultrasound
 system with 25-mHz scan head (VisualSonics, Toronto, ON, Canada). The “LV
 trace” feature was used to calculate LV structural and functional
 parameters from the LV parasternal short-axis M-mode view, which was
 recorded at the level of two papillary muscles. LV posterior wall
 thickness during diastole was measured on B-mode long-axis view. Vevo
 analysis software (VevoLab, VisualSonics) was used to conduct all
 measurements and calculations. Vevo software analysis was performed by a
 research team member blinded to study groups (H.W.).
 <strong>Serum chemistries</strong>
 Serum creatinine collected at study termination was analyzed via
 liquid chromatography-mass spectrometry at the O’Brien Center, University
 of Alabama at Birmingham (Birmingham, AL). Cystatin C was measured using
 the Invitrogen Cystatin C (CST3) Mouse ELISA Kit (EMCST3, Fisher
 Scientific). Total-form serum levels of the gut-derived uremic toxins
 p-cresyl sulfate, indoxyl sulfate and TMAO were measured using liquid
 chromatography with tandem mass spectrometry (HPLC-MS/MS) based on
 previously described methods. A 25 mL aliquot of serum was treated with
 200 uL of acetonitrile with 0.1% formic acid and internal standard for
 protein precipitation. The mixture was vortexed and centrifuged, and
 evaporated to dryness. The dried extract was reconstituted with 100 uL of
 25% acetonitrile. Standards and prepared samples were injected (10 ml)
 into the HPLC-MS/MS instrument, Waters Quattro Premier XE equipped with
 UPLC. For indoxyl sulfate and p-cresyl sulfate, hydrochlorothiazide was
 used as the internal standard and analysis was performed in negative
 ionization mode. For TMAO, salbutamol was used as the internal standard
 and analysis was performed in positive ionization mode.
 <strong>RNA sequencing</strong>
 Total RNA was isolated from left ventricular heart tissue using
 TRIzol® reagent (Life Technologies, Carlsbad, CA, USA), followed by an
 additional DNase I digestion to remove genomic DNA contamination. RNA
 samples were submitted to the UC Irvine Genomics Research and Technology
 Hub (GRTH) for sequencing with the NovaSeq 6000 platform S4 (Illumina, San
 Diego CA). RNA quality was verified using the Agilent Bioanalyzer Nano RNA
 chip (Agilent Technologies, Santa Clara CA) and Nanodrop (Thermo Fisher
 Scientific, Waltham MA). Samplesin the current study showed A260/A280
 absorbance ratios in the range 1.928-2.236. Library construction was
 performed according to the Illumina TruSeq mRNA stranded protocol using
 the Apollo 324 library prep system (Takara Bio, Mountain View CA). The
 input quantity for total RNA was 1µg and mRNA was enriched using oligo-dT
 magnetic beads. The enriched mRNA was chemically fragmented for three
 minutes. First-strand synthesis used random primers and reverse
 transcriptase to make cDNA. After second-strand synthesis, the cDNA was
 cleaned using AMPure XP beads, followed by end repair and 3’ adenylation.
 Illumina dual-indexed adapters were ligated on the ends and the
 adapter-ligated fragments were enriched by nine cycles of PCR. The
 resulting libraries were validated by qPCR (KAPA Library quantification
 kit, Kapa Biosystems, Wilmington MA) and sized with the Agilent
 Bioanalyzer DNA high-sensitivity chip. Library concentrations were
 normalized and then multiplexed together. The multiplexed libraries were
 sequenced using paired-end with read length 150 base pairs. Fastq files
 obtained from sequencing were inspected via FastQC then aligned to the
 current mouse genome build (mm10) using STAR. Counts matrices were then
 generated from aligned BAM files using summarizeOverlaps (Genomicfeatures)
 and differential expression was performed using DESeq2 with false
 discovery rate (FDR) adjusted P-value &lt;0.001. All plots were
 generated in R. Pathways derived from differentially expressed genes
 (DEGs) were identified using Gene Set Enrichment Analyses (GSEA) based on
 erichR focusing queries on Reactome, Gene Ontology and KEGG
 databases. <strong>Statistical
 analysis</strong> Data are presented as the mean
 ± standard error of the mean (SEM). Outliers were excluded using the ROUT
 test with Q=1%. Two-way analysis of variance (ANOVA) with Tukey’s multiple
 comparisons test were performed using GraphPad Prism 8.4 (GraphPad
 Software, San Diego, CA, USA). Šídák's multiple comparisons test was
 used to determine sex differences. Blood chemistries did not follow a
 Gaussian distribution per the Kolmogorov-Smirnov normality test, therefore
 nonparametric Spearman correlation analysis was used to analyze
 associations between toxin levels and echocardiographic parameters. Linear
 regression analysis was computed including regression equation, slope
 p-values, and R<sup>2</sup>. P-values &lt;0.05 were
 considered statistically significant.
description: # Data from: Cardiac dysfunction in C57Bl/6J mice with chronic kidney
 disease shows sex-specific effects: Comparison of dietary adenine and 5/6
 nephrectomy models
 [https://doi.org/10.5061/dryad.zcrjdfnps](https://doi.org/10.5061/dryad.zcrjdfnps) ## Description of the data and file structure Male and female adult C57Bl/6J mice were randomly assigned to control, adenine-induced CKD, and 5/6 nephrectomy CKD groups. Echocardiography was performed on all mice at age 17 weeks (5 weeks after CKD induction). Serum creatinine, cystatin C, and gut-derived uremic toxins were analyzed at study termination, and RNA sequencing of left ventricle tissue was performed and analyzed. Fastq files obtained from sequencing were inspected via FastQC then aligned to the current mouse genome build (mm10) using STAR. Counts matrices were then generated from aligned BAM files using summarizeOverlaps (Genomicfeatures) and differential expression was performed using DESeq2 with false discovery rate (FDR) adjusted P-value &lt;0.001. All plots were generated in R. Pathways derived from differentially expressed genes (DEGs) were identified using Gene Set Enrichment Analyses (GSEA) based on erichR focusing queries on Reactome, Gene Ontology, and KEGG databases. Files listed: 1. MASTERFILE_echo_mice_10.2025_Dryad.csv Contains all raw data collected in this study, including raw and adjusted serum creatinine, cystatin C, levels of uremic toxins, parameters tested in cardiac echocardiogram including: * heart rate (BPM). * diameter during systolic and diastolic (mm), * volumes during systolic and diastolic (uL), * stroke volume (uL), ejection fraction (%), * fractional shortening (%), * cardiac output (ml/min), * blood pressure during systolic and diastolic (mmHg). 2. F_full_pathyway_enrichment_file_for_CKD_over_CTL_Dryad_10.2025.csv Contains data collected for pathway enrichment ***in females*** comparing CKD over CTL based on RNA sequencing. Variables listed incluing: * ontology, * GO ID, * Description of pathways involved, * set size, * enrichment score, * NES value, * p value, * adjusted p value, * q value, * rank, * leading edge, * core enrichment list. 3. M_full_pathway_enrichment_file_for_CKD_over_CTL_Dryad_10.2025.csv Contains data collected for pathway enrichment ***in males*** comparing CKD over CTL based on RNA sequencing. Variables listed include * Ontology, * GO ID, * Description of pathways involved, * set size, * enrichment score, * NES value, * p value, * adjusted p value, * q values, * rank, * leading edge, * core enrichment list. 4. FDESeq2_full_results_CKD_over_CTL_Dryad_10.2025.csv Contains data collected ***in female mice*** for RNA sequencing, including * Gene name, * base mean, * log2 fold change, * lfcSE, * stat, * p values * adjusted p values.  5. M_DESeq2_full_results_CKD_over_CTL_Dryad_10.2025.csv Contains data collected ***in male mice*** for RNA sequencing, including * Gene name, * base mean, * log2 fold change, * lfcSE, * stat, * p values * adjusted p values.  6. gene_intersection_from_study.pdf (Zenodo) Contains figures presenting up-regulated (red) and down-regulated (blue) gene intersection between male and female mice.