Intestinal dysbiosis and gut-derived toxins in chronic kidney diseases (CKD) are associated with vascular injury. This study examined the relationship between gut dysbiosis and cerebral microhemorrhages (CMH) in young and aged CKD mice (3 vs 16 months of age) in both sexes. CKD was induced in C57BL/6J mice using a nephrotoxic adenine diet. Serum creatinine, trimethylamine N-oxide (TMAO), indoxyl sulfate (IS), and p-cresyl sulfate (pCS) were measured. CMH was quantified via brain histology, and gut microbial sequencing was analyzed from fecal pellets. Creatinine and uremic toxins were elevated in both young and aged CKD mice compared to controls, and microbial populations were altered by age, sex, and CKD status. Age was the most significant factor in microbial variance, with higher levels of IS and pCS in aged CKD mice. Aged male mice had significantly higher creatinine, TMAO, and IS than aged females. Males had higher CMH counts than females, and aged CKD males had the highest CMH burden. Age modified the relationship between uremic toxins and CMH burden, with creatinine, TMAO, and IS correlating with increased CMH in aged animals. In conclusion, gut dysbiosis in CKD is modulated by sex and age, and gut-derived uremic toxins including TMAO and IS may contribute to vascular injury and CMH development.

Animal model

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. Tubulointerstitial nephritis was induced via nephrotoxic adenine diet in adult C57BL/6J mice (Figure 5). Young mice (10-12 weeks old, Jackson Laboratories, Bar Harbor, ME)) and aged mice (16 months old, National Institute on Aging aged rodent colony) were fed a diet containing 0.2% adenine for 18 days (Dyet# 611732, Dyets Inc., Bethlehem, PA), placed back on regular chow for 2 weeks, and then re-exposed to adenine diet for 1 week to maintain CKD 11,13. Control (CTL) age-matched animals were maintained on regular feed for the duration of the experiment. Studies were done in both male and female animals.

Tissue collection

At study termination, the chest cavity was opened, and blood was collected via cardiac puncture under inhaled isoflurane anesthesia and centrifuged for serum collection (BD microtainer with clot activator, catalog# 2675185, Thermo Fisher Scientific). Perfusion was done for 5 min with ice-cold phosphate-buffered saline (PBS) at a flow rate of 7-8 mL/min via a 26-gauge needle in the left ventricle, followed by brain collection. Fecal pellets were collected from the descending colon, snap frozen and stored at -80°C for gut microbiota analysis.

Stool microbiome sequencing

Two stool pellets per animal were transported in 500 μL of RNA/DNA Shield™ (Catalog# R1101, (Zymo Research, Irvine, CA) to the Zymo Research laboratory (Irvine, CA). Extraction of microbial DNA was performed with a bead-beating step using the ZymoBIOMICS®-96 MagBead DNA Kit. DNA samples were prepared for targeted sequencing with the Quick-16STM NGS Library Prep Kit with custom designed Primer set V3-V4 (Zymo Research). Control Samples: The ZymoBIOMICS® Microbial Community Standard was used as a positive control for each DNA extraction, and a negative control (i.e., blank extraction control) was included to assess the level of bioburden carried by the wet-lab process. The sequencing library PCR products were quantified with qPCR fluorescence readings, pooled based on equal molarity, and cleaned up with the Select-a-Size DNA Clean & ConcentratorTM (Zymo Research), then quantified with TapeStationÒ (Agilent Technologies, Santa Clara, CA) and QubitÒ (Thermo Fisher Scientific, Waltham, WA). The final library was sequenced on IlluminaÒ MiSeqTM with a v3 reagent kit (600 cycles). The sequencing was performed in 10% PhiX spike-in. At the UCI Microbiome Center, the sequence data were quality checked and demultiplexed in QIIME 2 29 with rarefaction at 4300 sequences/sample, and was clustered into 100% Operational Taxonomic Units (OTUs, a.k.a. Exact Sequence Variants, ESVs) and analyzed for alpha- and beta-diversity metrics within the Vegan package in R  30. 

Quantification of serum uremic toxins

Serum creatinine was measured using capillary electrophoresis at the O’Brien Kidney Research Core Center (UT Southwestern, Dallas, TX). Serum levels of total (free and protein-bound) gut-derived uremic toxins were quantified using Xevo Tandem Quadrupole Mass Spectrometer (TQ Abs-MS instrument, Waters Quattro Premier XE equipped with UPLC). A 20 μL aliquot of serum was treated with 200 μL of acetonitrile with 0.1% formic acid and internal standards (hydrochlorothiazide 2 μg/mL and salbutamol 5 ng/mL) for protein precipitation. The mixture was vortexed and centrifuged, and the supernatant was evaporated to dryness. The dried extract was reconstituted with 100 µL of 25% acetonitrile.** **Standards and prepared samples were injected (10 μL) into the TQ Abs-MS instrument. For indoxyl sulfate (catalog# I3875, Sigma) and p-cresyl sulfate (p-tolyl sulfate, catalog# P2091, Fisher Scientific), the internal standard used was hydrochlorothiazide (catalog# 5001437615, Fisher Scientific) and analysis was performed in negative ionization mode with buffer A2 (10 mM ammonium formate+0.05% formic acid) and B2 (100% methanol). For TMAO (catalog# 317594, Sigma), the internal standard used was salbutamol (catalog# S8260, Sigma) and analysis was performed in positive ionization mode. Standard curves were generated in 25% acetonitrile with hydrochlorothiazide 4 g/mL and salbutamol 10 ng/mL (highest standard 10,000 ng/mL). The buffer of the positive mode is A1 (0.2 % acetic acid, 2 % acetonitrile in water), and B1 (100% acetonitrile + 0.2 % acetic acid). The transition (m/z) values are indoxyl sulfate 211.97→80.36, p-cresyl sulfate 186.94→107.30, TMAO 76→59, hydrochlorothiazide 296.96→270.08, salbutamol 240→148.

Brain microhemorrhage histology

One brain hemisphere was sectioned into 20-μm coronal sections. Sections were collected every 200 um and stained with Prussian blue to detect hemosiderin (a marker of iron deposition within CMH) 31-33. Nuclear fast red was used as a counterstain. Digitized images were analyzed using NIH ImageJ software by an observer blinded to the experimental groups. For each mouse, CMH number and total CMH surface area (mm2) were normalized to total brain surface area (cm2). Average CMH size (mm2) was calculated per animal.

Statistical analysis

Data were analyzed using GraphPad Prism 9 (GraphPad Software, Lo Jolla, CA) and R version 4.4.2 (R Core Team, Vienna, Austria). ROUT with Q=1% was used to exclude outliers. Results are presented as mean ± SEM. Normality tests were performed using D’Agostino-Pearson. Šídák's multiple comparisons test was used to determine age, sex and CKD status effects via 3-way ANOVA. Correlation between continuous variables was determined using Spearman’s correlation coefficient (r). Gut microbial alpha- and beta-diversity metrics were analyzed within the Vegan package in R (Oksanen). Shannon diversity of gut microbiota was analyzed using a linear mixed effects model (nlme package in R) after accounting for cage housing (adjustment for animals housed in the same cage). P<0.05 was considered statistically significant. Beta diversity significance was tested using PERMANOVA with adonis as part of the vegan package in R. To check for dispersion differences, beta disper was used as a part of the vegan package in R.