Chronic Kidney Disease Increases Cerebral Microbleeds in Mouse And Man

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Wei Ling Lau1,2 & Ane C. F. Nunes 1 & Vitaly Vasilevko3 & David Floriolli4 & Long Lertpanit 1 & Javad Savoj 1 & Maria Bangash 1 & Zhihui Yao 1,5 & Krunal Shah6 & Sameen Naqvi 1 & Annlia Paganini-Hill6 & Nosratola D. Vaziri 1 & David H Cribbs3 & Mark Fisher6,7

Received: 25 August 2018 /Revised: 28 January 2019 /Accepted: 22 February 2019 /Published online: 4 May 2019

# The Author(s) 2019

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Abstract

Brain microbleeds are increased in chronic kidney disease (CKD) and their presence increases the risk of cognitive decline and stroke. We examined the interaction between CKD and brain microhemorrhages (the neuropathological substrate of microbleeds) in mouse and cell culture models and studied the progression of microbleed burden on serial brain imaging from humans. Mouse studies: Two CKD models were investigated: adenine-induced tubulointerstitial nephritis and surgical 5/6 nephrectomy. Cell culture studies: bEnd.3 mouse brain endothelial cells were grown to confluence, and monolayer integrity was measured after exposure to 5– 15% human uremic serum or increasing concentrations of urea. Human studies: Progression of brain microbleeds was evaluated on serial MRI from control, pre-dialysis CKD, and dialysis patients. Microhemorrhages were increased 2–2.5-fold in mice with CKD independent of higher blood pressure in the 5/6 nephrectomy model. IgG staining was increased in CKD animals, consistent with increased blood-brain barrier permeability. Incubation of bEnd.3 cells with uremic serum or elevated urea produced a dose-dependent drop in trans-endothelial electrical resistance. Elevated urea induced actin cytoskeleton derangements and decreased claudin-5 expression. In human subjects, the prevalence of microbleeds was 50% in both CKD cohorts compared with 10% in age-matched controls. More patients in the dialysis cohort had increased microbleeds on follow-up MRI after 1.5 years. CKD disrupts the blood-brain barrier and increases brain microhemorrhages in mice and microbleeds in humans. Elevated urea alters the actin cytoskeleton and tight junction proteins in cultured endothelial cells, suggesting that this mechanism explains (at least in part) the microhemorrhages and microbleeds observed in animal and human studies.

Keywords Chronic kidney disease. Microbleeds. Mouse model. Endothelial cell culture. BrainMRI

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Introduction

One of the most significant stroke neurology discoveries in recent years is the emergence of chronic kidney disease to have an impact that goes well beyond traditional risk factors such as hypertension and diabetes [10, 11]. Given the high prevalence (about 50%) of cerebral microbleeds and cognitive impairment in patients with advanced CKD [2–4, 6, 7], this relationship deserves further study.

Cerebral microbleeds are small foci of hemosiderin–iron demonstrable on magnetic resonance imaging (MRI), believed to reflect underlying cerebral microhemorrhages [12, 13], and indicative of heightened risk for stroke, both hemorrhagic and ischemic [9, 14]. Specific MRI sequences (gradient echo and susceptibility-weighted imaging) demonstrate these focal areas of signal loss in brain parenchyma measuring ≤ 10 mm [12, 15]. Cerebral microbleeds are age-dependent, with prevalence approaching 20% by age 65 [16]. In addition to age, hypertension and cerebral amyloid angiopathy are the best-described risk factors for the development of microbleeds [13, 17]. In late-stage CKD, microbleeds are present in up to 50% of the population [2–4].

Concurrently, cognitive impairment is both more prevalent and more severe at lower levels of kidney function [18], reaching a prevalence of 30–70% in chronic dialysis patients [6, 7]. In a cross-sectional analysis of 338 hemodialysis patients aged 55 years and older with age-matched controls, 34% of dialysis patients had severe cognitive impairment compared with 12% of controls [6]. Another 35% of the dialysis cohort had moderate cognitive impairment [6].

Several studies in non-CKD cohorts have demonstrated a strong association between cerebral microbleeds and declining cognitive function [19–22]. Epidemiologic data support the co-existence of MRI microbleed burden and cognitive dysfunction in end-stage renal disease (ESRD) patients [8, 23]. Moreover, a recent report of 28 chronic dialysis patients with serial brain MRI showed an association between new microbleeds and a decline in mini-mental state examination (MMSE) score [4]. Further, ESRD patients have a 3- to 4-fold higher incidence rate of both ischemic and hemorrhagic strokes compared with the general population [5]. In a cohort of Japanese hemodialysis patients who were stroke-free at baseline, the presence of cerebral microbleeds was an independent predictor of intracerebral hemorrhage during a 5-year follow-up period [9].

Here we report results from studies in mice and in CKD patients. We found increased brain microhemorrhages in CKD mice and describe impaired endothelial tight junction and ac- tin cytoskeleton disruption as potential mechanisms for microhemorrhage formation in the CKD milieu. Retrospective analysis of serial brain MRI from non-CKD, pre-dialysis, and chronic hemodialysis subjects confirmed ESRD as a significant risk factor for the progression of microbleed burden.

Methods

Mice Experiments

Experimental Animals and Treatment Groups

Two CKD mouse models were investigated. (1) Adenine tubulointerstitial nephritis model: Male C57BL/6J mice from Jackson Laboratories (Bar Harbor, ME) aged 10– 12 weeks were fed a diet containing 0.2% adenine for 18 days to induce chronic interstitial nephropathy, placed back on regular chow for 2 weeks, and then re-exposed to adenine diet for 1 week to maintain CKD (Fig. 1a). Control mice were maintained on regular chow. (2) 5/6 nephrectomy model: Male C57BL/6J mice aged 10 weeks underwent two-stage surgery at Jackson Laboratories that involved left partial nephrectomy followed by right total nephrectomy 1 week later. Animals were delivered to the lab 1 week after the

second surgery. These two models were utilized to determine the effect of hypertension; hypertension is a hallmark of the 5/6 nephrectomy model, whereas adenine- CKD is non-hypertensive [24].

CKD animals were randomized to no lipopolysaccharide (LPS) or LPS injections. (In pilot studies, we de- termined that a longer duration of uremia was needed to detect a higher burden of spontaneous microhemorrhages in non-treated CKD animals; mice on adenine diet for only 10 days beyond the initial 18 days exposure had 3.0 ± 0.4 microhemorrhages per cm2.)

Five weeks after initial CKD induction and at an equivalent age in controls, mice were given intraperitoneal (i.p.) LPS injections (Salmonella enterica serotype Typhimurium, L6511-10MG, Sigma-Aldrich, St. Louis, MO) to induce brain microbleeds. LPS was administered in three doses, 1 mg/kg at 0, 6, and 24 h [25]. LPS-treated mice were given hydration with subcutaneous saline injections two-to-three times per day for 3 days after LPS treatment. Blood pressure (BP) was measured 2 days before LPS injections via tail-cuff plethysmography (CODA-S2 multi-channel, Kent Scientific). All experiments were approved by the University of California, Irvine Institutional Animal Care, and Use Committee.

Tissue Harvest and Blood Chemistries

Mice were euthanized 1 week after LPS injections or at an equivalent age in non-treated animals by exsanguination using cardiac puncture under inhaled isoflurane anesthesia. A 26-gauge needle was used as a cannula and inserted into the left ventricle, and ice-cold PBS solution was applied at a flow rate of 7–8 ml/min. After

perfusion for 5 min, the left brain hemisphere was snap-frozen for Western blot. The right brain hemisphere and both kidneys were fixed overnight in 4% paraformaldehyde and then stored in cold PBS prior to sectioning. Serum was aliquoted for blood chemistries. Blood urea nitrogen (BUN) was measured using the colorimetric kit from BioAssay Systems (Hayward, CA). Serum creatinine was measured using capillary electrophoresis at the O’Brien Kidney Research Core Center (UT Southwestern, Dallas, TX).

Detection of Microhemorrhages

The brains were mounted in 1.5% agarose and sectioned with a vibratome to generate coronal sections (40 μm). Every 5th section was collected for Prussian blue staining to detect microhemorrhages [26]. Prussian blue staining was performed using freshly prepared 5% potassium hexacyanoferrate-tri hydrate and 10% hydrochloric acid. Twenty minutes later, sections were rinsed in water and counterstained with nuclear fast red, dehydrated, and coverslipped. Microhemorrhages were identified at × 20 magnification as purple-blue deposits counted by three injections were given at0,6 and 24h. Subcutaneous saline hydration was given for 3 days after LPS injections. Tail blood pressure (BP) was measured prior to LPS injections. Mice were sacrificed 1 week after LPS injections. b Representative H&E stained kidney sections demonstrating adenine-induced tubulointerstitial injury CKD mice (right panel) compared with normal kidney from CTL animal (left panel), × 20 magnification. Scale bar = 100 μm

independent observers, and then the mean was calculated. Images of the observed positively stained sections were captured using a photomicroscope (Nikon Eclipse, Japan) for three animals per group to calculate the microhemorrhage area. Whole slide images were scanned and the free ImageJ software (version 10.2) from the National Institutes of Health (www.imagej.nih.gov/ij/) was used to

calculate total brain surface area. The number of microhemorrhages was normalized to total brain surface area per animal.

Western Blotting

Brain hemispheres for protein analysis were snap-frozen at the time of tissue collection and homogenized in ice-cold Tissue Extraction Reagent I (Thermo Fisher Scientific) supplemented with protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN). Protein concentrations were measured with a BCA protein assay kit (Thermo Fisher Scientific). SDS-PAGE gel electrophoresis was done with 100 μg of protein per sample. Proteins were transferred onto PVDF membranes and then blocked for

1 h in 5% non-fat dry skim milk prepared in TBS-T buffer(10 mM Tris–HCl, 150 mM NaCl, and 0.1% Tween-20) and incubated with primary antibodies targeting claudin-5 (diluted 1:200, Sigma-Aldrich SAB4502981), occludin (diluted 1:200, Invitrogen 711500, Thermo Fisher Scientific), and normalized to GAPDH internal control (diluted 1:10,000, Abcam, Cambridge, MA) in 5% non-fat milk TBS-T overnight at 4 °C. The blots were then incubated with the respective anti-rabbit or anti-mouse secondary antibodies for 2 h at room temperature. After washing with TBS-T, bands were detected using the Luminescent Image Analyzer LAS-3000 (Fujifilm Life Science, Stamford, CT).

Immunohistochemistry

To detect blood-brain barrier (BBB) leakage, sections were incubated with biotinylated anti-mouse IgG secondary antibody at 1:100 for 1 h at room temperature (Vector Laboratories, Burlingame, CA, USA). Following washing with PBS, sections were incubated for 30 min with avidin-biotin-peroxidase complex (Vector Laboratories) at a dilution of 1:200. Staining was developed using 3′,3′- diaminobenzidine (DAB, Vector Laboratories) as chromo- gen. Percent area stained with IgG was analyzed using ImageJ software in non-LPS CTL and CKD brains. A threshold for positive IgG staining was chosen by manually evaluating a control animal for IgG staining, and this threshold was then kept equal for all included animals [27].

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Cell Culture Experiments

Brain Endothelial Cell Culture and Serum Treatment

Immortalized mouse bEnd.3 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA). Endothelial cell phenotype was confirmed via immunostaining for von Willebrand factor.

Cell cultures were incubated in high-glucose complete Dulbecco’s modified Eagle’s medium (DMEM, ATCC 30-2002) containing 25 mM glucose supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in a humidified incubator at 37 °C in an atmosphere of 5% CO2 and 95% air. Cells were used at passage 5 for all experiments. The cells were seeded at 5× 106 density on 12-well polyester Transwell inserts (0.4 μm pore, Costar) and confluence was achieved at 24–48 h. The culture medium was then changed to expose cells to different concentrations of FBS, human normal serum (HNS), or uremic serum (CKD) from dialysis patients. TEER readings were measured using the EVOM2 volt/Ohm Meter (World Precision Instruments, Sarasota, FL) at three locations per well to obtain an average. Experiments continued for 21 days, and the culture medium was refreshed every 3–4 days. NHS and uremic serum were from previously banked samples obtained after IRB approval and informed consent.

Urea Cell Culture Experiments

To examine the effects of urea (the most abundant retained toxin in CKD), bEnd.3 cells were incubated in high glucose DMEM with 10% FBS alone or in medium supplemented with 42 or 72 mg/dL (70 or 120 μm) urea (Sigma- Aldrich). These urea concentrations approximate the pre-and post-hemodialysis values generally found in ESRD patients, and are considered to be clinically relevant [28]. At the conclusion of a 24-h incubation period, the TEER was measured and cells were harvested and processed for Western blot analysis. For visualization of the actin cytoskeleton, bEnd.3 cells were grown on glass cover slips and exposed to the above urea concentrations for 24 h, fixed for 10 min in chilled 4% formalin/PBS, and then processed for immunofluorescence staining using actistain 488 fluorescent phalloidin with DAPI nuclear stain (catalog# PHDG1-A, Cytoskeleton Inc., Denver, CO). Triplicate slides were done per group, and three frames were imaged per slide on ImageJ software to calculate the area of phalloidin staining normalized to the DAPI area.

Western Blotting

bEnd.3 cells from the urea experiments were pelleted, then lysed in Tissue Extraction Reagent I (Thermo Fisher Scientific) supplemented with protease inhibitor cocktail (Roche Applied Science). Protein concentrations were measured with a BCA protein assay kit, and Western blotting was done as described above with claudin-5 (diluted 1:500) and occludin (diluted 1:500) normalized to GAPDH internal control (diluted 1:20,000). Western blots were repeated at least three times for each sample.

Human Studies

Retrospective Brain MRI Chart Review

Electronic medical records between January 1, 2008, and December 31, 2014, at the University of California-Irvine Medical Center were screened using the Honest Broker system and UCReX (University of California Research Exchange) after IRB approval. Pre-dialysis CKD (n =8) and chronic hemodialysis patients (n =9) who had at least two brain MRI scans at separate time points were identified (of 10 hemodialysis patients initially identified, 1 was removed from the final analysis due to missing images in the radiology PACS system). Controls with normal kidney function (n = 10) were manually matched to the hemodialysis CKD patients by gender and age ± 5 years.

Review of MRI for Cerebral Microbleeds

Microbleeds were counted by an attending neuroradiologist (DF) and analyzed for progression over time. Brain MRI with T2*-weighted and susceptibility-weighted im- aging (SWI) and FLAIR (fluid-attenuated inversion recovery) images were performed on 1.5T and 3T MRI scanners. Layer thickness for SWI sequences was performed at 2 mm, with an interlayer interval of 0. Microbleeds were counted based on 2-mm axial SWI. FLAIR was per-

formed to detect white matter lesions at 3 mm slice thickness, also with an interlayer interval of 0.

Statistical Analysis

There were no data outliers upon screening with the Grubbs’ test (extreme studentized deviate method, http://graphpad. com/quick calls/grubbs1/). Differences among groups in mice and in man were compared by chi-square (Fisher’s exact) tests for categorical variables and t-tests and ANOVA for continuous variables. Continuous data are reported as mean ± SEM. For mouse data, we performed both a Kruskal–Wallis non-parametric and a one-way ANOVA with Tukey HSD tests. Two-way ANOVA (CKD-yes/no and LPS- yes/no) was used to test CKD interaction. Differences among groups were considered significant if P < 0.05. Figures were generated using GraphPad Prism 4 software (GraphPad Software, San Diego CA).

Results

Survival with LPS Treatment

CKD mice treated with LPS had an 80% survival rate. Only mice that survived to 1 week after LPS treatment were

included in the final analyses. All mice in other groups survived until the end of the experiment.

CKD Significantly Increased Brain Microhemorrhages

Burden After LPS Treatment

Animals with adenine-induced and 5/6 nephrectomy CKD showed significantly elevated blood urea nitrogen (BUN) and serum creatinine values compared with CTL animals (Table 1). Tail BP was significantly higher in nephrectomy-CKD animals compared with CTL and adenine-CKD animals (Table1); however, microbleed counts in the two CKD groups were similar. H&E staining confirmed adenine-induced tubulointerstitial in- jury in the kidneys from CKD mice (Fig. 1b).

Mean brain microhemorrhage burden was 2.0 ± 0.5 per cm2 in non-LPS CTL mice and was increased 2–2.5-fold in non- LPS CKD animals (adenine-CKD mice 4.5 ± 0.9 per cm2;

nephrectomy-CKD mice 4.2 ± 1.1 per cm2) (Table 1; Fig. 2a). For the non-LPS groups, an increase in microhemorrhages was significant for adenine-CKD compared with CTL but not for nephrectomy-CKD. LPS treatment increased microhemorrhage formation to the same degree in CTL and CKD animals by