Introduction
One of the most significant stroke neurology discoveries in recent years is the emergence of chronic kidney disease (CKD) as an independent risk factor for early cognitive decline and cerebrovascular disease (reviewed in [
1]), especially cerebral microbleeds and stroke [
2‐
9]. While CKD and cerebrovascular disease share common risk factors, CKD appears 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 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 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 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 actin 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 progression of microbleed burden.
Discussion
In this paper, we report an increased prevalence of cerebral microhemorrhages in CKD mice and of microbleeds in CKD patients. CKD animals had evidence of BBB disruption, and cerebral microhemorrhages were increased over 2-fold compared with control mice. In the human study, ~ 50% of CKD patients (both in pre-dialysis and dialysis groups) had MRI-demonstrable microbleeds compared with 10% of controls. In our mouse studies, the highest microhemorrhage load was observed with LPS treatment in the adenine-CKD model, while development of microhemorrhages appeared to be independent of hypertension. The latter finding was consistent with our MRI studies of microbleeds in humans. Cell culture studies demonstrated significant impairment of the brain endothelial barrier upon exposure to uremic serum, with up to 65% drop in TEER in the 15% CKD serum group. Incubation with increasing concentrations of urea, the most abundant retained toxin in CKD [
29], demonstrated disruption of the actin cytoskeleton and decreased tight junction proteins as potential mechanisms to explain the endothelial barrier dysfunction.
CKD has an impact on cerebrovascular disease risk that appears to go well beyond traditional risk factors such as hypertension and diabetes [
10,
11]. Brain microbleeds are present in up to 50% of patients with advanced CKD [
2‐
4] and correlate with cognitive dysfunction [
4,
8,
19‐
23] and increased risk of hemorrhagic stroke [
9]. Our group recently proposed CKD-specific pathways that could promote cerebral small vascular disease via disruption of blood flow autoregulation and BBB integrity, such as increased vascular calcification, systemic inflammation, and uremic toxins [
1]. Our tissue culture studies demonstrate that exposure to urea in concentrations similar to those present in dialysis patients produced reduced expression of tight junction protein claudin-5 and disruption of the actin cytoskeleton. These pathways appear to explain, at least in part, the increased microhemorrhages due to increased BBB permeability observed in the CKD animals and patients. Of note, CKD has previously been associated with a marked depletion of tight junction proteins (75–80% decreased expression) in gut epithelial cells from experimental animals [
30]. Subsequent in vitro studies revealed the central role of urea in the CKD-induced disruption of intestinal epithelial barrier structure and function [
28]. Taken together, these observations suggest that urea toxicity is not limited to endothelial cells alone. Our data add to the growing body of evidence for direct adverse effects of uremic toxins on the vascular endothelium; other groups have reported that the gut-derived bacterial metabolites indoxyl sulfate and
p-cresyl sulfate induce oxidative stress in cultured endothelial cells [
31,
32].
LPS is an established acute inflammatory stimulus used in rodent models [
33] that binds to toll-like receptor 4 [
34] to induce BBB damage [
35] and brain endothelial dysfunction [
36,
37]. LPS has been used to amplify and study microbleeds pathophysiology in young adult and aged mice [
25,
26,
38]. In our current animal studies, LPS treatment increased microhemorrhage formation to the same degree in CTL and CKD animals, by approximately 2-fold (Table
1; Fig.
2a). In CTL mice, average microhemorrhage count was increased from 2 to 3.6 per cm
2 with LPS, whereas in CKD mice, the increase was from 4.5 to 10.3 per cm
2. We conclude that the uremic brain is predisposed to more severe injury after an acute inflammatory event due to pre-existing injured BBB.
The actin cytoskeleton reversibly polymerizes between globular (G-actin) and filamentous (F-actin) configurations. F-actin is important for cell adhesion, division, and apoptosis; it is modulated by actin-binding proteins, which are in turn regulated by Rho GTPases [
39,
40]. Actin reorganization, from its cortical distribution into stress fibers, is a key component of the endothelial response to inflammation [
41]. Our cell culture studies implicated elevated urea in the pathogenesis of decreased F-actin expression, with fragmentation of cortical fibers and appearance of radial stress fibers (Fig.
4c). Further studies are needed to clarify the role of actin cytoskeletal disruption in microbleeds pathophysiology in vivo.
Although hypertension is a strong predictor of cerebral microbleeds in the general population [
42], we found that hypertension did not modify presence of microbleeds in our CKD animal and human subjects (Tables
1 and
2). Our study is limited by small sample size; however, our results are consistent with prior cross-sectional reports in which CKD was a risk factor for brain microbleeds, independent of hypertension, age, and diabetes mellitus [
10,
11]. The degree of hypertension in the 5/6 nephrectomy animals was mild; however, our findings are consistent with the study by Passos et al. in which induction of significant hypertension in wild-type mice via infusion of angiotensin II and L-N
G-nitroarginine methyl ester (systolic 180 mmHg) did not significantly raise microhemorrhage counts [
43]. While prevalence of brain microbleeds was similar in the two CKD patient groups (approximately 50%), analysis of consecutive brain MRIs demonstrated microbleed progression in three of four hemodialysis patients as compared to zero of four pre-dialysis patients (follow-up interval of ~ 1.5 years). Hemodialysis patients may be particularly predisposed to microbleed formation due to inter- and intradialytic BP fluctuations, regional gut ischemia leading to increased endotoxin translocation (which drives systemic inflammation), and use of heparin anticoagulation during dialysis therapy [
1,
44,
45].
The major limitation of our cell culture work is the use of an immortalized cell line, with known limitation of brain-specific properties [
46]. Further cell culture studies using, for example, primary brain endothelial cells are needed to confirm our findings. Our human study, a retrospective analysis of patients with serial brain MRIs, has the limitations of patient identification using hospital database diagnosis and procedure codes and of small sample size.
In summary, CKD increased brain microbleeds in both hypertensive and non-hypertensive mouse models. Uremic serum, and urea alone, disrupted the cultured brain endothelial cell monolayer, thus supporting a mechanistic role for uremic toxins affecting BBB permeability and promoting microhemorrhages. Human brain MRI studies confirmed increased prevalence of microbleeds in CKD patients compared with age-matched non-CKD controls, and hemodialysis patients in particular were noted to develop new microbleeds over a 1.5-year follow-up period. More studies are warranted to further characterize factors in the CKD milieu that promote brain microhemorrhages.
Compliance with Ethical Standards
All experiments were approved by the University of California, Irvine Institutional Animal Care and Use Committee.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.