Longitudinal Changes to Tight Junction Expression and Endothelial Cell Integrity in a Mouse Model of Sterile Corneal Inflammation

Introduction: Cerebral microbleeds are more common in chronic kidney disease (CKD) and dialysis patients compared to the general population. Diminished kidney function alone appears to be a risk factor for microbleeds, independent of age and hypertension. Microbleed burden in CKD patients is associated with increased risk of future hemorrhagic stroke and with cognitive dysfunction. The mechanisms that drive uremic microbleed formation are unclear. Hypothesis: We hypothesized that CKD mice are predisposed to develop cerebral microhemorrhages (the pathologic substrate of microbleeds), and that a standardized inflammatory stimulus (lipopolysaccharide, LPS) will amplify microhemorrhage burden in CKD mice compared to non-CKD controls (CTL). We also hypothesized that uremia induces depletion of tight junction proteins, altering blood-brain barrier integrity and representing a potential mechanism of microbleed formation. Methods: Animal groups included CTL (n=3), CKD (n=3), CTL+LPS (n=5) and CKD+LPS (n=5). CKD induction in male C57BL/6 mice was achieved via nephrotoxic adenine diet x18 days. Two weeks following CKD induction, CKD and control mice were treated with LPS 1 mg/kg i.p. dosed at 0, 6 and 24 hours. Brains were harvested one week after LPS injections and 40-micron sections were stained using Prussian blue to identify microhemorrhages. Immunohistochemistry was performed for the blood-brain barrier tight junction protein claudin-5. Results: CKD mice had significantly elevated blood urea nitrogen, and tubulointerstitial fibrosis was present on kidney histology. Total number of microhemorrhages per brain was 2.3±1.5 (mean ± standard error of the mean) for CTL mice, 8.3±1.5 for CKD mice, 23.2±4.2 for CTL+LPS mice, and 27.6±6.2 for CKD+LPS mice (p<0.05 for CKD+LPS vs. CTL). Immunostaining showed decreased claudin-5 expression in CKD mice compared to CTL. Conclusions: We have generated a mouse model that will facilitate future mechanistic studies in the field of uremic microbleeds. Our initial findings suggest that CKD alters blood-brain barrier integrity and that inflammation amplifies development of microbleeds in CKD.

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Inhibition of inwardly rectifying K+ channels by cGMP in pulmonary vascular endothelial cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00469.2001 ◽

2002 ◽

Vol 283 (2) ◽

pp. L297-L304 ◽

Cited By ~ 13

Author(s):

Larissa A. Shimoda ◽

Laura E. Welsh ◽

David B. Pearse

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Vascular Endothelial Cells ◽

Cell Types ◽

Protective Mechanism ◽

Cell Integrity ◽

Barrier Dysfunction ◽

Whole Cell Patch Clamp ◽

Inward Currents ◽

Inwardly Rectifying

Endothelial barrier dysfunction is typically triggered by increased intracellular Ca2+concentration. Membrane-permeable analogs of guanosine 3′,5′-cyclic monophosphate (cGMP) prevent disruption of endothelial cell integrity. Because membrane potential ( E m), which influences the electrochemical gradient for Ca2+ influx, is regulated by K+ channels, we investigated the effect of 8-bromo-cGMP on E m and inwardly rectifying K+ (KIR) currents in bovine pulmonary artery and microvascular endothelial cells (BPAEC and BMVEC), using whole cell patch-clamp techniques. Both cell types exhibited inward currents at potentials negative to −50 mV that were abolished by application of 10 μM Ba2+, consistent with KIR current. Ba2+ also depolarized both cell types. 8-Bromo-cGMP (10−3 M) depolarized BPAEC and BMVEC and inhibited KIR current. Pretreatment with Rp-8-cPCT-cGMPS or KT-5823, protein kinase G (PKG) antagonists, did not prevent current inhibition by 8-bromo-cGMP. These data suggest that 8-bromo-cGMP induces depolarization in BPAEC and BMVEC due, in part, to PKG-independent inhibition of KIR current. The depolarization could be a protective mechanism that prevents endothelial cell barrier dysfunction by reducing the driving force for Ca2+ entry.

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