Renal glomerular and tubular responses to glutaraldehyde- polymerized human hemoglobin

Hemoglobin-based oxygen carriers (HBOCs) are being developed as oxygen and volume replacement therapeutics, however, their molecular and cellular effects on the vasculature and different organ systems are not fully defined. Using a guinea pig transfusion model, we examined the renal glomerular and tubular responses to PolyHeme, a highly characterized glutaraldehyde-polymerized human hemoglobin with low tetrameric hemoglobin content. PolyHeme-infused animals showed no major changes in glomerular histology or loss of specific markers of glomerular podocytes (Wilms tumor 1 protein, podocin, and podocalyxin) or endothelial cells (ETS-related gene and claudin-5) after 4, 24, and 72 h. Relative to sham controls, PolyHeme-infused animals also showed similar expression and subcellular distribution of N-cadherin and E-cadherin, two key epithelial junctional proteins of proximal and distal tubules, respectively. In terms of heme catabolism and iron-handling responses, PolyHeme induced a moderate but transient expression of heme oxygenase-1 in proximal tubular epithelium and tubulointerstitial macrophages that was accompanied by increased iron deposition in tubular epithelium. Contrary to previous findings with other modified or acellular hemoglobins, the present data show that PolyHeme does not disrupt the junctional integrity of the renal glomerulus and tubular epithelium, and triggers moderate activation of heme catabolic and iron sequestration systems likely as part of a renal adaptive response

Transcriptional Suppression of Renal Antioxidant Enzyme Systems in Guinea Pigs Exposed to Polymerized Cell-Free Hemoglobin

Hemoglobin-based oxygen carriers (HBOCs) are being developed as oxygen and plasma volume-expanding therapeutics though their potential to promote oxidative tissue injury has raised safety concerns. Using a guinea pig exchange transfusion model, we examined the effects of polymerized bovine hemoglobin (HbG) on the transcriptional regulation, activity, and expression of the renal antioxidant enzymes; superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). HbG infusion downregulated the mRNA levels for genes encoding SOD isoforms 1-3, GPx1, GPx3, GPx4, and CAT. This transcriptional suppression correlated with decreased enzymatic activities for SOD, CAT, and GPx. Immunostaining revealed decreased protein expression of SOD1, CAT, and GPx1 primarily in renal cortical tubules. DNA methylation analyses identified CpG hypermethylation in the gene promoters for SOD1-3, GPx1, GPx3, and GPx4, suggesting an epigenetic-based mechanism underlying the observed gene repression. HbG also induced oxidative stress as evidenced by increased renal lipid peroxidation end-products and 4-HNE immunostaining, which could be the result of the depleted antioxidant defenses and/or serve as a trigger for increased DNA methylation. Together, these findings provide evidence that the renal exposure to HbG suppresses the function of major antioxidant defense systems which may have relevant implications for understanding the safety of hemoglobin-based products

Anthrax Lethal Toxin Downregulates Claudin-5 Expression in Human Endothelial Tight Junctions

<div><p></p><p>Vascular leakage pathologies such as pleural effusion and hemorrhage are hallmarks of anthrax pathogenesis. We previously reported that anthrax lethal toxin (LT), the major virulence factor of anthrax, reduces barrier function in cultured primary human microvascular endothelial cells. Here, we show that LT-induced barrier dysfunction is accompanied by the reduced expression of the endothelial tight junction (TJ) protein claudin-5 but no change in the expression of other TJ components occludin, ZO-1, ZO-2, or the adherens junction (AJ) protein VE-cadherin. The downregulation of claudin-5 correlated temporally and dose-dependently with the reduction of transendothelial electrical resistance. LT-induced loss of claudin-5 was independent of cell death and preceded the appearance of actin stress fibers and altered AJ morphology. Pharmacological inhibition of MEK-1/2, two kinases that are proteolytically inactivated by LT, showed a similar reduction in claudin-5 expression. We found that LT reduced claudin-5 mRNA levels but did not accelerate the rate of claudin-5 degradation. Mice challenged with LT also showed significant reduction in claudin-5 expression. Together, these findings support a possible role for LT disruption of endothelial TJs in the vascular leakage pathologies of anthrax.</p></div

Time-dependent reduction of claudin-5.

<p>(A) Cells were treated with medium alone or the combination of 100 ng/ml LF +500 ng/ml PA. Claudin-5 expression was analyzed by Western blot in whole cell lysates collected after the indicated treatment times. Claudin-5 expression was normalized to tubulin and presented relative to control. Means ± SE for a minimum of three separate experiments are shown. *, <i>p</i><0.05 versus control. (B) Immunofluorescence analysis of claudin-5. Cells were treated with medium alone, inactive mutant LT, or LT as described above for 48 hours. Monolayers were stained for claudin-5 (red) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062576#s2" target="_blank">Materials and Methods</a>. Nuclei were counterstained with Hoechst 33342 (blue). Images are representative of three separate experiments (400x magnification).</p

Claudin-5 expression and inhibitors of proteosome, lysosome, and matrix metalloproteinases (MMPs).

<p>(A) Cells were treated with medium alone or the combination of 100 ng/ml +500 ng/ml PA for 18 hours prior to the addition of the proteosome inhibitor MG132 (1 µM) for an additional 12 hours. Whole cell lysates were analyzed for claudin-5, MEK-1, and the accumulation of ubiquitinated proteins by Western blot. Representative immunoblots of three separate experiments are shown. Claudin-5 expression was normalized to tubulin and presented relative to control. Means ± SE for a minimum of three separate experiments are shown. (B) Cells were treated as indicated above for 18 hours prior to the addition of lysosome inhibitors, chloroquine (CQ, 20 µM) or E-64 (10 µg/ml) plus pepstatin A (10 µg/ml) (EP), or the broad spectrum MMP inhibitor marimastat (MST, 100 µM) for an additional 24 hours. Whole cell lysates were analyzed for claudin-5 and MEK-1 by Western blot. Claudin-5 expression was normalized to tubulin and presented relative to control. Means ± SE for a minimum of three separate experiments are shown. *, <i>p</i><0.05 versus control, #, <i>p</i><0.05 versus LT alone.</p

LT reduces claudin-5 expression but does not down-regulate other TJ proteins or VE-cadherin.

<p>Cells were treated with medium alone, 100 ng/ml LF, 500 ng/ml PA, inactive mutant LF_E687C+PA, or PA combined with increasing concentrations of LF for 48 hours. (A) Whole cell lysates were analyzed for claudin-5, ZO-1, ZO-2, occludin, and VE-cadherin. Tubulin served as a loading control. Representative immunoblots of three separate experiments are shown. (B) Claudin-5 expression was normalized to tubulin and presented relative to control. Means ± SE for a minimum of three separate experiments are shown. *, <i>p</i><0.05 versus control.</p

LT reduces claudin-5 expression in mouse liver.

<p>Mice were injected with PBS (non-treated, NT) or LT (50 µg LF +50 µg PA). (A) Claudin-5 immunofluorescence analysis in frozen liver sections from NT and LT-72 h mice as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062576#s2" target="_blank">Materials and Methods</a>. Reduced sinusoidal claudin-5 staining observed in LT-treated mice compared to NT mice (top panels, 200x magnification). Diffused claudin-5 staining also observed in larger hepatic blood vessels in LT-treated mice (bottom panels, 600x magnification). Nuclei were counterstained with Hoechst 33342 (blue). (B) Western blot analyses of claudin-5, VE-cadherin, PARP (full length and cleaved), and caspase 3 (full length and cleaved) in liver whole cell extracts collected from NT and LT mice after 24 and 72 hours. Representative immunoblots of three different animals per group are shown. Claudin-5 or VE-cadherin expression was normalized to tubulin and presented relative to control. Means ± SE for a minimum of three separate animals are shown. *, <i>p</i><0.05 versus control.</p

Claudin-5 downregulation is independent of cell death.

<p>Cells were pretreated with the caspase inhibitors, z-VAD-fmk (20 µM) or DEVD-fmk (20 µM) for 30 min prior to incubation with LT (100 ng/ml LF +500 ng/ml PA) for 72 hours. (A) Whole cell lysates were analyzed for claudin-5, cleaved caspase 3, and MEK-1 by Western blot. Tubulin served as the loading control. Arrowheads denote the activated p17/p12 fragments of caspase 3. Blots are representative of three separate experiments. (B) Cell viability and necrosis visualized by calcein AM and propidium iodide staining in control- and LT (1000 ng/ml LF +500 ng/ml PA)- treated cells after 72 hours. Images are representative of three separate experiments (100x magnification).</p

Effect of MEK and MAPK inhibitors on claudin-5 expression.

<p>Cells were treated with medium alone, 10 µM U0126, 10 µM SP600125, or 20 µM SB208580 for 48 hours. (A) Whole cell lysates were analyzed for claudin-5, VE-cadherin, and the phosphorylated and total forms of ERK 1/2, HSP27, and c-Jun by Western blot. Tubulin served as the loading control. Representative immunoblots of three separate experiments are shown. (B) Claudin-5 expression was normalized to tubulin and presented relative to control. Means ± SE for a minimum of three separate experiments are shown. *, <i>p</i><0.05 versus control.</p