Nath, Karl A.

The present study examined the heme oxygenase (HO) system in an in vivo murine model of pathological shear stress induced by partial carotid artery ligation. In this model, along with upregulation of vasculopathic genes, HO-1 is induced in the endothelium and adventitia, whereas HO-2 is mainly upregulated in the endothelium. Within minutes of ligation, NF-κB, a transcription factor that upregulates vasculopathic genes and HO-1, is activated. Failure to express either HO-1 or HO-2 exaggerates the reduction in carotid blood flow and exacerbates vascular injury. After artery ligation, comparable induction of HO-2 occurred in HO-1+/+and HO-1−/−mice, whereas HO-1 induction was exaggerated in HO-2−/−mice compared with HO-2+/+mice. Upregulation of HO-1 by an adeno-associated viral vector increased vascular HO-1 expression and HO activity and augmented blood flow in both ligated and contralateral carotid arteries. Acute inhibition of HO activity decreased flow in the ligated carotid artery, whereas a product of HO, carbon monoxide (CO), delivered by CO-releasing molecule-3, increased carotid blood flow. In conclusion, in the partial carotid artery ligation model of pathological shear stress, this study provides the first demonstration of 1) upregulation and vasoprotective effects of HO-1 and HO-2 and the vasorelaxant effects of CO as well as 2) vascular upregulation of HO-1 in vivo by an adeno-associated viral vector that is attended by a salutary vascular response. Induction of HO-1 may reside in NF-κB activation, and, along with induced HO-2, such upregulation of HO-1 provides a countervailing vasoprotective response in pathological shear stress in vivo.

The mechanism of heme oxygenase-1 gene ( ho-1) activation by heme in immortalized rat proximal tubular epithelial cells was examined. Analysis of the ho-1promoter identified the heme-responsive sequences as the stress-response element (StRE), multiple copies of which are present in two enhancer regions, E1 and E2. Electrophoretic mobility shift assays identified Nrf2, MafG, ATF3, and Jun and Fos family members as StRE-binding proteins; binding of Nrf2, MafG, and ATF3 was increased in response to heme. Dominant-negative mutants of Nrf2 and Maf, but not of c-Fos and c-Jun, inhibited basal and heme-induced expression of an E1-controlled luciferase gene. Heme did not affect the transcription activity of Nrf2, dimerization between Nrf2 and MafG, or the level of MafG, but did stimulate expression of Nrf2. Heme did not influence the level of Nrf2 mRNA but increased the half-life of Nrf2 protein from ∼10 min to nearly 110 min. These results indicate that heme promotes stabilization of Nrf2, leading to accumulation of Nrf2 · MafG dimers that bind to StREs to activate the ho-1 gene.

This study examined the effect of hemin on the expression of heme oxygenase-1 (HO-1) and monocyte chemoattractant protein-1 (MCP-1) in immortalized rat proximal tubular epithelial cells (IRPTCs). Hemin elicited a dose- and time-dependent induction of HO-1 and MCP-1 mRNA. HO activity contributed to MCP-1 mRNA expression at early time points (4–6 h) because inhibition of HO activity by zinc protoporphyrin (ZnPP) prevented hemin-induced expression of MCP-1 mRNA. Catalytically active intracellular iron was markedly increased in hemin-treated IRPTCs and contributed to the induction of HO-1 and MCP-1 mRNA because an iron chelator blocked hemin-induced upregulation of both genes, whereas a cell-permeant form of iron directly induced these genes. N-acetylcysteine completely blocked hemin-induced expression of HO-1 and MCP-1 mRNA, thereby providing added evidence for redox regulation of expression of these genes. The redox-sensitive transcription factor NF-κB was recruited in hemin-induced upregulation of MCP-1 because two different compounds that abrogate the activation of NF-κB (TPCK and BAY 11-7082) completely blocked hemin-induced upregulation of MCP-1 mRNA. In contrast to this HO-mediated induction of MCP-1 through redox-sensitive, iron-dependent, and NF-κB-involved pathways observed after 4–6 h, hemin also elicited a delayed induction of MCP-1 at 18 h through HO-independent pathways. We conclude that hemin is a potent inducer of MCP-1 in IRPTCs: HO-dependent, heme-degrading pathways lead to an early, robust, and self-remitting induction of MCP-1, whereas HO-independent mechanisms lead to a delayed expression of MCP-1.

Key Points Heme, released from hemoglobin, elicits vaso-occlusion in transgenic sickle mice via endothelial TLR4 signaling. Heme/TLR4 signaling activates NF-κB and triggers vaso-occlusion through Weibel-Palade body degranulation and adhesion molecule expression.

Activation of the renin-angiotensin-aldosterone system plays a critical role in the development of chronic renal damage in patients with renovascular hypertension. Although angiotensin II (Ang II) promotes oxidative stress, inflammation, and fibrosis, it is not known how these pathways intersect to produce chronic renal damage. We tested the hypothesis that renal parenchymal cells are subjected to oxidant stress early in the development of RVH and produce signals that promote influx of inflammatory cells, which may then propagate chronic renal injury. We established a reproducible murine model of RVH by placing a tetrafluoroethhylene cuff on the right renal artery. Three days after cuff placement, renal tissue demonstrates no histologic abnormalities despite up regulation of both pro- and anti-oxidant genes. Mild renal atrophy was observed after seven days and was associated with induction of Tnfα and influx of CD3+ T cells and F4/80+ macrophages. By 28 days, kidneys developed severe renal atrophy with interstitial inflammation and fibrosis, despite normalization of plasma renin activity. Based on these considerations, we propose that renal parenchymal cells initiate a progressive cascade of events leading to oxidative stress, interstitial inflammation, renal fibrosis, and atrophy.

The proinflammatory vasculotoxic effects of intravascular hemolysis are modulated by plasma hemoglobin and heme clearance via the haptoglobin/CD163 system and the hemopexin/CD91 system, respectively, and detoxification through the heme oxygenase/ferritin system. However, sudden or excessive hemolysis can overwhelm these protective systems leading to heme interacting with cells of the vasculature. Heme presents a damage-associated molecular pattern to the innate immune system. Heme is an extracellular inflammatory signaling molecule with strict binding specificity for TLR4 on monocyte/macrophages, endothelial, and other cells. The resulting TLR4 signaling cascade rapidly leads to intracellular oxidative stress and an inflammatory response. Heme also induces a cytoprotective response that includes Nrf2 responsive genes such as heme oxygenase-1, ferritin, haptoglobin, hemopexin, and other antioxidant response genes. It is the balance between the pro-inflammatory/vasculotoxic effects of plasma hemoglobin/heme and the cytoprotective responses that ultimately determines the pathophysiologic outcome in patients.

Heme oxygenase-2 (HO-2), the constitutive isoform of the heme-degrading enzyme heme oxygenase, may serve as an anti-inflammatory vasorelaxant, in part, by generating carbon monoxide. Arteriovenous fistulas (AVFs) are employed as hemodialysis vascular accesses because they provide an accessible, high-blood-flow vascular segment. We examined the role of vascular expression of HO-2 in AVF function. An AVF was created in mice by anastomosing the carotid artery to the jugular vein. HO-2 expression was detected by immunohistochemistry in the intact carotid artery, mainly in endothelial cells and smooth muscle cells; expression of HO-2 protein and mRNA was modestly increased in the artery of the AVF. Creating an AVF in HO-2−/−mice compared with an AVF in HO-2+/+mice led to markedly reduced AVF blood flow and increased numbers of nonfunctioning AVFs. The impairment of AVF function in the setting of HO-2 deficiency could not be ascribed to either preexisting intrinsic abnormalities in endothelium-dependent and endothelium-independent relaxation of the carotid artery in HO-2-deficient mice or to impaired vasorelaxant responses in the intact carotid artery in vivo. HO-1 mRNA was comparably induced in the AVF in HO-2+/+and HO-2−/−mice, whereas the AVF in HO-2−/−mice compared with that in HO-2+/+mice exhibited exaggerated induction of matrix metalloproteinase (MMP)-9 but similar induction of MMP-2. HO-2 deficiency also led to lower AVF blood flow when AVFs were created in uremia, the latter induced by subtotal nephrectomy. We conclude that HO-2 critically contributes to the adequacy of AVF blood flow and function.

Renal artery stenosis (RAS) is an important cause of chronic renal dysfunction. Recent studies have underscored a critical role for CCL2 (MCP-1)-mediated inflammation in the progression of chronic renal damage in RAS and other chronic renal diseases. In vitro studies have implicated p38 MAPK as a critical intermediate for the production of CCL2. However, a potential role of p38 signaling in the development and progression of chronic renal disease in RAS has not been previously defined. We sought to test the hypothesis that inhibition of p38 MAPK ameliorates chronic renal injury in mice with RAS. We established a murine RAS model by placing a cuff on the right renal artery and treated mice with the p38 inhibitor SB203580 or vehicle for 2 wk. In mice treated with vehicle, the cuffed kidney developed interstitial fibrosis, tubular atrophy, and interstitial inflammation. In mice treated with SB203580, the RAS-induced renal atrophy was reduced (70% vs. 39%, P < 0.05). SB203580 also reduced interstitial inflammation and extracellular matrix deposition but had no effect on the development of hypertension. SB203580 partially blocked the induction of CCL2, CCL7 (MCP-3), CC chemokine receptor 2 (CCR2), and collagen 4 mRNA expression in the cuffed kidneys. In vitro, blockade of p38 hindered both TNF-α and TGF-β-induced CCL2 upregulation. Based on these observations, we conclude that p38 MAPK plays a critical role in the induction of CCL2/CCL7/CCR2 system and the development of interstitial inflammation in RAS.

Age increases the risk for ischemic acute kidney injury (AKI). We questioned whether a similar age-dependent injury occurs following exposure to hemoglobin, a known nephrotoxin. Old mice (∼16 mo old), but not young mice (∼6 mo old), when administered hemoglobin, exhibited marked elevation in blood urea nitrogen (BUN) and serum creatinine, and acute tubular necrosis with prominent tubular cast formation. The aged kidney exhibited induction of heme oxygenase-1 (HO-1) and other genes/proteins that may protect against heme-mediated renal injury, including ferritin, ferroportin, haptoglobin, and hemopexin. Old mice did not evince induction of HO-2 mRNA by hemoglobin, whereas a modest induction of HO-2 mRNA was observed in young mice. To determine the functional significance of HO-2 in heme protein-induced AKI, we administered hemoglobin to relatively young HO-2+/+and HO-2−/−mice: HO-2−/−mice, compared with HO-2+/+mice, exhibited greater renal dysfunction and histologic injury when administered hemoglobin. In addition to failing to elicit a protective system such as HO-2 in response to hemoglobin, old mice exhibited an exaggerated maladaptive response typified by markedly greater induction of the nephrotoxic cytokine IL-6 (130-fold increase vs. 10-fold increase in mRNA in young mice). We conclude that aged mice, unlike relatively younger mice, are exquisitely sensitive to the nephrotoxicity of hemoglobin, an effect attended by a failure to induce HO-2 mRNA and a fulminant upregulation of IL-6. Age thus markedly augments the sensitivity of the kidney to heme proteins, and HO-2 confers resistance to such insults.

Vascular access dysfunction causes morbidity in hemodialysis patients. This study examined the generation and pathobiological significance of superoxide anion in a rat femoral arteriovenous fistula (AVF). One week after AVF creation, there was increased production of superoxide anion accompanied by decreased total superoxide dismutase (SOD) and Cu/Zn SOD activities and induction of the redox-sensitive gene heme oxygenase-1. Immunohistochemical studies of nitrotyrosine formation demonstrated that peroxynitrite, a product of superoxide anion and nitric oxide, was present in increased amounts in endothelial and smooth muscle cells in the AVF. Because uncoupled NOS isoforms generate superoxide anion, and NOS coupling requires tetrahydrobiopterin (BH4) as a cofactor, we assessed NOS uncoupling by determining the ratio of BH4to dihydrobiopterin (BH2); the BH4-to-BH2ratio was markedly attenuated in the AVF. Because Src is a vasculopathic signaling species upstream and downstream of superoxide anion, such expression was evaluated; expression of Src and phosphorylated Src was both markedly increased in the AVF. Expression of NADPH oxidase (NOX) 1, NOX2, NOX4, cyclooxygenase (COX) 1, COX2, p47phox, and p67phoxwas all unchanged, as assessed by Western analyses, thereby suggesting that these proteins may not be involved in increased production of superoxide anion. Finally, administration of tempol, a superoxide anion scavenger, decreased neointima formation in the juxta-anastomotic venous segment and improved AVF blood flow. We conclude that the AVF exhibits increased superoxide anion generation that may reflect the combined effects of decreased scavenging by SOD and increased generation by uncoupled NOS, and that enhanced superoxide anion production promotes juxta-anastomotic stenosis and impairs AVF function.