Ferritin plays a central role in iron metabolism and is made of 24 subunits of 2 types: heavy chain and light chain. The ferritin heavy chain (FtH) has ferroxidase activity that is required for iron incorporation and limiting toxicity. The purpose of this study was to investigate the role of FtH in acute kidney injury (AKI) and renal iron handling by using proximal tubule-specific FtH-knockout mice (FtH PT-/-mice). FtH PT-/-mice had significant mortality, worse structural and functional renal injury, and increased levels of apoptosis in rhabdomyolysis and cisplatin-induced AKI, despite significantly higher expression of heme oxygenase-1, an antioxidant and cytoprotective enzyme. While expression of divalent metal transporter-1 was unaffected, expression of ferroportin (FPN) was significantly lower under both basal and rhabdomyolysis-induced AKI in FtH PT-/-mice. Apical localization of FPN was disrupted after AKI to a diffuse cytosolic and basolateral pattern. FtH, regardless of iron content and ferroxidase activity, induced FPN. Interestingly, urinary levels of the iron acceptor proteins neutrophil gelatinase-associated lipocalin, hemopexin, and transferrin were increased in FtH PT-/-mice after AKI. These results underscore the protective role of FtH and reveal the critical role of proximal tubule FtH in iron trafficking in AKI.

Autophagy is a tightly regulated, programmed mechanism to eliminate damaged organelles and proteins from a cell to maintain homeostasis. Cisplatin, a chemotherapeutic agent, accumulates in the proximal tubules of the kidney and causes dose-dependent nephrotoxicity, which may involve autophagy. In the kidney, cisplatin induces the protective antioxidant heme oxygenase-1 (HO-1). In this study, we examined the relationship between autophagy and HO-1 during cisplatin-mediated acute kidney injury (AKI). In wild-type primary proximal tubule cells (PTC), we observed a time-dependent increase in autophagy after cisplatin. In HO-1 Ϫ/Ϫ PTC, however, we observed significantly higher levels of basal autophagy, impaired progression of autophagy, and increased apoptosis after cisplatin. Restoring HO-1 expression in these cells reversed the autophagic response and inhibited apoptosis after treatment with cisplatin. In vivo, although both wild-type and HO-1-deficient mice exhibited autophagosomes in the proximal tubules of the kidney in response to cisplatin, HO-1-deficient mice had significantly more autophagosomes, even in saline-treated animals. In addition, ecdysone-induced overexpression of HO-1 in cells led to a delay in autophagy progression, generated significantly lower levels of reactive oxygen species, and protected against cisplatin cytotoxicity. These findings demonstrsate that HO-1 inhibits autophagy, suggesting that the heme oxygenase system may contain therapeutic targets for AKI.

The cardioprotective inducible enzyme heme oxygenase-1 (HO-1) degrades prooxidant heme into equimolar quantities of carbon monoxide, biliverdin, and iron. We hypothesized that HO-1 mediates cardiac protection, at least in part, by regulating mitochondrial quality control. We treated WT and HO-1 transgenic mice with the known mitochondrial toxin, doxorubicin (DOX). Relative to WT mice, mice globally overexpressing human HO-1 were protected from DOX-induced dilated cardiomyopathy, cardiac cytoarchitectural derangement, and infiltration of CD11b+ mononuclear phagocytes. Cardiac-specific overexpression of HO-1 ameliorated DOX-mediated dilation of the sarcoplasmic reticulum as well as mitochondrial disorganization in the form of mitochondrial fragmentation and increased numbers of damaged mitochondria in autophagic vacuoles. HO-1 overexpression promotes mitochondrial biogenesis by upregulating protein expression of NRF1, PGC1α, and TFAM, which was inhibited in WT animals treated with DOX. Concomitantly, HO-1 overexpression inhibited the upregulation of the mitochondrial fission mediator Fis1 and resulted in increased expression of the fusion mediators, Mfn1 and Mfn2. It also prevented dynamic changes in the levels of key mediators of the mitophagy pathway, PINK1 and parkin. Therefore, these findings suggest that HO-1 has a novel role in protecting the heart from oxidative injury by regulating mitochondrial quality control.

Historically, heme oxygenase-1 (HO-1) has been viewed as a cytoprotective protein, ameliorating the effects of inflammatory cellular damage. The demonstration, however, of a beneficial role for HO-1 in allograft protection from acute and chronic rejection, 1-3 strongly suggests an important function of this enzyme in both innate and adaptive immune responses. In the original description of a mouse model of HO-1 deficiency, Poss and Tonegawa 4 noted an age-related overgrowth of the CD4 ϩ T-cell population, suggesting impaired regulation of T-cell proliferation. Our previous work assessing immune function in HO-1 Ϫ/Ϫ mice supported this notion because it showed a predominance of Th1-type cytokine secretion [eg, interleukin (IL)-1, interferon-␥, tumor necrosis factor-␣, IL-6] from splenocytes after polyclonal stimulation of T cells, implying that HO-1 activity is important in modulation of lymphocyte activation.5 This is of particular interest given reports that HO-1 is constitutively expressed in the CD4 ϩ CD25 ϩ subset of Treg cells, 6 and that HO-1 levels increase even further after T-cell stimulation.7 Furthermore, the report by Song and colleagues 8 demonstrated that carbon monoxide (CO), a byproduct of HO activity, has a very strong inhibitory effect on CD3-activated T-cell proliferation. Previously, we proposed a hypothetical model for the immunomodulatory effects of HO-1 in the maintenance of peripheral tolerance based on then available evidence that CO produced in regulatory T (Treg) cells may be an integral component of the suppression of T-cell activation by Treg cells in the presence of effector T (Teff) cells and antigen-presenting cells (APCs). 9The purpose of the present study was to analyze the role of HO-1 in Treg-mediated suppression. As a first step, we performed a phenotypic analysis of lymphocytes obtained from the peripheral lymphoid organs of HO-1 Ϫ/Ϫ mice for potential abnormalities. In the second step, we explored the functional significance of these findings in a series of T-cell proliferation/suppression assays. Finally, we examined the possibility that HO-1 exSupported by the National Institutes of Health (grants K08 AI 57362 to M.H.K., and R01 DK 075532 and R01 HL068157 to A.A).

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Inflammation culminating in fibrosis contributes to progressive kidney disease. Crosstalk between the tubular epithelium and interstitial cells regulates inflammation by a coordinated release of cytokines and chemokines. Here we studied the role of heme oxygenase-1 (HO-1) and the heavy subunit of ferritin (FtH) in macrophage polarization and renal inflammation. Deficiency in HO-1 was associated with increased FtH expression, accumulation of macrophages with a dysregulated polarization profile, and increased fibrosis following unilateral ureteral obstruction in mice; a model of renal inflammation and fibrosis. Macrophage polarization in vitro was predominantly dependent on FtH expression in isolated bone marrow-derived mouse monocytes. Utilizing transgenic mice with conditional deletion of FtH in the proximal tubules (FtHPT−/−) or myeloid cells (FtHLysM−/−), we found that myeloid FtH deficiency did not affect polarization or accumulation of macrophages in the injured kidney compared to wild-type (FtH+/+) controls. However, tubular FtH deletion led to a marked increase in pro-inflammatory macrophages. Furthermore, injured kidneys from FtHPT−/− mice expressed significantly higher levels of inflammatory chemokines and fibrosis compared to kidneys from FtH+/+ and FtHLysM−/− mice. Thus, there are differential effects of FtH in macrophages and epithelial cells, which underscores the critical role of FtH in tubular-macrophage crosstalk during kidney injury.

Heme oxygenase-1 (HO-1) is a cytoprotective enzyme that catalyzes the breakdown of heme to biliverdin, carbon monoxide, and iron. The beneficial effects of HO-1 expression are not merely due to degradation of the pro-oxidant heme but are also credited to the by-products that have potent, protective effects, including antioxidant, anti-inflammatory, and prosurvival properties. This is well reflected in the preclinical animal models of injury in both renal and nonrenal settings. However, excessive accumulation of the by-products can be deleterious and lead to mitochondrial toxicity and oxidative stress. Therefore, use of the HO system in alleviating injury merits a targeted approach. Based on the higher susceptibility of the proximal tubule segment of the nephron to injury, we generated transgenic mice using cre-lox technology to enable manipulation of HO-1 (deletion or overexpression) in a cell-specific manner. We demonstrate the validity and feasibility of these mice by breeding them with proximal tubule-specific Cre transgenic mice. Similar to previous reports using chemical modulators and global transgenic mice, we demonstrate that whereas deletion of HO-1, specifically in the proximal tubules, aggravates structural and functional damage during cisplatin nephrotoxicity, selective overexpression of HO-1 in proximal tubules is protective. At the cellular level, cleaved caspase-3 expression, a marker of apoptosis, and p38 signaling were modulated by HO-1. Use of these transgenic mice will aid in the evaluation of the effects of cell-specific HO-1 expression in response to injury and assist in the generation of targeted approaches that will enhance recovery with reduced, unwarranted adverse effects.