Catalytic iron and acute kidney injury

Abstract

Acute kidney injury (AKI) is a common and often devastating condition among hospitalized patients and is associated with markedly increased hospital length of stay, mortality, and cost. The pathogenesis of AKI is complex, but animal models support an important role for catalytic iron in causing AKI. Catalytic iron, also known as labile iron, is a transitional pool of non-transferrin-bound iron that is readily available to participate in redox cycling. Initial findings related to catalytic iron and animal models of kidney injury have only recently been extended to human AKI. In this review, we discuss the role of catalytic iron in human AKI, focusing on recent translational studies in humans, assay considerations, and potential therapeutic targets for future interventional studies.

acute kidney injury (aki) is a common and often devastating condition associated with markedly increased hospital length of stay, mortality, and cost (10, 53, 54). An important role for iron in the pathogenesis of AKI has long been appreciated. Iron, the most abundant transitional metal in the body, has the capacity to readily accept and donate electrons, cycling reversibly between ferrous (Fe²⁺) and ferric (Fe³⁺) forms. This property, which is essential for its functions as a critical component of cytochromes, oxygen-binding molecules (e.g., hemoglobin), and many enzymes, also makes iron dangerous in excess because it can catalyze the formation of free radicals, which can damage macromolecular components of cells.

Catalytic iron, also known as labile iron, is a transitional pool of extracellular and intracellular iron that is not transferrin bound, but rather loosely bound to albumin or low-molecular-weight metal complexing groups, such as citrate, acetate, malate, phosphate, and adenine nucleotides (17, 25, 51). Although plasma catalytic iron is only a small portion of total body iron (Fig. 1), it represents a pool of “free iron” that is readily available to participate in redox cycling. By facilitating the conversion of hydrogen peroxide to free-radical ions, a process known as the Fenton reaction, catalytic iron causes oxidative damage to cellular membranes, proteins, and DNA (23, 28). In this review, we discuss the role of catalytic iron in AKI, focusing on recent translational studies in humans, assay considerations, and potential therapeutic targets for future studies.

Fig. 1.

Distribution of cellular and extracellular iron. Extracellular (plasma) iron represents a small portion (∼3 mg) of total body iron (∼3,000–5,000 mg), the vast majority of which is intracellular (e.g., stored in ferritin, hemoglobin, and other cellular iron-containing proteins). Under normal physiological conditions, plasma iron is transferrin bound. In some disorders, a small portion of plasma iron is non-transferrin-bound iron (NTBI). The latter is either bound to albumin or to low-molecular-weight chelates, such as citrate. A portion of circulating NTBI is readily available to participate in redox cycling and is referred to as plasma catalytic iron or labile plasma iron (LPI).

Catalytic Iron in Animal Models of AKI

Two broad lines of evidence support a role for catalytic iron in the pathogenesis of AKI: catalytic iron levels are increased in the kidneys of animals with AKI, and iron chelators are protective (50). Specifically, catalytic iron has been implicated in AKI resulting from a wide range of insults, including ischemia-reperfusion injury (2), aminoglycosides (55), cisplatin (4), rhabdomyolysis (3), hemoglobinuria (43), and iodinated radiocontrast (24). In many of these models, pretreatment with an iron chelator prevents or attenuates AKI (4, 44, 55, 56). Until recently, the relevance of these findings to human AKI was unknown.

Catalytic Iron in Human AKI

Unlike animal models of kidney injury, the study of catalytic iron in human AKI has largely relied on measurement of catalytic iron levels in blood samples (methodology discussed below), since kidney tissue is unavailable in most patients with AKI. Robust animal data demonstrating a causal role for catalytic iron in AKI suggest that elevated plasma catalytic iron levels could serve as an important biomarker and potential mediator of AKI in humans. This hypothesis was recently tested in a series of prospective cohort studies that evaluated plasma and serum catalytic iron levels in three commonly encountered AKI clinical settings: cardiac surgery-associated AKI, critical illness-associated AKI, and contrast-induced AKI.

Cardiac surgery-associated AKI.

Leaf et al. (32) measured plasma catalytic iron levels, along with plasma free hemoglobin, total iron, transferrin, and ferritin, in 250 adult patients who underwent cardiac surgery with cardiopulmonary bypass (CPB). Each of these plasma iron markers was measured preoperatively, at the end of CPB, and on postoperative days 1 and 3. Plasma catalytic iron levels, but not other iron markers, rose significantly at the end of CPB (Fig. 2A) and were directly associated with CPB time and number of packed red blood cell (pRBC) transfusions. In multivariate analyses adjusted for age and preoperative estimated glomerular filtration rate, patients in the highest compared with lowest quartile of catalytic iron on postoperative day 1 had a greater odds of in-hospital death or need for renal replacement therapy (death/RRT), AKI, and several other adverse events postoperatively (Fig. 2B).

Fig. 2.

Plasma catalytic iron levels and adverse outcomes following cardiac surgery. A: plasma catalytic iron levels are significantly higher at the end of cardiopulmonary bypass (CPB) compared with preoperative (Preop) levels. †P < 0.001 and †††P < 0.05 for within-group comparison to preoperative levels. Furthermore, catalytic iron levels at the end of CPB and on postoperative day 1 (POD#1) are higher in patients who develop in-hospital death or need for renal replacement therapy (death/RRT). *P < 0.01 for between-group comparisons. Bars represent median (25th–75th interquartile range). B: patients with catalytic iron levels in the highest compared with lowest quartile on POD#1 have a greater risk of death/RRT, AKI, and in-hospital mortality after adjusting for age and preoperative estimated glomerular filtration rate. *P < 0.05. [Adapted from Leaf et al. (32) with permission.]

Critical illness-associated AKI.

In a separate study conducted by the same group (33), plasma catalytic iron levels were measured on intensive care unit day 1 in 121 critically ill patients admitted to medical or surgical intensive care units. Again, higher plasma catalytic iron levels were associated with a greater risk of death/RRT, AKI, and other adverse events. These associations were independent of age, estimated glomerular filtration rate, and number of pRBC transfusions.

Contrast-induced AKI.

In the largest study of catalytic iron and human AKI conducted to date, Lele et al. (34) measured serum catalytic iron levels in 806 patients with acute coronary syndrome who underwent coronary angiography. Catalytic iron levels were measured at baseline and at 24 and 48 h after exposure to iodinated radiocontrast. Patients with vs. without contrast-induced AKI had higher catalytic iron levels at 24 and 48 h.

Measurement of Catalytic Iron in Human Blood Samples

Catalytic iron can be measured in plasma or serum by two different methods: the bleomycin-detectable catalytic iron (BDI) assay, and the labile plasma iron (LPI) assay. A summary of the characteristics of the two assays is shown in Table 1. The BDI assay relies on the principle that the antitumor antibiotic, bleomycin, degrades DNA in the presence of catalytic iron and reducing agent (ascorbate). The DNA degradation products react with thiobarbituric acid to form a pink chromogen, which is quantified spectrophotometrically (16, 22). The LPI assay relies on the conversion of the nonfluorescent oxidation-sensitive probe, dihydrorhodamine, into the fluorescent rhodamine, in the presence of catalytic iron and reducing agent (ascorbate). Comparison of the generated fluorescence in the presence or absence of an iron chelator (deferrioxamine or deferiprone) confers specificity for exchangeable or chelatable iron (9, 15).

Table 1.

Comparison of the bleomycin-detectable catalytic iron assay vs. the labile plasma iron assay

	BDI	LPI
Reference assay	Halliwell et al. (22); Evans and Halliwell (16)	Esposito et al. (15)
Agents	Ascorbate, bleomycin, DNA, and thiobarbituric acid	Ascorbate and dihydrorhodamine
Method of detection	Colorimetric	Fluorescence
Lower limit of detection	Variable*	Variable‡
Interassay CV	Variable†	Variable‡
Commercially available	No	Yes (Aferrix Ltd., Tel-Aviv, Israel)
Used in clinical AKI studies	Yes (Refs. 32–34)	No
Used in clinical studies of iron-overload disorders	Yes (Refs. 8, 12)	Yes (Refs. 7, 9)

BDI, bleomycin-detectable catalytic iron; LPI, labile plasma iron; CV, coefficient of variation.

^*Depending on the laboratory, with 0.03 μmol/l reported by Leaf et al. (32).

^†Depending on the laboratory, with <10% reported by Leaf et al. (32).

^‡Depending on the laboratory or the commercial kit used (12).

In recent studies among patients undergoing hematopoetic stem cell transplantation, and among patients with iron-overload disorders, the BDI and LPI assays were compared and provided similar results (12, 27). Interestingly, in these studies both DBI and LPI were related to transferrin saturation (TSAT), with BDI and LPI mostly undetectable at TSAT < 80%. However, in patients undergoing cardiac surgery, as well as in critically ill patients, Leaf et al. detected elevated plasma concentrations of BDI at much lower TSATs (32, 33). It is unknown whether this is due to differences in the BDI vs. LPI assays, or differences in patient populations.

Therapeutic Targets

A schematic of the proposed pathophysiology of iron-associated AKI is provided in Fig. 3. Notably, the complex nature of iron homeostasis provides a variety of opportunities for pharmacological intervention. Targets that are particularly attractive for further study include iron chelators, haptoglobin, ferritin, and inducers of heme oxygenase-1 (HO-1) and the CD163/CD91 scavenger receptors.

Fig. 3.

Proposed pathophysiological mechanisms of catalytic iron-induced AKI. 1: Hemolysis, which can occur through endogenous or exogenous processes, results in the release of free hemoglobin (Hgb) into the plasma. 2: Free Hgb is bound by haptoglobin, and the complex is taken up by monocytes and macrophages via the scavenger receptor, CD163. CD163 can also facilitate receptor-mediated endocytosis of free Hgb, even in the absence of haptoglobin (48). 3: Once internalized, Hgb is broken down into heme, which is degraded further by heme oxygenase-1 (HO-1) into carbon monoxide (CO), biliverdin, and Fe²⁺. The latter is oxidized into Fe³⁺ and sequestered by ferritin, which is upregulated by HO-1 (41). 4: Free Hgb not sequestered by haptoglobin may be oxidized into free heme in the circulation. 5: Free heme is sequestered by hemopexin and taken up by hepatocytes, vascular smooth muscle cell (VSMCs), and other cell types via the scavenger receptor, CD91, also known as low density lipoprotein receptor-related protein 1 (LRP1). 6: When the ability of haptoglobin and hemopexin to scavenge free Hgb and free heme is overwhelmed, respectively, nonenzymatic degradation (i.e., in the absence of HO-1) may release catalytic iron from heme. 7: This catalytic iron catalyzes the formation of free radicals, which can damage macromolecular components of cells, resulting in AKI (5). 8: Even in the absence of catalytic iron generation, free Hgb and free heme may contribute to AKI by a variety of mechanisms, including nitric oxide (NO) sequestration, which results in vasoconstriction (14), oxidant-mediated cellular damage (46), vascular injury (30), and induction of apoptosis in the presence of other cytotoxic agonists, such as tumor necrosis factor (TNF) (49). Key targets for potential therapeutic intervention are colored in red. CPB, cardiopulmonary bypass; DIC, disseminated intravascular coagulation; HUS, hemolytic uremic syndrome; TTP, thrombotic thrombocytopenic purpura.

Iron chelation.

Iron chelators form a complex with iron and thereby promote its excretion through the urinary or biliary pathways. In various experimental models of AKI, pretreatment with the iron chelator, deferoxamine, protects animals against a wide variety of nephrotoxic insults (4, 44, 55, 56). Furthermore, administration of apotransferrin or neutrophil gelatinase-associated lipocalin, both endogenous iron-binding molecules, protects against kidney injury in animal models of renal ischemia-reperfusion injury (13, 36, 39). However, only a few small studies have attempted to extend these findings from animal AKI models to humans.

Menasche et al. (38) evaluated the effects of intravenous deferoxamine vs. placebo in 24 adult patients undergoing cardiac surgery with CPB. Although the study was underpowered to detect differences in rates of AKI between the two groups, isolated neutrophils from patients treated with deferoxamine produced fewer superoxide radicals than those from control patients. In a follow-up study by the same group, deferoxamine-treated patients undergoing cardiac surgery had lower plasma lipid peroxidation parameters than the control group, suggesting a potentially beneficial effect on iron-mediated oxidative stress (37).

Paraskevaidis and colleagues (45) randomly assigned 45 patients undergoing coronary artery bypass grafting to receive an 8-h intravenous infusion of deferoxamine, beginning immediately after induction of anesthesia, vs. placebo. Compared with placebo, deferoxamine-treated patients had lower peak levels of oxygen free-radical production, as well as higher left ventricular ejection fraction at 12 mo follow-up. Rates of AKI were similar between the two groups (45).

Lastly, Fraga et al. (18) randomly assigned 30 critically ill patients with sustained hypotension to receive combination therapy with deferoxamine and N-acetylcysteine vs. placebo. While the incidence of AKI was similar between the two groups, deferoxamine/N-acetylcysteine-treated patients had lower serum creatinine levels at hospital discharge compared with controls. A larger follow-up study by the same group was recently completed (NCT00870883), and results are awaited.

While these pilot studies appear promising, additional well-designed and adequately powered studies of iron chelation for the prevention and/or treatment of AKI are needed. One of the critical considerations for such studies will be the choice of iron chelator. The parenteral iron chelator, deferoxamine, was the drug used in most of the preclinical AKI models and has been used in humans for nearly half a century for the treatment of chronic iron overload disorders. In contrast, two oral iron chelators, deferasirox and deferiprone, became available more recently. Deferasirox and deferiprone have lower iron-binding affinities than deferoxamine (22.5 and 19.9 pM, respectively, compared with 26.6 pM), but their greater lipid solubility and enhanced intracellular penetrance suggest greater potential for chelation of cytosolic iron compared with deferoxamine. However, deferasirox has been associated with AKI (26), whereas deferiprone is excreted primarily via the urinary rather than the biliary pathway, potentially limiting its efficacy in patients with impaired renal function.

Haptoglobin.

By increasing the efficiency of free hemoglobin sequestration, administration of haptoglobin could attenuate hemoglobin/iron-mediated nephrotoxicity in clinical settings associated with hemolysis (e.g., cardiac surgery). This strategy was effective in preventing hemoglobin-induced AKI in guinea pigs (1), as well as pRBC transfusion-induced AKI in a murine model (21). In humans, therapeutic administration of a plasma-derived haptoglobin product developed by Benesis in Japan was evaluated in several small studies with mixed results (47). However, this product is not currently available.

Ferritin.

Ferritin is a key protein that has the capacity to sequester large amounts of intracellular iron and thereby protects against oxidant-mediated cellular damage (5). Recent animal studies have highlighted the importance of renal proximal tubular heavy chain ferritin expression in regulating renal inflammation and AKI (6, 57). Additionally, it has long been recognized that the renoprotective effects of HO-1, a key cytoprotective protein, may be largely attributable to upregulation of ferritin (41). In humans, there is conflicting epidemiological data on the role of ferritin in AKI. A pilot study of 30 patients found that low preoperative serum ferritin levels were associated with a greater incidence of AKI following CPB surgery (11). However, a larger subsequent study (n = 120) failed to confirm this association (52). Whether pharmacological targeting of ferritin could enhance the efficiency of catalytic iron sequestion and thereby improve renal outcomes remains to be studied.

HO-1.

HO-1 is the rate-limiting enzyme in the breakdown of heme into carbon monoxide, iron, and biliverdin. A wealth of data from animal models support its relevance as a cytoprotective enzyme in AKI (40). Additionally, a recent study found that common genetic polymorphisms in the HO-1 gene promoter, which are known to be associated with HO-1 expression and activity, are associated with postoperative AKI in patients undergoing cardiac surgery (31). Thus development of novel therapeutic agents that induce renal and extrarenal (e.g., monocyte) HO-1 expression as a strategy for AKI prevention has been a topic of great interest (35). Unfortunately, most of the known inducers of HO-1 (e.g., free heme, endotoxin, H₂O₂) are compounds that may be unsafe for administration to humans.

CD163/CD91.

Finally, targeted upregulation of the scavenger receptors, CD163 and CD91, could increase the efficiency of hemoglobin and heme removal from the circulation, respectively, and thereby attenuate heme/iron-mediated AKI. Glucocorticoids are known inducers of CD163 cell surface expression on monocytes (20) and CD91 expression on macrophages (42). Interestingly, a recent study found that glucocorticoids may be beneficial in preventing severe AKI among patients undergoing cardiac surgery (29). However, since this was a post hoc study, the findings should be interpreted with caution; moreover, a more recent study found no benefit of glucocorticoids in the prevention of cardiac surgery-associated AKI (19).

In conclusion, a wealth of data on catalytic iron in animal models of AKI have only recently begun to be translated to human AKI. Additional studies are needed to establish whether plasma catalytic iron may serve as a useful biomarker of AKI in humans. Moreover, adequately powered clinical trials are needed to assess whether interventions that target iron regulatory pathways may improve clinical outcomes in human AKI.

GRANTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant K23DK106448 (to D. E. Leaf).

DISCLOSURES

D. W. Swinkels is an employee of Radboudumc that offers high-quality NTBI and LPI assays on a fee-for-service basis (http://www.radboud-ironcenter.com/diagnostics/ntbi-lpi).

AUTHOR CONTRIBUTIONS

D.E.L. conception and design of research; D.E.L. and D.W.S. analyzed data; D.E.L. prepared figures; D.E.L. drafted manuscript; D.E.L. and D.W.S. edited and revised manuscript; D.E.L. and D.W.S. approved final version of manuscript.