Hepcidin Mitigates Renal Ischemia-Reperfusion Injury ...

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Hepcidin Mitigates Renal Ischemia-Reperfusion Injury by Modulating Systemic Iron Homeostasis Yogesh Scindia,*† Paromita Dey,*† Abhinav Thirunagari,* Huang Liping,*† Diane L. Rosin,†‡ Matteo Floris,* Mark D. Okusa,*† and Sundararaman Swaminathan*† *Division of Nephrology, †Center for Inflammation, Immunity and Regenerative Medicine, and ‡Department of Pharmacology, University of Virginia Health System, Charlottesville, Virginia

ABSTRACT Iron-mediated oxidative stress is implicated in the pathogenesis of renal ischemia–reperfusion injury. Hepcidin is an endogenous acute phase hepatic hormone that prevents iron export from cells by inducing degradation of the only known iron export protein, ferroportin. In this study, we used a mouse model to investigate the effect of renal ischemia–reperfusion injury on systemic iron homeostasis and determine if dynamic modulation of iron homeostasis with hepcidin has therapeutic benefit in the treatment of AKI. Renal ischemia–reperfusion injury induced hepatosplenic iron export through increased ferroportin expression, which resulted in hepatosplenic iron depletion and an increase in serum and kidney nonheme iron levels. Exogenous hepcidin treatment prevented renal ischemia-reperfusion–induced changes in iron homeostasis. Hepcidin also decreased kidney ferroportin expression and increased the expression of cytoprotective H-ferritin. Hepcidininduced restoration of iron homeostasis was accompanied by a significant reduction in ischemia-reperfusion– induced tubular injury, apoptosis, renal oxidative stress, and inflammatory cell infiltration. Hepcidin–deficient mice demonstrated increased susceptibility to ischemia-reperfusion injury compared with wild-type mice. Reconstituting hepcidin-deficient mice with exogenous hepcidin induced hepatic iron sequestration, attenuated the reduction in renal H-ferritin and reduced renal oxidative stress, apoptosis, inflammation, and tubular injury. Hepcidin-mediated protection was associated with reduced serum IL-6 levels. In summary, renal ischemia– reperfusion injury results in profound alterations in systemic iron homeostasis. Hepcidin treatment restores iron homeostasis and reduces inflammation to mediate protection in renal ischemia–reperfusion injury, suggesting that hepcidin-ferroportin pathway holds promise as a novel therapeutic target in the treatment of AKI. J Am Soc Nephrol 26: 2800–2814, 2015. doi: 10.1681/ASN.2014101037

Renal ischemia–reperfusion injury (IRI) is a major cause of AKI and is associated with increased morbidity, mortality, and prolonged hospitalization.1,2 Acute ischemia leads to ATP depletion, tubular epithelial injury, and hypoxic cell death. Reperfusion further amplifies injury by promoting the formation of reactive oxygen species (ROS) and inducing leukocyte activation, infiltration, and inflammation.3–6 Iron and ROS are important mediators of cell death and the ensuing inflammatory response during the course of IRI.7,8 During ischemia, cytochromes translocate from the mitochondria into the cytosol.9,10 Heme protein catabolism results in increase in labile ferrous (Fe2+) iron.11–13 Fe2+ catalyzes the generation of tissue damaging hydroxyl radical (OH–) by interacting with superoxide anion (O–2) and hydrogen 2800

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peroxide (H2O2), both of which are increased during IRI.7,14 Labile iron’s contribution to tissue damage has been demonstrated not only in renal IRI15,16 but also in other models of AKI.17,18 Iron chelation is protective in diverse animals models of AKI.19 Received October 25, 2014. Accepted January 11, 2015. Published online ahead of print. Publication date available at www.jasn.org. Present address: Dr. Matteo Floris, Department of Nephrology and Dialysis, G. Brotzu Hospital, Cagliari, Italy Correspondence: Dr. Sundararaman Swaminathan, Division of Nephrology, University of Virginia Health Sciences Center, PO Box 800133, Charlottesville, VA 22908-0133. Email: [email protected] Copyright © 2015 by the American Society of Nephrology

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Iron (Fe2+) is exported from cells by ferroportin, the only known iron export protein.20 Ferroportin is significantly expressed on macrophages, hepatocytes, and proximal and distal renal tubular cells.21–23 To sustain the physiologic iron requirement but avoid iron toxicity, systemic iron balance is regulated by hepcidin (hepatic antimicrobial peptide [HAMP]), an endogenous peptide hormone produced by hepatocytes.23,24 Hepcidin induces covalently modification of ferroportin, which leads to its internalization and lysosomal degradation, and thereby prevents cellular iron egress.25,26 Hepcidin is acutely and positively regulated during iron imbalance25 and inflammation27,28 and has antibacterial properties,29,30 whereas hypoxia and erythropoiesis negatively regulate its expression.31 Although human studies have indicated a positive correlation between increased urinary hepcidin levels and protection against AKI, its direct role has not been examined in any models of AKI.32 Here, we present evidence that renal IRI induces dynamic changes in renal and extrarenal iron homeostasis. Hepcidin-deficient mice sustain more severe renal injury after IRI and synthetic hepcidin mitigates renal IRI.

RESULTS Hepcidin Prevents Renal Ischemia-Reperfusion– Induced Iron Dyshomeostasis

There is a dynamic and continuous exchange of iron between hepatosplenic compartments and plasma. This process is regulated by hepcidin through its modulation of reticuloendothelial (splenic macrophages and hepatic kupffer cells) ferroportin expression. Renal IRI increases serum iron levels; however, no previous studies have characterized the dynamic changes in iron distribution between different organs after renal IRI. Here, we first confirmed the previous observations that renal IRI increases serum iron levels compared with sham-operated animals (Figure 1A). Hepcidin treatment significantly reduced the IRI-induced increase in serum iron (Figure 1A) and kidney nonheme iron content (Figure 1B). Hepatosplenic nonheme iron levels were significantly lower after IRI than in the sham group and hepcidin pretreated mice demonstrated significantly higher hepatosplenic nonheme iron content than IRI or sham groups (Figure 1, C and D). To determine if increased serum iron levels after renal IRI could result in an iron-dependent increase in hepcidin synthesis, we measured hepcidin transcripts and protein in liver and serum, respectively. Renal IRI resulted in significant upregulation of liver hepcidin gene expression and increased serum hepcidin levels compared with sham mice, and these were prevented by hepcidin treatment before IRI (Figure 1, E and F). Collectively, our observations indicate that IRI induces dynamic changes in iron metabolism characterized by hepatosplenic iron depletion, systemic iron mobilization, kidney nonheme iron accumulation, and post-IRI hepcidin induction. Hepcidin treatment before IRI effectively prevents these changes. J Am Soc Nephrol 26: 2800–2814, 2015

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Hepcidin Mitigates IRI, Reduces Acute Tubular Necrosis, and Improves Renal Function

We examined the functional significance of hepcidin-mediated modulation of iron homeostasis on IRI. Renal IRI produced a marked increase in plasma creatinine that was reduced by a single bolus of 50 or 100 mg hepcidin (intraperitoneally) given 24 hours before IRI (Figure 2B). Because there were no significant differences between the effects of 50 and 100 mg hepcidin, all further experiments were performed using the 50 mg hepcidin. We next established the timing of hepcidin injection that would offer maximum protection against IRI (Figure 2A). Although treatment at both 2 and 8 hours before IRI significantly reduced kidney injury, the protection was less than that observed with hepcidin treatment 24 hours before IRI (Figure 2B). In a separate experiment, to examine if hepcidin has renoprotective effects after the onset of reperfusion injury, we injected hepcidin 30 minutes after IRI. We observed a small but significant decrease in plasma creatinine (3.160.09 IRI group versus 2.660.05 hepcidin injected 30 minutes postIRI group; P,0.01). Therefore, hepcidin was most effective when injected 24 hours before IRI, and the benefit was partial when administered after the onset of reperfusion injury (Figure 2B). Quantification of the tubular injury by stereological analysis of hematoxylin and eosin-stained kidney sections correlated well with renal function and histology (Figure 2C). As compared to sham operated mice (Figure 2D, Supplemental Figure 1A), the kidneys of untreated IRI mice showed extensive tubular necrosis in the S3 segment in the outer stripe of the outer medulla and extending to the S1 and S2 segment of the proximal tubule in the deep cortex (Figure 2E, Supplemental Figure 1B). However, hepcidin-treated mice had negligible necrosis and luminal debris in the S1 and S2 segments of the proximal tubules and substantially reduced tubular damage and casts that were restricted to the S3 segment in the outer medulla (Figure 2F, Supplemental Figure 1C). Hepcidin pretreatment also reduced the kidney injury marker neutrophil gelatinase–associated lipocalin (NGAL) (Supplemental Figure 1D). Collectively, these findings demonstrate a protective role of hepcidin in renal IRI, amounting to approximately 70% reduction in injury as measured by plasma creatinine. Hepcidin Decreases Ischemia-Induced Renal Epithelial Apoptosis

Apoptosis is a major mediator of tubular cell death after renal IRI.33 To investigate if hepcidin-mediated protection in renal IRI is mediated through modulation of epithelial apoptosis, we measured apoptosis-related genes in kidneys of the three experimental groups. Compared with sham-operated mice, caspase-3 expression was significantly increased in the kidneys of untreated IRI mice, and this was prevented by hepcidin (Figure 3A). Expression of Bcl-2, an antiapoptotic gene with known protective properties in renal IRI,34 was significantly Iron Dynamics and Kidney Injury

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Hepcidin Treatment Reduces Renal IRI-Induced Oxidative Stress and Inflammation

Oxidative stress-induced apoptotic cell death is believed to be one of the major pathways involved in the pathogenesis of kidney IRI.35,36 Iron is known to play a central role in oxidative tissue injury through its ability to catalyze the redox cycle. Iron chelation and antioxidants attenuate IRI.37,38 Because hepcidin treatment directly modulates IRI-induced iron dyshomeostasis, we examined whether the reduction in injury is associated with lowering of oxidative stress. 4-hydroxynonenal (4-HNE), an aldehyde product of membrane lipid peroxidation, is used as a tissue marker of oxidative stress.39 Kidneys of untreated IRI mice showed prominent 4-HNE immunoreactivity. The pattern of staining mirrored renal injury distribution, being most intense in the medulla with lesser intensity in the corticomedullary junction and deep cortex. In contrast, kidneys of hepcidin-treated mice showed significantly reduced 4-HNE immunoreactivity (Figure 4A, Supplemental Figure 2, A and B). Apoptosis and ROS generation are early events after renal IRI, and they are followed by the infiltration of immune cells, which leads to further amplification of kidney pathology after IRI.6,40,41 Therefore, we investigated whether hepcidin treatment is Figure 1. Hepcidin pretreatment prevents renal IRI-induced dysregulation of systemic associated with decreased kidney infiltrairon homeostasis. Mice were subjected to sham operation or 26 minutes of kidney tion of inflammatory cells. IRI resulted ischemia followed by 24 hours of reperfusion. Hepcidin (50 mg, intraperitoneally) was in a large increase in CD45+ cells in kidney administered 24 hours before IRI. Serum iron was measured by ELISA and normalized to sample volume (A). Nonheme iron was measured after acid digestion of the kidneys compared with sham; however, hepcidin (B), spleen (C), and liver (D), normalized to tissue weight and expressed as micrograms treatment reduced renal inflammatory cell inper gram of tissue. Liver hepcidin expression was measured by quantitative real-time filtration by almost half (Figure 4, B and C). PCR and normalized to glyceraldehyde-3-phosphate dehydrogenase (E). Hepcidin Consistent with prior studies, we found that + hi + hi content in the serum was measured by ELISA (F). □, Sham; ○, IRI; ▽, Hepcidin+IRI. neutrophils (CD45 CD11b Ly6G Ly6C *P,0.05; **P,0.01; ***P,0.005; ****P,0.001. Data points are plotted as mean6SEM cells) were the most abundant immune cells (n=4–5 per group). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. in the kidneys after IRI. Hepcidin treatment significantly reduced IRI-induced renal neutrophil infiltration (Figure 4C). Collectively, these observations demonstrate that hepcidin treatment reduces reduced in kidneys after IRI and hepcidin treatment rerenal IRI-associated oxidative stress and inflammation. stored Bcl-2 expression to levels seen in sham-operated mice (Figure 3B). These observations were further supported by terminal-deoxynucleotidyl transferase-mediated digoxigeninHepcidin Decreases IRI-Induced Renal Ferroportin and Increases the Expression of Cytoprotective H-Ferritin deoxyuridine nick-end labeling (TUNEL) assay. We did not The primary known function of hepcidin is to induce ferroportin detect any TUNEL-positive cells in kidney sections of shamdegradation and increase intracellular iron stores. In turn, this operated mice (data not shown). There was an increase in TUNEL reactivity in renal tubules after IRI (Figure 3, C and D), results in the induction of H-ferritin, an iron binding ferroxidase with cytoprotective function.42 To determine the involvement of which was greatly reduced in hepcidin-treated kidneys (Figure 3, this pathway in hepcidin-mediated protection, we measured the E and F). 2802

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minutes ischemia, 24 hours reperfusion), which did not result in a rise in plasma creatinine in wild-type (WT) mice. However, plasma creatinine was significantly elevated in hepcidin knockout (Hamp2/2) mice after subIRI (Figure 6A). We next treated Hamp2/2 mice with a single 50 mg bolus of hepcidin, 24 hours before renal ischemia, and found that hepcidin reconstitution rescued these mice from IRI (Figure 6A). Renal histology and the tubular injury scores paralleled creatinine (Figure 6B). There were a large number of casts and necrotic tubules in the S1, S2, and S3 segments in Hamp2/2 kidneys after subIRI. In contrast, casts and necrotic tubules were remarkably less in hepcidin-treated Hamp2/2 mice after subIRI, and the occasional necrotic tubules were restricted to the deep medullary region (Figure 6, C–E, Supplemental Figure 3, A–C). NGAL expression paralleled changes in creatinine and histology (Supplemental Figure 3D). Because Hamp2/2 mice exhibit chronic iron overload, we investigated if protection induced by reconstitution of hepcidin was associated with changes in systemic iron levels. Compared with sham mice, there Figure 2. Hepcidin pretreatment mitigates kidney injury after renal IRI. Experimental was a paradoxic trend toward lowering of design and time course of treatment (A). Plasma creatinine was measured 24 hours after serum iron in Hamp2/2 mice after IRI. IRI in vehicle or hepcidin-injected (50 or 100 mg/mouse, intraperitoneally) mice and However, hepcidin reconstitution significompared with sham-operated mice (B). Tubular injury score (semiquantitative) of cantly lowered iron even further (Figure hematoxylin and eosin–stained kidneys was calculated for each section as mentioned in the Concise Methods section (C). Representative morphology (by hematoxylin and 6F). Changes in serum iron were associated eosin staining) of kidneys after sham operation (D), IRI (E), and hepcidin-treated IRI (50 with increased hepatic sequestration of 2/2 mice, mg/mouse, 24 hours before IRI) (F). Arrow denotes edge of the kidney section. IRI nonheme iron after IRI in Hamp resulted in severe tubular injury originating from the medullary region (S3) and ex- and this was further amplified by hepcidin tending all the way to the deep cortex (S1 and S2), whereas with hepcidin treatment, treatment (Figure 6G). Although Prussian injury was restricted to occasional tubules in the S3 segment of the medullary region. blue staining demonstrated qualitative Scale bar is 100 mm. □, Sham; ○, IRI; ▪, Hepcidin+IRI (-2Hr); △, Hepcidin+IRI (-8Hr); increases in splenic iron content in hepcidin▽, Hepcidin+IRI (-24Hr); ◇, 100 mg Hepcidin+IRI (-24Hr). Data are represented as treated Hamp 2 /2 IRI mice, we did not mean6SEM. *P,0.05; ***P,0.005; ****P,0.001 (n=4–5 per group). observe differences in the nonheme iron content of the spleen and kidneys of sham, untreated, and hepcidin-treated Hamp2/2 expression of these proteins and found that increase in renal mice after IRI (data not shown). Taken together, our data ferroportin after IRI was prevented by pretreatment with hepcidin show that hepcidin deficiency significantly increases (Figure 5, A and B). H-ferritin showed a decreasing trend after the kidney’s susceptibility to IRI and that reconstitution IRI compared with sham levels. However, hepcidin treatment with hepcidin at least partially restores iron homeostasis resulted in significantly higher H-ferritin after IRI (Figure 5, and results in mitigation of IRI. C and D). Hepcidin Reconstitution Preserves H-Ferritin and Hepcidin Deficiency Increases Susceptibility to AKI Prevents Apoptosis Given the significance of hepcidin and ferroportin regulation Similar to the observations in WT mice, a decrease in kidney in renal IRI, we hypothesized that genetic hepcidin deficiency H-ferritin was observed after IRI in Hamp2/2 mice, and this would aggravate IRI. To reveal exacerbation of injury, we was partially restored by reconstitution with hepcidin (Figure subjected mice to a subthreshold ischemic injury (subIRI) (24 7, A and B). We next measured apoptosis-related genes and J Am Soc Nephrol 26: 2800–2814, 2015

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Hepcidin Reconstitution Restores Protection against IRI-Induced Oxidative Stress and Inflammation in Hamp2/2 Mice

To evaluate the role of ROS in mediating injury in Hamp2/2 mice, we stained kidneys for 4-HNE. Immunoreactivity for 4-HNE, which was not observed in sham-operated Hamp2/2 mice, increased dramatically in these mice after IRI, particularly in the papilla, inner medulla, and along the corticomedullary junction. However, hepcidinreconstituted animals demonstrated greatly reduced immunoreactivity to 4-HNE (Figure 8, A–C). These observations suggest that protection mediated by hepcidin in Hamp2/2 mice is associated with reduced IRI-induced oxidative stress. Substantial neutrophil (7/4 antigen positive cells) and CD11b+ cell infiltration was detected in kidneys of Hamp2/2 mice after IRI (Figure 8, D and F), but it was not detected in sham-operated Hamp2/2 mice (data not shown); this was almost completely prevented by hepcidin-reconstitution in Hamp2/2 IRI mice (Figure 8, E and G). The reduction in inflammation correlated well with the lack of apoptosis and ROS in the protected animals. Figure 3. Hepcidin reduces apoptosis in the kidneys of mice after IRI. Hepcidin treatment protects kidneys from IRI-induced apoptosis [(A)–(F)]. Mice receiving hepcidin (50 mg, intraperitoneally) 24 hours before IRI had a lower transcriptional activation of proapoptotic caspase-3 (A) and better preservation of antiapoptotic Bcl-2 (B); n=3–5. Data for panels (A) and (B) are represented as mean6SEM of 2DDCT values normalized to glyceraldehyde-3phosphate dehydrogenase. TUNEL reactivity was used to assay apoptosis after renal IRI; fluorescein-dUTP-labeled apoptotic cells. Untreated IRI mice showed severe apoptosis in the cells of the corticomedullary region [(C)–(D)] that was reduced by hepcidin pretreatment [(E)–(F)]; scale bar is 200 mm [(C) and (E)] and 100 mm [(D) and (F)]. □, Sham; ○, IRI; ▽, Hepcidin+IRI. *P,0.05; **P,0.01; ***P,0.001. Data points are plotted as mean6 SEM of triplicates. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

indicators. Caspase-3 was significantly upregulated in Hamp2/2 mice after IRI, but this was prevented by a single dose of hepcidin (Figure 7C). Expression of the antiapoptotic gene Bcl-2 was significantly lower in kidneys of Hamp2/2 IRI mice, and hepcidin treatment prevented this change (Figure 7D). We observed a large number of TUNEL-positive cells, especially in the corticomedullary region of the kidneys of Hamp2/2 mice after IRI. Hepcidin reconstitution dramatically reduced the number of TUNEL-positive cells (Figure 7, E–G, Supplemental Figure 4, A–C). Collectively, these findings demonstrate that renal IRI in the setting of hepcidin deficiency is associated with reduced cytoprotective H-ferritin levels and increased epithelial apoptosis. Hepcidin reconstitution preserves H-ferritin and prevents apoptosis. 2804


Hepcidin Prevents IRI-Induced Ferroportin Upregulation and Splenic Iron Depletion

The spleen plays an important role in the pathophysiology of renal IRI.43–45 Splenic macrophages play a sentinel role in systemic iron homeostasis. Because splenic iron content decreases after renal IRI, we examined the expression of ferroportin in splenic macrophages. Renal IRI resulted in an upregulation of ferroportin in the splenic F4/80+ red pulp macrophages in both WT and Hamp2/2 mice, and hepcidin treatment prevented this (Figure 9A). In sham-operated Hamp2/2 mice, because of chronic hepcidin deficiency, basal splenic ferroportin expression was much higher than in the WT mice. As expected, its expression did not further increase after IRI. However, hepcidin reconstitution reduced ferroportin expression in Hamp2/2 IRI mice as compared to sham-operated Hamp 2/2 mice (Figure 9A). Because hepcidin-induced ferroportin downregulation is expected to result in intracellular iron retention, we hypothesized that this would lead to an increase in H-ferritin synthesis. Compared with sham-operated mice, there was no significant change in H-ferritin levels in spleens of WT or Hamp2/2 mice J Am Soc Nephrol 26: 2800–2814, 2015

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Figure 4. Hepcidin reduces oxidative stress and infiltration of CD45 positive cells and neutrophils in the kidney after IRI. Reactivity to 4-HNE was used as a measure of oxidative stress in the kidney after IRI (A). Renal IRI resulted in generation of ROS-mediated oxidative stress as shown by strong reactivity to 4-HNE (brown immunoperoxidase labeling) in the corticomedullary region. This was reduced by hepcidin pretreatment. Representative stitched images from two different experiments with four to five each; blue, methylene blue counterstain; scale bar is 200 mm. Infiltration of immune cells (CD45+ as a percentage of total cells) and neutrophils (Ly6G+Ly6C+cells within CD45+ CD11b+gates) in kidneys of sham, IRI, and hepcidin-treated IRI mice was studied by FACS [(B) and (C)]. Representative gates depict CD45, CD11b, and Ly6G and Ly6C populations (B). Hepcidin reduced the IRI-induced infiltration of total CD45 cells and neutrophils (C). Data are from one of three different experiments with four to five mice in each group. □, Sham; ○, IRI; ▽, Hepcidin+IRI. **P,0.001; ***P,0.005; ****P,0.0001. Data points are plotted as mean6SEM.

after IRI. However, hepcidin-treated WTand Hamp2/2 IRI mice showed a significant increase in H-ferritin (Figure 9, B and C). Therefore, renal IRI results in an increase in splenic ferroportin expression. Hepcidin treatment prevents this to induce splenic iron accumulation and an iron-dependent increase in H-ferritin. J Am Soc Nephrol 26: 2800–2814, 2015

Iron and Hepcidin Have Contrasting Effects on IL-6 Production

IL-6 is a pleiotropic cytokine that has been linked with the pathogenesis of renal IRI.46,47 To investigate if hepcidinmediated protection in renal IRI was associated with changes Iron Dynamics and Kidney Injury

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chelators and NGAL. Iron-chelating agents are thought to be beneficial in renal IRI because of their iron binding effects in intra- and extracellular spaces.15,37,48 Conversely, NGAL was shown to be of benefit in AKI through its ability to deliver iron to viable cells to enhance recovery. 49 Our findings not only demonstrate the role of iron in IRI but also reveal a more profound role of iron export and extrarenal iron homeostasis in renal pathophysiology. The implication of iron in pathogenesis of AKI is rapidly unfolding. A recent study Figure 5. Hepcidin prevents IRI-induced upregulation of ferroportin and H-ferritin demonstrated worse outcome of AKI after degradation. Expression of ferroportin in the kidney membrane fraction and H-ferritin deletion of H-ferritin from renal proximal in whole kidney lysates were measured by quantitative western blots. Equal protein tubules. 50 Similarly, in a liver ischemia loading was confirmed using Ponceau staining for ferroportin and with glyceraldehydemodel, H-ferritin suppressed inflammation 3-phosphate dehydrogenase (GAPDH) for H-ferritin blots, respectively. Hepcidin and had a direct antiapoptotic effect on hepprevented IRI-induced upregulation of ferroportin [(A) and (B)], and loss of H-ferritin [(C) 42 and (D)] in the kidney. Representative blots from three independent experiments are atocytes. Each2+molecule of H-ferritin can shown. □, Sham; ○, IRI; ▽, Hepcidin+IRI. Quantitation of ferroportin and H-ferritin bind to 4500 Fe ions, making it an impor51 was carried out using densitometry software; values were normalized to Ponceau or tant endogenous iron chelator. Sequestra2+ tion of Fe by H-ferritin is known to prevent GAPDH, respectively, and are expressed as mean6SEM. *P,0.05; **P,0.01. iron-mediated formation of ROS and cell injury. In our study, hepcidin treatment increased renal H-ferritin levels after IRI. Therefore, by limiting in IL-6, we measured the serum IL-6 levels by ELISA. Renal IRI the availability of free intracellular iron in renal cells, H-ferritin resulted in a significant increase in serum IL-6 in both WTand could potentially reduce renal ROS and the ensuing injury. This is Hamp2/2 mice, which was prevented by hepcidin treatment supported by our finding that lipid peroxidation (a marker of (Figure 10, A and B). To explore the possibility that free iron iron-mediated injury/ferroptosis) is reduced in hepcidin-treated might directly regulate IL-6 production in immune cells, we mice subjected to IRI.52 treated splenocytes from WT mice with ferrous ammonium chloride and measured secreted IL-6. Treatment with iron Previous studies have suggested an important role of the significantly increased splenocyte IL-6 secretion compared spleen43–45 and liver53 in the pathophysiology of renal IRI. We with untreated ones (Figure 10C). These findings suggest demonstrate that IRI induces hepatosplenic iron export, which that excess splenocyte-free iron may increase the production likely contributes to the pathogenesis of renal IRI. Splenic macof IL-6, a cytokine implicated in the pathogenesis of renal IRI. rophages along with hepatocytes are the main storage sites and sources of iron in the body.54 Because hepcidin acts rapidly on splenic macrophages,55 under our experimental conditions, contributions from the spleen are likely to be more critical. Hepcidin DISCUSSION induced splenic ferroportin degradation and H-ferritin inducIn this study we present several novel observations related to tion, thereby causing splenic iron retention. These changes prevent systemic iron mobilization after IRI and limit its availability dynamic changes in renal and extrarenal iron homeostasis after to participate in ROS generation in the kidney. Despite having IR, and identify a novel renoprotective effect of exogenous high splenic ferroportin expression after IRI, both untreated WT hepcidin. We demonstrate that renal IRI results in a systemic and Hamp2/2 mice did not demonstrate a decrease in H-ferritin. iron mobilization from the hepatosplenic compartments and is associated with kidney iron accumulation. Hepcidin prevents This could be attributed to an iron-independent, cytokinethese changes by inducing ferroportin degradation, H-ferritin mediated induction of H-ferritin.56 The mechanism of parainduction, and effective hepatosplenic iron sequestration. doxical increase in splenic ferroportin despite ongoing tissue Hepcidin-induced restoration of iron homeostasis is associinjury after IRI is unclear. One could speculate that renal hypoxia ated with reduced ROS, apoptosis, and inflammation and after IRI triggers stress erythropoietic response,57 which in turn nearly 70% improvement of renal function after IRI. We also could result in signals that induce ferroportin-dependent macprovide evidence that hepcidin deficiency in mice severely rophage iron export. increases susceptibility to renal IRI and that hepcidin reAnother novel observation is the increase in endogenous constitution restores protection. hepatic and serum hepcidin after renal IRI. Hepatic hepcidin The beneficial effect of modulating iron metabolism in AKI production increases after endotoxemia and inflammation.58 has been demonstrated in previous studies that used iron Similarly, hepcidin levels increase after liver IRI.59 Therefore, 2806


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Our data in Hamp2/2 mice does not completely mimic the findings in WT mice, where the serum iron levels decreased and liver iron content increased after renal IRI. However, these mice are constitutively iron overloaded because of the lack of hepcidin.60 It has been demonstrated that hepatic ferritin content in patients with hemochromatosis is higher than normal individuals.61 Hence, liver ferritin could potentially act as a sink for excess iron released after kidney injury. Treating these animals with hepcidin could further increase ferritin content in the liver to facilitate iron sequestration after renal injury. CD45+ immune cells infiltrate the kidney after reperfusion, with neutrophils playing a major role in ensuing pathologic response.6,40 Hepcidin treatment reduced the infiltration of total CD45+ cells, most of which were neutrophils. Whether hepcidin affects the chemotaxis of immune cells to the kidney or the reduction in infiltration is a consequence of less epithelial injury needs further study. Inflammatory cytokines, such as IL-6, are deleterious to the outcome of renal IRI.46,47 It is made locally by injured epithelial cells and by infiltrating macrophages.62 Interestingly, IL-6 is also a powerful inducer of hepcidin.63 Hepcidin treatment prevented the IRIinduced increase in systemic IL-6 levels. Furthermore, our in vitro data suggest an Figure 6. Renal IRI is exacerbated in hepcidin-deficient mice and mitigated by re- iron-dependent increase in splenic IL-6 secreconstitution with exogenous hepcidin. Mice were subjected to subIRI (24 minutes is- tion could be an important source. In support chemia), which was not sufficient to increase plasma creatinine significantly in WT mice of our findings, De Domenico et al. showed after 24 hours of reperfusion but produced a large increase in creatinine in Hamp2/2 mice. that iron-loaded bone marrow macrophages Hepcidin reconstitution (50 mg hepcidin, intraperitoneally, 24 hours before IRI) rescued treated with hepcidin produced less IL-6 the Hamp2/2 mice from IRI (A). Tubular injury score of hematoxylin and eosin–stained and TNF-a on stimulation with LPS. 64 kidney sections (B). Data are represented as mean6SEM. ****P,0.001 (n=3–5 per Collectively, these observations suggest group). Representative morphology of Hamp2/2 kidneys (by hematoxylin and eosin staining) in sham (C), IRI (D), and hepcidin plus IRI (E) mice. Arrow denotes edge of the that hepcidin treatment reduces IRI-induced kidney section. IRI resulted in extensive tubular necrosis and cast formation throughout IL-6 production through modulation of iron the kidney, whereas hepcidin-treated mice showed remarkably few injured tubules. Scale metabolism. Our studies demonstrate a novel renal bar is 100 mm. Serum iron was measured by ELISA and normalized to sample volume (F). Hepcidin reconstitution significantly reduced serum iron in Hamp2/2 IRI mice compared protective effect of hepcidin. We propose that with both untreated Hamp2/2 IRI and Hamp2/2 sham-operated mice. Nonheme iron was hepcidin mediates protection by acting not measured after acid digestion of liver and normalized to tissue weight (G). Hepcidin- only on the renal cells but also through its treated Hamp2/2 IRI mice had significantly higher hepatic nonheme iron compared with effects on extrarenal iron homeostasis and both untreated Hamp2/2 IRI and Hamp2/2 sham-operated mice. ○, WT; c, Hamp2/2 inflammation. The protective effects of hepSham; ▪, Hamp2/22IRI; ▲, Hamp2/22Hepcidin2IRI. *P,0.05; **P,0.01; ***P,0.001. cidin are mediated through ferroportin degData points are plotted as mean6SEM. radation, subsequent H-ferritin induction, and sequestration of free iron. Further studies under settings of both sterile (IRI) and nonsterile acute inflam- are required to investigate the relative contribution of renal and mation, hepcidin is induced to facilitate iron sequestration and extrarenal ferroportin in mediating the protective effects of hepcidin. thereby prevent iron-dependent oxidant injury. J Am Soc Nephrol 26: 2800–2814, 2015


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Figure 7. Hepcidin treatment mitigates renal H-ferritin degradation in hepcidin-deficient mice and is associated with reduced apoptosis. Expression of H-ferritin in whole kidney lysates was measured by quantitative western blots. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to confirm equal protein loading and normalize H-ferritin. IRI resulted in a significant reduction in H-ferritin in the kidneys of both untreated and hepcidin-treated Hamp2/2 mice compared with sham Hamp2/2 mice [(A) and (B)]. Hepcidin-treated Hamp2/2 IRI mice had significantly higher H-ferritin levels compared with untreated Hamp2/2 IRI mice. *P,0.05; **P,0.01; ***P,0.001. Hepcidin reconstitution protects Hamp2/2 kidney from IRI-induced apoptosis. Hepcidin-treated Hamp2/2 IRI mice had lower transcriptional activation of proapoptotic caspase-3 (C) and higher levels of antiapoptotic Bcl-2 (D); n=3–4. c, Hamp2/2 Sham; ▪, Hamp2/22IRI; ▼, Hamp2/22Hepcidin2IRI. *P,0.05; **P,0.01. Data are represented as mean6SEM of 2DDCT normalized to GAPDH. TUNEL reactivity was used to assay apoptosis after renal IRI. Sham-operated Hamp2/2 mice did not show signs of apoptosis (E). Untreated Hamp2/2 IRI mice showed severe apoptosis in the cells of the corticomedullary region (F), which was markedly reduced in the hepcidin-treated Hamp2/2 IRI mice (G). Representative images from two different experiments with four to five each are depicted. Scale bar is 200 mm.

CONCISE METHODS Mice and Surgical Protocol All experiments were performed in accordance with the National Institutes of Health and Institutional Animal Care and Use Guidelines. The Animal Care and Use Committee of the University of Virginia approved all procedures and protocols. Male 8- to 10-week-old C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME) and hepcidin knockout 2808


mice (Hamp2/2), a kind gift from Dr. Sophie Vaulont (National Institute of Health and Medical Research, Paris, France) and Dr. Cindy Roy (Johns Hopkins University), were used for all experiments. Kidney ischemia– reperfusion surgery was performed as previously reported.45 The core temperature of the mice was maintained between 34°C and 36°C using a heating pad. After anesthetizing the mice with an intraperitoneal injection of ketamine (120 mg/kg), xylazine (12 mg/kg), and atropine (0.324 mg/kg), bilateral flank incisions were made, and both renal pedicles were exposed J Am Soc Nephrol 26: 2800–2814, 2015

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Blood and Tissue Samples Before euthanasia, animals were anesthetized and blood was drawn from the axilla, after which tissues were harvested. All the tissue slices were fixed with 10% neutral-buffered formalin for paraffin embedding and periodate-lysine-paraformaldehyde fixative (PLP) to be frozen in optimal cutting temperature compound or snap frozen in liquid nitrogen for subsequent mRNA extraction.

Renal Function Plasma creatinine, determined using a modified Jaffe colorimetric assay,45 was used as an indicator of renal function.

Histology and Stereologic Analysis of Tissue Morphology

Kidneys were fixed in buffered formalin for 48 hours, embedded in paraffin, sectioned (3-mm thickness), and stained with hematoxylin and eosin. The extent of acute tubular necrosis was assessed in an unbiased, systematic manner using design-based stereology to achieve statistically accurate random sampling of kidney sections. The investigator was blinded to the experimental identity of the sections. Sections were imaged by using a Zeiss Axio Imager Z1/ApoTome Microscope fitted with motorized focus drives and motorized XYZ microscope stage and integrated to a workstation running Stereo Investigator software (MBF Bioscience, Figure 8. Hepcidin reconstitution of hepcidin-deficient mice reduces renal IRI-induced Williston, VT). The area fraction fractionator oxidative stress and results in decreased infiltration of immune cells. 4-HNE immu- probe was used for stereologic analysis of the noreactivity was used as an indicator of oxidative stress in the kidneys of Hamp2/2 mice fractional area of the medulla (the region of after IRI. Sham-operated Hamp2/2 mice did not show reactivity to 4-HNE (A). Renal IRI the kidney most susceptible to ischemic injury) in Hamp2/2 mice resulted in increased oxidative stress as indicated by the extensive occupied by tubular necrosis. The following 4-HNE immunoreactivity in the papilla, deep medulla, and corticomedullary regions parameters were defined: counting frame (B). Kidneys of hepcidin-treated Hamp2/2 IRI mice showed very little 4-HNE immu- (4003400 mm), sample grid (8003800 mm), noreactivity, indicative of reduced oxidative stress (C). Representative images from and grid spacing (85 mm). These values were two different experiments with four to five mice each are depicted; brown, 4-HNE determined empirically such that adequate immunoperoxidase labeling; blue, methylene blue counterstain. Scale bar is 200 mm. Immunofluorescence labeling of kidneys showed large infiltration of neutrophils (D) (7/ numbers of sample sites were visited and ade4; green) and CD11b cells (F) (CD11b; red) in untreated Hamp2/2 IRI mice. Infiltration quate numbers of markers (indicating injured of neutrophils (E) and CD11b cells (G) was dramatically reduced in hepcidin-treated tubules) were acquired, in keeping with accepted Hamp2/2 IRI mice (arrows). Nuclei were stained with DAPI (blue). Scale bar is 100 mm. counting rules for stereology. A total of 16.060.6 (mean6SEM) grid sites were evaluated per section; the sampling fraction was 25% of a total average area of 5.15310660.33 mm2 for each kidney section. Injured and cross-clamped for 26 minutes (severe IRI) or 24 minutes (subIRI). tubules were identified on the basis of the presence of cast formation, Clamps were then removed, and kidneys were allowed to reperfuse for 24 tubule dilation, and/or tubular epithelial denucleation. To better hours. Ambient postoperative air temperature was maintained between represent the degree of injury, which was extensive in some groups 30°C and 32°C during the entire reperfusion period. Sham-operated aniand not adequately reflected in the fractional area of injury, a mals underwent bilateral flank incisions without clamping of renal pedicles. weighted injury score was calculated for each section as follows: Twenty-four hours later, mice were euthanized, and tissues were harvested. [(the fraction of area occupied by casts and severely degenerated Animals were injected with dH2O (vehicle) or hepcidin (50–100 mg/mouse, tubules)32]+(the fraction of area occupied by mild to moderately intraperitoneally; Peptide International) 2, 8, or 24 hours before IRI. injured tubules). In some experiments hepcidin was injected 30 minutes after IRI. J Am Soc Nephrol 26: 2800–2814, 2015


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Figure 9. Hepcidin treatment prevents renal IRI-induced upregulation of splenic ferroportin and increased expression of cytoprotective H-ferritin. Immunofluorescence labeling of spleen indicated upregulation of ferroportin (green) in F4/80+ macrophages (red) after renal IRI (A: middle panel). Hepcidin treatment resulted in degradation of ferroportin, which could be detected in only a few scattered F4/80+ cells in the red pulp region (A: right panel). Scale bar is 100mm. Ferroportin expression is constitutively high in spleens of Hamp2/2 mice compared with WT mice (A: left panel). Expression of H-ferritin in spleen lysates was measured by quantitative western blots using b-actin as the loading control. Hepcidin increased splenic H-ferritin levels in both WT (B) and Hamp2/2 (C) mice after renal IRI. Representative blots from three independent experiments are shown. B (WT) and C (Hamp2/2). c, Sham; ▪, IRI; ▲, Hepcidin+IRI. Quantitation of H-ferritin was carried out using densitometry software and is expressed as mean6SEM. *P, 0.05 (B); **P,0.005 (C).

Serum Iron, Hepcidin, and IL-6 Measurement Serum iron (Abcam, Inc.), hepcidin (Intrinsic LifeSciences), and IL-6 levels (eBioscience) were measured using commercial ELISA kits, as per manufacturers’ instructions.

Nonheme Iron Assay Nonheme iron content (microgram of iron per gram of tissue) was measured as described previously,65 with slight modifications. Briefly, tissues were weighed, finely cut, and incubated for 20 hours at 65°C in a solution of 3 M HCl/0.61 M TCA. After cooling to room temperature, 2810


the acid extract was spun at 12,000 rpm, 0.1 ml of the supernatant was incubated with 5 chromogen solution (1.86 mM bathophenanthroline sulfonate/143 mM thioglycolic acid) for 20 minutes, and OD was measured at 535 nm. A standard curve was generated using an iron standard solution (Ricca Iron AA standard) against water as the blank. Nonheme iron content was calculated using the equation described.65

Flow Cytometry Single-cell suspensions from the kidney were prepared using standard methods. Briefly, kidney was cut into small pieces and digested with

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Figure 10. High iron levels after IRI are associated with an increase in IL-6 that is reduced by hepcidin. Serum IL-6 levels were measured by ELISA. Hepcidin administration significantly reduced IRI-induced serum IL-6 in both WT and Hamp2/2 mice [(A) and (B)] (n=3–5). In vitro treatment of primary mouse splenocytes with 10 mM ferrous ammonium citrate (Fe) for 4 hours induced higher IL-6 secretion compared with saline-treated ones (C). A (WT) and B (Hamp2/2). c, Sham; ▪, IRI; ▲, Hepcidin+IRI. Untreated splenocytes (c); Fe treated splenocytes (▪). Representative data of two independent experiments. Data points are plotted as mean6SEM. **P,0.01.

collagenase (type 2; Worthington) for 20 minutes at 37°C. The digested kidney was then passed serially through a 100- and 50-mm sieve to collect the cell suspension. The cells were then incubated with antiCD16/32 (Fc block, clone 93; eBioscience, La Jolla, CA) and stained with APC-eFlour 780-conjugated anti-CD45 (30-F11), PE-conjugated anti-CD11b (M1/70), APC-conjugated anti-Ly6G (RB6–8C5), and eFlour 450-conjugated anti-Ly6C (HK1.4) (eBioscience). Flow cytometry data were acquired using BD FACSCalibur (BD Biosciences, San Jose, CA) with Cytek eight-color flow cytometry upgrade (Cytek Development, Fremont, CA) and analyzed with FlowJo software 9.0 (TreeStar, Inc., Ashland, OR). There were 100–200,000 events/samples acquired. Neutrophils were identified as CD11b+Ly6G+Ly6C+ cells within the CD45+ gate.

TUNEL Assay Apoptotic cells in the kidney were detected by TUNEL assay following the manufacturer’s protocol (Roche Diagnostics, Mannheim, Germany). Briefly, cryostat sections (3 mm) of PLP-fixed kidneys were immersed in 3% hydrogen peroxide-methanol mixture (10 minutes) and permeabilized with cold 0.1% Triton-X100 and 0.1% sodium acetate (2 minutes). DNA fragments in apoptotic cells were then labeled with fluorescein-conjugated dUTP by terminal transferase (Roche Diagnostics) for 60 minutes at room temperature in dark. The fluorescein-labeled apoptotic cells were imaged using a Zeiss Axiovert 200 microscope with ApoTome imaging and AxioVision 4.6 software (Carl Zeiss GmbH).

Immunohistochemistry

Paraffin-embedded kidney sections (3 mm) were stained for 4-HNE using standard protocols. Briefly, sections were deparaffanized in xylenes, rehydrated in a series of ethanol rinses (100%–70%), and then washed in distilled water. Sections were then incubated in 3% H2O2 in methanol for 20 minutes. After treating with avidin and biotin for 15 minutes each (Avidin/Biotin Blocking Kit; Vector Laboratories) the sections were incubated in blocking buffer containing 10% donkey serum in 0.1 M J Am Soc Nephrol 26: 2800–2814, 2015

sodium phosphate buffer pH 7.4 (phosphate buffer) at room temperature for 30 minutes. Sections were incubated in 4-HNE antibody (1:1000 in 1% BSA/phosphate buffer; Abcam, Inc.) overnight at 4°C. Sections were washed three times with phosphate buffer for 5 minutes each and incubated with biotinylated donkey anti-goat secondary antibody (1:400 dilution; Vector Laboratories) for 1 hour. After three washes in phosphate buffer, sections were incubated with ABC ready-to-use reagent (VECTASTAIN Elite ABC Kit; Vector Laboratories) for 30 minutes. After another three washes with phosphate buffer, the sections were incubated with 3,39-diaminobenzidine for 5 minutes followed by washing with distilled water. The sections were counter-=stained with 1% methylene blue solution, washed in water, and dehydrated with xylene. Sections were imaged by using a Zeiss Axio Imager Z2/ApoTome Microscope fitted with motorized focus drives and motorized XYZ microscope stage and integrated to a workstation running Stereo Investigator software, version 10.51 (MBF Bioscience, Williston, VT).

Immunofluorescence Three-micron PLP-fixed cryostat-cut kidney sections were used for the immunofluorescence detection of neutrophils and CD11b+ cells. Briefly, the kidney sections were air dried and incubated with 0.3% Triton3100/ 10% horse serum in PBS for 30 minutes. After washing the sections with PBS, anti-CD16/32 antibody was added to block FC receptors. This was followed by incubation with FITC-labeled antineutrophil antibody (7/4, 1:30; Cedarlane, Burlington, NC) and PE-labeled CD11b (M1/70, 1:30; eBioscience) in 10% horse serum/PBS for 1.5 hours. The sections were then washed three times in PBS and mounted with ProLong Gold antifade agent with DAPI (Life Technologies).

Iron Stimulation of Splenocytes Splenocytes were harvested from 10-week-old B6 male mice. After excising the spleen, red blood cells were lysed, and the remaining splenocytes were seeded at 13106 cells/ml in 24-well tissue culture plates and cultured under 5% CO2 at 37°C for 24 hours. The culture medium Iron Dynamics and Kidney Injury

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consisted of RPMI 1640 medium supplemented with 10% FCS, 100 U/ml penicillin, 100 mg/ml streptomycin, 1% nonessential amino acids (Gibco/Life Technologies, Foster City, CA), 2 mM L-glutamine (SigmaAldrich), and 50 mM 2-mercaptoethanol. After resting for 24 hours, the cells were treated with 10 mM ferric ammonium chloride for 4 hours. The cell culture supernatants were harvested, and IL-6 levels were measured by ELISA (eBioscience).

Western Blot Analysis Snap-frozen tissue sections were homogenized in Tris-Triton tissue lysis buffer containing complete protease inhibitor cocktail (Halt Protease and Phosphatase Inhibitor Cocktail; Thermo Fisher Scientific, Rockford, IL) using a Dounce Homogenizer. Ferroportin was measured in membrane fractions isolated from the whole kidney lysate using a membrane isolation kit (Thermo Fisher Scientific). H-ferritin was measured in whole kidney and spleen lysates. Protein content in the homogenate was estimated using the Pierce BCA protein estimation kit (Thermo Fisher Scientific). Twenty to thirty micrograms of protein per sample were loaded on a 10% NuPage Bis-Tris gel under reducing conditions. The resolved proteins were transferred onto a nitrocellulose membrane (LI-COR Biosciences, Lincoln, NE) and probed with goat anti-mouse H-ferritin (Santa Cruz Biotechnology) and rabbit anti-mouse ferroportin (Novus Biologics) antibodies (concentrations). The primary antibodies were detected with donkey anti-goat Alexa 800 and goat anti-rabbit Alexa 800 antibodies (LI-COR). Mouse monoclonal glyceraldehyde-3-phosphate dehydrogenase and b-actin (Abcam, Inc.) were used as the loading controls and detected using donkey anti-mouse Alexa 680 antibody (LI-COR). Quantitation of data was performed using densitometry software (LI-COR).

Real-Time PCR For RNA isolation, frozen tissues were resuspended in RLT buffer (Qiagen, Valencia, CA) and homogenized using the TissueLyser system (Qiagen). Total RNA from tissue homogenates was purified using the RNAeasy mini kit (Qiagen) following the manufacturer’s instructions. Then 1 mg of RNA was used to synthesize cDNA using the iScript cDNA synthesis kit (BioRad, Hercules, CA). The cDNA template was mixed with iTAQ SYBR green universal super mix (Bio-Rad), and quantitative PCR was carried out on a CFX Connect system (Bio-Rad). Predesigned primers for NGAL, caspase-3, and Bcl-2 were purchased from Bio-Rad. Glyceraldehyde-3phosphate dehydrogenase was amplified in parallel and used as the reference gene in quantification. Data are expressed as relative gene expression and were calculated using the 2 [2DDC(T)] method.

Statistical Analyses GraphPad Prism 6 (GraphPad) was used to analyze and present the data. Data were analyzed, after transformation if needed to generate a normal distribution, by t test, one- or two-way ANOVA with post hoc analysis as appropriate. P,0.05 was used to indicate significance.

ACKNOWLEDGMENTS We gratefully acknowledge Dr. Sophie Vaulont and Dr. Cindy Roy for providing us with hepcidin knockout mice and we thank Dr. Michael Forbes and Mr. Saleh Mohammad for their help with 2812


immunehistochemical and immunofluorescence staining of kidney sections and the University of Virginia Research Histology core for hematoxylin and eosin staining.

DISCLOSURES None.

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