Markers of oxidative/nitrosative stress and inflammation in lung tissue of rats exposed to different intravenous iron compounds

Abstract

Iron deficiency anemia is a frequent complication in clinical conditions such as chronic kidney disease, chronic heart failure, inflammatory bowel disease, cancer, and excessive blood loss. Given the ability of iron to catalyze redox reactions, iron therapy can be associated with oxidative stress. The lung is uniquely susceptible to oxidative stress, and little is known about the effects of intravenous iron treatment in this organ. This study characterized changes in markers of oxidative/nitrosative stress and inflammation in the lung of non-iron deficient, non-anemic rats treated with five weekly doses (40 mg iron per kg body weight) of low molecular weight iron dextran (LMWID), iron sucrose (IS), ferric carboxymaltose (FCM), ferumoxytol (FMX), iron isomaltoside 1000 (IIM), or saline (control). Rats treated with LMWID, FMX, or IIM showed significant changes in most measures of oxidative/nitrosative stress, inflammation, and iron deposition compared to the saline-treated controls, with greatest changes in the LMWID treatment group. Increases in products of lipid peroxidation (thiobarbituric acid reactive substances) and protein nitrosation (nitrotyrosine) were consistent with increases in the activity of antioxidant enzymes (catalase, Cu,Zn-SOD, GPx), decreases in antioxidative capacity (reduced:oxidized GSH ratio), increased levels of transcription factors involved in the inflammatory pathway (NF-κB, HIF-1α), inflammatory cytokines (TNF-α, IL-6), adhesion molecules (VCAM-1), markers of macrophage infiltration (ED-1), and iron deposition (Prussian blue, ferritin). Since changes in measured parameters in FCM- or IS-treated rats were generally modest, the results suggest that FCM and IS have a low propensity to induce lung inflammation. The relevance of these findings to clinical safety profiles of the tested intravenous iron products requires further investigation.

Keywords:

• intravenous iron
• oxidative stress
• nitrosative stress
• inflammation
• lung
• rodent model

Supplementary materials

Figure S1 NF-κB₆₅ immunohistochemistry.

Notes: Arrows indicate alveolar cells with positive staining for NF-κB. Original magnification ×200. Scale bar 50 μm. Cell types and tissue are indicated as alveolar sac (AS), and alveolar cells (AC). IIM, iron isomaltoside 1000 (Monofer^®, Pharmacosmos A/S, Holbæk, Denmark); IS, iron sucrose (Venofer^®, American Regent, Shirley, NY, USA); FCM, ferric carboxymaltose (Ferinject^®, Vifor (International) Ltd., St Gallen, Switzerland); FMX, ferumoxytol (Feraheme^®, AMAG Pharmaceuticals Inc., Lexington, MA, USA); LMWID, low molecular weight iron dextran (Infed^®, Watson Pharma Inc., Morristown, NJ, USA); control, saline treatment.

Figure S2 TNF-α immunohistochemistry.

Notes: Arrows indicate localization of TNF-α in alveolar cells. Original magnification ×200. Scale bar 50 μm. Cell types and tissue are indicated as alveolar sac (AS), and alveolar cells (AC). IIM, iron isomaltoside 1000 (Monofer^®, Pharmacosmos A/S, Holbæk, Denmark); IS, iron sucrose (Venofer^®, American Regent, Shirley, NY, USA); FCM, ferric carboxymaltose (Ferinject^®, Vifor (International) Ltd., St Gallen, Switzerland); FMX, ferumoxytol (Feraheme^®, AMAG Pharmaceuticals Inc., Lexington, MA, USA); LMWID, low molecular weight iron dextran (Infed^®, Watson Pharma Inc., Morristown, NJ, USA); control, saline treatment.

Figure S3 IL-6 immunohistochemistry.

Notes: Arrows indicate localization of IL-6 in alveolar cells. Original magnification ×200. Scale bar 50 μm. Cell types and tissue are indicated as alveolar sac (AS), and alveolar cells (AC). IIM, iron isomaltoside 1000 (Monofer^®, Pharmacosmos A/S, Holbæk, Denmark); IS, iron sucrose (Venofer^®, American Regent, Shirley, NY, USA); FCM, ferric carboxymaltose (Ferinject^®, Vifor (International) Ltd., St Gallen, Switzerland); FMX, ferumoxytol (Feraheme^®, AMAG Pharmaceuticals Inc., Lexington, MA, USA); LMWID, low molecular weight iron dextran (Infed^®, Watson Pharma Inc., Morristown, NJ, USA); control, saline treatment.

Figure S4 VEGF immunohistochemistry.

Notes: Arrows indicate presence of VEGF not only in endothelial cells’ localization but also in alveolar cells in the different groups. Original magnification ×200. Scale bar 50 μm. Cell types and tissue are indicated as alveolar sac (AS). IIM, iron isomaltoside 1000 (Monofer^®, Pharmacosmos A/S, Holbæk, Denmark); IS, iron sucrose (Venofer^®, American Regent, Shirley, NY, USA); FCM, ferric carboxymaltose (Ferinject^®, Vifor (International) Ltd., St Gallen, Switzerland); FMX, ferumoxytol (Feraheme^®, AMAG Pharmaceuticals Inc., Lexington, MA, USA); LMWID, low molecular weight iron dextran (Infed^®, Watson Pharma Inc., Morristown, NJ, USA); control, saline treatment.

Figure S5 VCAM-1 immunohistochemistry.

Notes: Arrows indicate presence of VCAM in endothelial cells, alveolar cells, and also in lung interstitium in the different groups. Original magnification ×200. Scale bar 50 μm. Cell types and tissue are indicated as alveolar sac (AS). IIM, iron isomaltoside 1000 (Monofer^®, Pharmacosmos A/S, Holbæk, Denmark); IS, iron sucrose (Venofer^®, American Regent, Shirley, NY, USA); FCM, ferric carboxymaltose (Ferinject^®, Vifor (International) Ltd., St Gallen, Switzerland); FMX, ferumoxytol (Feraheme^®, AMAG Pharmaceuticals Inc., Lexington, MA, USA); LMWID, low molecular weight iron dextran (Infed^®, Watson Pharma Inc., Morristown, NJ, USA); control, saline treatment.

Figure S6 Ferritin L chain immunohistochemistry.

Notes: Arrows indicate localization of ferritin L in alveolar cells and other cells of the different groups. Original magnification ×200. Scale bar 50 μm. Cell types and tissue are indicated as alveolar sac (AS). IIM, iron isomaltoside 1000 (Monofer^®, Pharmacosmos A/S, Holbæk, Denmark); IS, iron sucrose (Venofer^®, American Regent, Shirley, NY, USA); FCM, ferric carboxymaltose (Ferinject^®, Vifor (International) Ltd., St Gallen, Switzerland); FMX, ferumoxytol (Feraheme^®, AMAG Pharmaceuticals Inc., Lexington, MA, USA); LMWID, low molecular weight iron dextran (Infed^®, Watson Pharma Inc., Morristown, NJ, USA); control, saline treatment.

Figure S7 Ferritin H chain immunohistochemistry.

Notes: Arrows indicate localization of ferritin H in alveolar cells of the different groups. Original magnification ×200. Scale bar 50 μm. Cell types and tissue are indicated as alveolar sac (AS). IIM, iron isomaltoside 1000 (Monofer^®, Pharmacosmos A/S, Holbæk, Denmark); IS, iron sucrose (Venofer^®, American Regent, Shirley, NY, USA); FCM, ferric carboxymaltose (Ferinject^®, Vifor (International) Ltd., St Gallen, Switzerland); FMX, ferumoxytol (Feraheme^®, AMAG Pharmaceuticals Inc., Lexington, MA, USA); LMWID, low molecular weight iron dextran (Infed^®, Watson Pharma Inc., Morristown, NJ, USA); control, saline treatment.

Acknowledgments

The study was financially supported by Vifor (International) Ltd. Scientific writing support was provided by Lee Mizzen, Taija Koskenkorva-Frank (both Vifor Pharma) and Walter Fürst (SFL Regulatory Affairs & Scientific Communication, funded by Vifor Pharma). Support for graphics and illustrations were provided by Tracey Nichols (Tracey Nichols Graphic Design Ltd.).

Disclosure

Jorge E Toblli has received research grants and consultancy fees from Vifor (International) Ltd., Switzerland. Gabriel Cao, Jorge F Giani, Fernando P Dominici, and Margarita Angerosa have no conflicts of interest to report.