Sensitivity to Iron‐Mediated Pro‐Inflammatory Signaling in NZBWF1 Mice, a Model of Systemic Lupus Erythematosus

Abstract

IntroductionThe autoimmune disease systemic lupus erythematosus predominantly affects women and can lead to lupus nephritis (LN) as a common and severe complication in approximately 50% of patients. The F1 hybrid of New Zealand Black (NZB) X New Zealand White (NZW) mice (NZBWF1) is a well‐established mouse model to study LN. Our previous data indicated that the onset of albuminuria in female 34‐week NZBWF1 mice was associated with renal iron accumulation, and iron may contribute to renal injury. This study tests the hypothesis that 34‐week NZBWF1 lupus mice are sensitized to iron's injurious and pro‐inflammatory effects compared to 8‐week normal NZBWF1 mice. As a means to differentiate effects of age as opposed to development of lupus, parallel experiments were performed in 8 and 34‐week‐old NZW mice as healthy controls.Methods and ResultsFemale 8‐week (pre‐autoimmune) and 34‐week NZBWF1 (autoimmune, pre‐albuminuric) mice together with age‐matched healthy control NZW mice underwent 24 h urine collection to confirm non‐albuminuric status. Mice were then were injected i.v. with a single dose of iron‐sucrose (2 mg elemental iron) or vehicle (saline) and sacrificed 24 h later. Plasma blood urea nitrogen (BUN) was measured to determine renal health, SRY‐Box Transcription Factor 9 (SOX‐9)‐ positive cells were quantified in kidney sections as an early tubular injury marker, and plasma monocyte chemoattractant protein‐1 (MCP‐1) was measured by ELISA as an inflammatory marker. Lupus (NZBWF1) and control mice (NZW) were compared within strains by age (8‐week vs. 34‐week) and treatment (Vehicle vs. Treatment). All statistical analyses were done by 2‐way ANOVA (n=7‐12/group), and post‐hoc testing was done using Bonferroni. Iron‐sucrose treatment significantly increased plasma MCP‐1 concentrations in the NZBWF1 and the control NZW mice (PTreatment < 0.01). There was no significant effect of age on this effect for NZW mice (PTreatment*Age = 0.2); however, in NZBWF1 mice, the iron‐sucrose induced increase in MCP‐1 was significantly greater in 34‐week compared to 8‐week mice (PTreatment*Age < 0.05; P<0.05 by posthoc test). BUN was significantly increased with age in both strains (PAge<0.05), but there was no significant effect of iron‐sucrose (PTreatment>0.05). The numbers of SOX‐9+ tubular cells increased in the 34‐week NZBWF1 mice compared to 8‐week mice (Page<0.05), consistent with early stages of renal injury, but iron sucrose treatment did not show any significant effect (PTreatment= 0.9).ConclusionOur data suggest that at the autoimmune but pre‐albuminuric stage, 34‐week NZBWF1 mice show increased pro‐inflammatory responsiveness to iron‐mediated stress. Further studies to determine whether the enhanced pro‐inflammatory effects of iron contribute to pathology in lupus nephritis are ongoing.