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. 2018 May 1;9(5):550.
doi: 10.1038/s41419-018-0568-z.

Endogenous Hepcidin Synthesis Protects the Distal Nephron Against Hemin and Hemoglobin Mediated Necroptosis

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Free PMC article

Endogenous Hepcidin Synthesis Protects the Distal Nephron Against Hemin and Hemoglobin Mediated Necroptosis

Rachel P L van Swelm et al. Cell Death Dis. .
Free PMC article

Abstract

Hemoglobinuria is associated with kidney injury in various hemolytic pathologies. Currently, there is no treatment available and its pathophysiology is not completely understood. Here we studied the potential detrimental effects of hemoglobin (Hb) exposure to the distal nephron (DN). Involvement of the DN in Hb kidney injury was suggested by the induction of renal hepcidin synthesis (p < 0.001) in mice repeatedly injected with intravenous Hb. Moreover, the hepcidin induction was associated with a decline in urinary kidney injury markers 24p3/NGAL and KIM1, suggesting a role for hepcidin in protection against Hb kidney injury. We demonstrated that uptake of Hb in the mouse cortical collecting duct cells (mCCDcl1) is mediated by multi-protein ligand receptor 24p3R, as indicated by a significant 90% reduction in Hb uptake (p < 0.001) after 24p3R silencing. Moreover, incubation of mCCDcl1 cells with Hb or hemin for 4 or 24 h resulted in hepcidin synthesis and increased mRNA expression of markers for oxidative, inflammatory and ER stress, but no cell death as indicated by apoptosis staining. A protective role for cellular hepcidin against Hb-induced injury was demonstrated by aggravation of oxidative, inflammatory and ER stress after 4 h Hb or hemin incubation in hepcidin silenced mCCDcl1 cells. Hepcidin silencing potentiated hemin-mediated cell death that could be diminished by co-incubation of Nec-1, suggesting that endogenous hepcidin prevents necroptosis. Combined, these results demonstrate that renal hepcidin synthesis protects the DN against hemin and hemoglobin-mediated injury.

Conflict of interest statement

RvS and DS are managing director and medical director, respectively, of the “Hepcidinanalysis.com” initiative, which aims to serve the scientific and medical communities with high-quality hepcidin measurements (www.hepcidinanalysis.com). The remaining authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. Kidney injury markers in Hb-treated mice.
Mice treated with a weekly i.v. injection of saline (control; n = 5) or Hb (n = 4) for 8 weeks demonstrated increased urinary kidney injury markers 24p3 and KIM1 compared to control (a). Increased renal mRNA expression of Hamp after 8 weeks of Hb treatment, accompanied by increased IL-6, 24p3, H-ferritin, and L-ferritin (b). Data in panel A was analyzed with Two-way ANOVA with Bonferroni post-hoc test; data in panel B with Student’s t-test. * = p < 0.05; ** = p < 0.01; and *** = p < 0.001 compared to control
Fig. 2
Fig. 2. Uptake of Hb by 24p3R in mCCDcl1 cells.
Uptake of Alexa-546 Hb in mCCDcl1 cells after 4 h incubation (a) with Alexa-546 Hb alone (Alexa-546 Hb, 1 nM), 4 °C, and with co-incubations of unlabeled excess Hb (+excess Hb, 100 nM) or 24p3 (+24p3, 1 nM). Silencing of 24p3R in mCCDcl1 cells resulted in a 60% reduction of mRNA expression level (b) and 95% reduction of protein levels as assessed by immunostaining (c) compared to scrambled siRNA (scrmbl). MCCDcl1 cells treated with 24p3R siRNA demonstrated a 90% reduction in 5 nM Alexa-546 Hb uptake after 1 h (d). Panel A: N = 2 experiments in duplicate; uptake of Alexa-546 Hb was quantified in 3–4 images per sample; scale bar = 30 µm, *** = p < 0.001 compared to Alexa-546 Hb analyzed by One-way ANOVA with Bonferroni’s Multiple Comparison Test. Panel B–D: N = 3–4 experiments in duplicate; fluorescence was quantified in 3–4 images per sample; scale bar = 20 µm, ** = p < 0.01; *** = p < 0.001 compared to scrmbl siRNA, analyzed by Student’s t-test
Fig. 3
Fig. 3. Hb-mediated induction of hepcidin synthesis, cell death and cellular stress.
Immunostaining of hepcidin mCCDcl1 cells after 24 h hemoglobin (Hb) incubation and Hamp mRNA expression levels in mCCDcl1 cells after 4 h and 24 h Hb incubation (a). Absence of Hb-induced cell death modes necrosis (PI) and apoptosis (Annexin-V FITC) in mCCDcl1 cells incubated with Hb for 48 h (b). H2O2 (30 min) served as positive control for necrosis and Doxorubicin (DOX, 24 h) for apoptosis. Incubation with Hb for 4 h (white bars) and 24 h (grey bars) resulted in significant dose-dependent increased mRNA expression of Ho-1, L-ferritin, H-ferritin, and C/ebpα, whereas Chop, Hif1α and IL-6 were moderately elevated (c). N = 3 experiments in duplicate; fluorescence was quantified in 3–4 images per sample. Scale bar panel A = 40 µm, scale bar panel B = 30 µm. * = p < 0.05; ** = p < 0.01; and *** = p < 0.001 compared to control (ctrl); analyzed by One-way ANOVA with Bonferroni’s multiple comparison test
Fig. 4
Fig. 4. Hemin-induced cellular stress.
Incubation of mCCDcl1 cells with hemin for 4 h (white bars) or 24 h (grey bars) resulted in significant and dose-dependent increases in mRNA expression of Hamp, Ho-1, L-ferritin, H-ferritin, C/ebpα, Chop, and IL-6, whereas mRNA expression of Hif1α was significantly reduced compared to control (ctrl). N = 3 experiments in duplicate. * = p < 0.05; ** = p < 0.01; and *** = p < 0.001 compared to control; analyzed by One-way ANOVA with Bonferroni’s multiple comparison test
Fig. 5
Fig. 5. Cellular stress after hepcidin silencing in mCCDcl1 cells.
MCCDcl1 cells treated with Hamp siRNA demonstrate a 90% reduction in Hamp mRNA expression level compared to their negative controls treated with RLUC siRNA (a). Expression levels of IL-6 and Chop are also significantly reduced. Hamp silenced mCCDcl1 cells (grey bars) show increased baseline oxidative stress (b) compared to RLUC silenced cells (white bars) and enhanced oxidative stress response after 4 h incubation with Hb (10 µM) or hemin (1 and 10 µM). Panel A: N = 6 experiments in duplicate; panel B: N = 2 experiments in duplicate. CellRox green fluorescence was quantified in 3–4 images per sample. Scale bar = 40 µm, merged = CellRox + DAPI. * = p < 0.05; ** = p < 0.01; and *** = p < 0.001; data in panel A was analyzed by Student’s t-test, data in panel B by One-way ANOVA with Bonferroni’s multiple comparison test
Fig. 6
Fig. 6. Hemin-induced necroptosis in hepcidin silenced mCCDcl1 cells.
Incubation with hemin for 4 h resulted in similarly increased Annexin V-FITC signal in both RLUC (white bars) and Hamp silenced (grey bars) mCCDcl1 cells, whereas PI was more increased in Hamp silenced mCCDcl1 cells. Co-incubation with the necroptosis inhibitor Nec-1, but not with ferroptosis inhibitor Fer-1 or apoptosis inhibitor zVAD-fmk, reduced hemin mediated PI signal in Hamp silenced cells, indicating necroptosis. N = 3–4 experiments in duplicate. * = p < 0.05; ** = p < 0.01; and *** = p < 0.001, analyzed by one-way ANOVA with Bonferroni’s multiple comparison test
Fig. 7
Fig. 7. Cellular stress in hepcidin silenced cells incubated with Hb or hemin.
MCCDcl1 cells treated with Hamp siRNA and incubated with hemoglobin (Hb, a) or hemin (b) for 4 h demonstrated significant changes in mRNA expression levels of Ho-1, L-ferritin, H-ferritin, C/ebpα, Chop, Hif1α, and IL-6 compared to cells treated with RLUC siRNA. Changes in mRNA expression levels after Hb or hemin incubated are depicted as fold change relative to their untreated controls in either RLUC or Hamp silenced mCCDcl1. N = 3 experiments in duplicate. * = p < 0.05; ** = p < 0.01; and*** = p < 0.001, analyzed by one-way ANOVA with Bonferroni’s multiple comparison test
Fig. 8
Fig. 8. Schematic representation of the proposed mechanisms involved in hepcidin-mediated protection against heme-mediated injury in mCCDcl1 cells.
The results suggest that Hb is taken up via the 24p3R in mCCDcl1 cells, after which the heme-group is liberated and catabolized by HO-1 to yield reactive iron. Initially, reactive iron is metabolized and safely stored (H-ferritin and L-ferritin), but when intracellular iron levels exceed the capacity for safe storage, excess iron may cause inflammation (IL-6), oxidative stress (Hif1α) and ER stress (Chop). These deleterious pathways can all lead to necroptosis, but simultaneous induction of hepcidin (Hamp) synthesis, possibly via C/ebpα, prevents cell death. The gene products typed in Italic represent the markers measured in the study

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