Iron therapy mitigates chronic kidney disease progression by regulating intracellular iron status of kidney macrophages

Abstract

Systemic iron metabolism is disrupted in chronic kidney disease (CKD). However, little is known about local kidney iron homeostasis and its role in kidney fibrosis. Kidney-specific effects of iron therapy in CKD also remain elusive. Here, we elucidate the role of macrophage iron status in kidney fibrosis and demonstrate that it is a potential therapeutic target. In CKD, kidney macrophages exhibited depletion of labile iron pool (LIP) and induction of transferrin receptor 1, indicating intracellular iron deficiency. Low LIP in kidney macrophages was associated with their defective antioxidant response and proinflammatory polarization. Repletion of LIP in kidney macrophages through knockout of ferritin heavy chain (Fth1) reduced oxidative stress and mitigated fibrosis. Similar to Fth1 knockout, iron dextran therapy, through replenishing macrophage LIP, reduced oxidative stress, decreased the production of proinflammatory cytokines, and alleviated kidney fibrosis. Interestingly, iron markedly decreased TGF-β expression and suppressed TGF-β-driven fibrotic response of macrophages. Iron dextran therapy and FtH suppression had an additive protective effect against fibrosis. Adoptive transfer of iron-loaded macrophages alleviated kidney fibrosis, validating the protective effect of iron-replete macrophages in CKD. Thus, targeting intracellular iron deficiency of kidney macrophages in CKD can serve as a therapeutic opportunity to mitigate disease progression.

Keywords: Chronic kidney disease; Fibrosis; Macrophages; Nephrology.

Figure 1. In mice with CKD, kidney macrophages display depletion of LIP associated with oxidative stress and inflammation.

(A) Schematic diagram of CKD induction by the 0.2% adenine diet. (B) Trichrome staining of control (CTR) and CKD kidneys. Scale bars, 2.5 mm. (C) Kidney iron content in 2 groups of mice at euthanasia. (D) Expression of ferritin heavy chain (FtH) protein in isolated kidney mononuclear cells (KMC, macrophages/monocytes, and lymphocytes) normalized by macrophage marker F4/80 and by GAPDH. KMCs were isolated using Ficoll density gradient centrifugation of kidney single-cell suspensions. (E) Kidney macrophage labile iron pool (LIP) and transferrin receptor 1 (TfR1 or CD71) expression in CTR and CKD groups. (F) ROS production and iNOS expression in kidney macrophages in CTR and CKD groups. (G) Production of proinflammatory cytokines IL-6, IL-1β, and TNF-α in kidney macrophages, CTR and CKD groups. (H) TGF-β expression in kidney macrophages in CTR and CKD groups. Error bars represent SEM. Data were analyzed using t test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. MFI, mean fluorescence intensity.

Figure 2. Myeloid-specific deletion of Fth1 improves iron deficiency of kidney macrophages and mitigates kidney fibrosis.

(A) Myeloid Fth1 deletion replenished the intracellular LIP and reduced TfR1 expression by kidney macrophages; n = 3 per group. One-tailed t test. *P < 0.05. (B) ROS and lipid peroxidation in kidney macrophages of CKD Fth1^LysM–/– and Fth1^LysM+/– CKD mice; n = 3–5 per group. (C) Expression of IL-1β and TNF-α by kidney macrophages in 2 groups of mice; n = 4–8 per group. (D) Fibrosis markers TGF-β and α–smooth muscle actin (α-SMA) in kidney macrophages of CKD Fth1^LysM–/– and Fth1^LysM+/– CKD mice; n = 5–9 per group. (E) Representative images of Masson’s trichrome staining of kidney sections in 2 groups of mice; the quantification is shown as kidney fibrosis score; n = 5 per group. (F) Biomarkers of the kidney function: urine albumin to creatinine ratio (ACR), blood urea nitrogen (BUN), and serum cystatin C in CKD Fth1^LysM–/– and Fth1^LysM+/– mice; n = 5 per group. ACR and cystatin C were measured after 4 weeks of adenine diet, BUN after 8 weeks of adenine diet. Scale bars, 100 μm. Error bars represent SEM. Data were analyzed using t test. *P < 0.05; **P < 0.01.

Figure 3. Iron dextran administration improves iron deficiency of kidney macrophages and reduces kidney macrophage oxidative stress and inflammation in mice with CKD.

(A) Schematic diagram of iron dextran administration in mice with adenine-induced CKD and (B) Western blot analysis of FtH protein levels in kidney tissue of CKD mice in the absence (CKD) and presence (CKD+Fe) of iron administration; n = 3 per group. (C) Perls Prussian blue staining for ferric iron (scale bars: 3 mm for low magnification, 100 μm for high magnification) and transmission electron microscopy (original magnification, 5,000×, first 2 images, and 30,000×, last image) of kidney tissue of CKD mice in the absence and presence of iron administration. Arrows point at iron-loaded lysosomes within kidney macrophages (Mϕ). (D) Kidney tissue iron content in 2 groups of mice (n = 8–18 per group). (E) Iron therapy increases LIP and decreases TfR1 expression in CKD kidney macrophages, thus improving their iron deficiency status; n = 7–16 per group. (F) ROS and iNOS expression in CKD kidney macrophages in the absence and presence of iron therapy; n = 5–13 per group. (G) Iron therapy inhibits production of proinflammatory cytokines IL-6, IL-1β, and TNF-α in CKD kidney macrophages. (H) TGF-β expression in CKD kidney macrophages is suppressed by iron therapy. Dashed gray lines indicate mean values of the respective parameters in control mice (E–H). Error bars represent SEM. Data were analyzed using t test. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4. Iron dextran therapy improves kidney fibrosis and kidney function in mice with CKD.

(A) Markers of kidney function: serum creatinine, BUN, serum cystatin C, and urine ACR in CTR mice and mice with CKD in the absence and presence of iron administration; n = 5–8 per group. (B) Representative images of Masson’s trichrome staining of kidney sections of CTR mice and mice with CKD in the absence and presence of iron administration; the quantification of blue-stained collagen is shown as kidney fibrosis score; n = 5–8 per group. Scale bars, 100 μm. (C) Western blot analysis of fibronectin and α–smooth muscle actin in kidney tissue of CTR mice and mice with CKD in the absence and presence of iron administration; n = 3 per group. Iron dextran was administered i.p. once a week, 0.5 g/kg (CKD Fe group). Error bars represent SD. Data were analyzed using ANOVA; *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 5. Effects of iron administration on kidney fibrosis in the UUO model.

(A) Mice were pretreated with iron dextran (0.5 g/kg/dose weekly) or PBS for 4 weeks prior to surgery. Kidneys were harvested 7 days after the UUO or sham surgeries. (B) LIP, TfR1, and (C) TGF-β expression in kidney macrophages (n = 3 per group). (D) Perls Prussian blue staining demonstrates ferric iron accumulation in the interstitial spaces in the obstructed kidneys of UUO+Fe mice. Scale bar, 100 μm. (E) Immunoblotting of kidney tissues for FtH, fibronectin, and α-SMA protein expression in the obstructed kidneys of UUO mice that received iron dextran injections (UUO+Fe) compared with 2 other groups of mice. (F) Histologic assessment of kidney fibrosis in 3 groups of mice with Masson’s trichrome staining; quantification (n = 6 per group) and representative images. Scale bars, 100 μm. Data were analyzed using t test (B and C) or ANOVA (E and F). Error bars represent SEM; *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 6. Iron administration and renal antioxidant response in mice with CKD.

(A) CKD was induced by a 0.2% adenine diet, and kidney tissues were processed for RNA sequencing. (B) Heatmap showing expression of top differentially expressed antioxidant genes in the kidneys of CTR mice, untreated CKD mice, and CKD mice that received weekly i.p. injections of iron dextran (CKD+Fe). Blue color represents low expression and red color high expression; *P < 0.05; **P < 0.01; ***P < 0.001 for differences between CKD and CKD+Fe groups. (C) Expression of selected genes catalase and heme binding protein 1; n = 4 per group. (D) Schematic diagram of α-tocopherol (vitamin E), an antioxidant, administration in CKD mice. (E) α-Tocopherol administration did not change LIP and TfR1 expression in kidney macrophages but (F) reduced their ROS and (G) TNF-α expression and (H) improved serum creatinine. Error bars represent SEM. *P < 0.05. RPKM, reads per kilobase of transcript per million mapped reads.

Figure 7. Iron attenuates profibrotic response to TGF-β in macrophages.

BMDMs were left untreated (Fe-) or exposed to 25 mM ferric ammonium citrate (Fe+) for 20 hours. (A) Perls Prussian blue staining for ferric iron in control and iron-loaded BMDMs. Scale bars, 100 μm. (B) Western blot analysis of FtH, (C) fibronectin, and iNOS proteins in control BMDMs and BMDMs exposed to iron and TGF-β1 alone (5 ng/mL) or combined. Statistical analysis and representative blots. Immunoblotting was repeated 3 times. Error bars represent SD. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 8. Adoptive transfer of iron-loaded macrophages limits kidney fibrosis in mice subjected to UUO.

(A) Schematic diagram of the adoptive transfer (AT) of iron-loaded macrophages into UUO mice. BMDMs were isolated from CD45.1⁺ mice and left untreated (control) or treated with 25 μM ferric ammonium citrate (iron-loaded) for 20 hours prior to AT. Kidney fibrosis was induced by UUO in CD45.2⁺ mice. CD45.2⁺ mice received intravenously control or iron-loaded CD45.1⁺ macrophages. Kidneys were harvested 5 days after AT. (B) The ratio between live and dead cells in control versus iron-loaded BMDMs prior to transfer was assessed by flow cytometry using DAPI; n = 4 per group. (C) Flow cytometry histogram and count of CD45.1⁺CD11b⁺F4/80⁺ macrophages in recipient CD45.2⁺ kidneys upon AT of control macrophages. (D) Assessment of UUO-induced kidney fibrosis by Masson’s trichrome staining after AT of control versus iron-loaded macrophages. (E) Expression of latent TGF-β and fibronectin in kidney tissues after AT of control versus iron-loaded macrophages. Scale bars, 100 μm. Error bars represent SD; n = 3 per group. Data were analyzed using t test. *P < 0.05.

Figure 9. Proposed mechanism of the effects of kidney macrophage iron status in kidney fibrosis.

(A) In healthy kidney macrophages, a balance between LIP (ferrous iron, Fe²⁺) and iron stores (ferritin-bound ferric iron, Fe³⁺) supports an adequate antioxidant response to oxidative stress and minimizes unwanted effects of ROS. (B) In kidney fibrosis, LIP of kidney macrophages, essential for the antioxidant response, is depleted. Consecutively, increased production of ROS, proinflammatory cytokines, and TGF-β mediates the profibrotic effects in kidney macrophages. (C) Blockade of FtH impairs conversion of Fe²⁺ to Fe³⁺ owing to the ferroxidase activity of FtH and replenishes LIP of kidney macrophages, which leads to reduced oxidative stress, reduced inflammation, and ultimately attenuation of fibrosis. (D) Similar to FtH blockade, increased iron supply via therapeutic iron administration replenishes LIP in kidney macrophages and reduces macrophage oxidative stress, inflammation, and production of TGF-β, which attenuates fibrosis.