- Email: [email protected]
Local hepcidin increased intracellular iron overload via the degradation of ferroportin in the kidney
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Local hepcidin increased intracellular iron overload via the degradation of ferroportin in the kidney Sai Pan a, Zhong-Ming Qian b, Shaoyuan Cui a, Delong Zhao a, Weiren Lan c, Xu Wang a, Xiangmei Chen a, * a
Department of Nephrology, Chinese PLA General Hospital, Chinese PLA Institute of Nephrology, Beijing Key Laboratory of Kidney Disease, State Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, Beijing, People’s Republic of China Laboratory of Neuropharmacology, Fudan University School of Pharmacy, Shanghai, 201203, People’s Republic of China c The Second Affiliated Hospital of Army Medical University, Chongqing, People’s Republic of China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 October 2019 Accepted 10 November 2019 Available online xxx
Background: Hepcidin is a key regulator of iron homeostasis. Some studies showed that exogenous hepcidin decreased the expression of divalent metal transporter (DMT1) rather than ferroportin(FPN1) to regulate renal iron metabolism. This study explored the effects of hepcidin synthesized by the kidney and its mechanism of iron regulation. Methods: In the in vivo experiments, mice were divided into a unilateral ureter obstruction (UUO) model group and a sham operation group, and mice in the UUO model group were sacrificed on days 1, 3, 5 and 7. The expression of renal hepcidin, FPN1, DMT1 and the retention of renal iron were studied. In the in vitro experiments, we overexpressed hepcidin in HK-2 cells. Then we tested the expression of renal hepcidin, FPN1, DMT1 and observed the production of intracellular ferrous ions. Results: Renal hepcidin expression was consistently higher in the UUO group than in the sham group from the first day. The expression of FPN1 gradually decreased, and the expression of DMT1 gradually increased in the UUO model. Intracellular ferrous ions significantly increased on the first day of the UUO model. In hepcidin overexpressed HK-2 cells, the expression of FPN1 was decreased, while the expression of DMT1 has no significant change. In addition, production of intracellular ferrous ions increased. Conclusion: local hepcidin can regulate iron metabolism in the kidney by adjusting the expression of FPN1. © 2019 Elsevier Inc. All rights reserved.
Keywords: Hepcidin Ferroportin Iron overload Kidney
1. Introduction The prevalence of chronic kidney disease (CKD) is high in both developing and developed countries [1,2]. CKD rose to 18th on the list of causes of the total number of global deaths in 2010 [3]. Notably, fibrosis is a pathological manifestation of all forms of CKD and irreversibly reduces kidney function [4]. Many studies have indicated that renal iron content is increased in patients with CKD [5,6] and animal models of CKD [7e9] and that dietary iron restriction or iron chelation reduces renal fibrosis. Excess iron in renal cells catalyzes the production of hydroxylation by the Fenton reaction, promoting lipid peroxidation, lysosomal damage, and
* Corresponding author. 28 Fuxing Road, Haidian District, Beijing, People’s Republic of China. E-mail address: [email protected] (X. Chen).
mitochondrial dysfunction, leading to apoptosis, ferroptosis and renal fibrosis [10e12]. Therefore, understanding the mechanism of kidney iron accumulation is important. Renal filtration and reabsorption of iron play an important role in the balance of iron homeostasis [13]. In patients with kidney diseases, renal tubules are exposed to a high concentration of iron owing to increased glomerular filtration of iron and iron-containing proteins, including hemoglobin, transferrin and neutrophil gelatinase-associated lipocalin (NGAL) [14,15]. Luminal proteinbound iron is endocytosed by transferrin receptor protein 1 (TFR1), transferrin receptor protein 2 (TFR2), cubilin and megalin [16]. Filtered free iron and iron released from proteins as a result of acidification of the filtrate as it passes along the nephron enter the tubular epithelial cells via the apical ion transporter DMT-1 (in both proximal and distal tubules) [17,18]. Hepcidin, which is mainly synthesized by hepatocytes, is a central iron regulator and induces internalization and degradation
https://doi.org/10.1016/j.bbrc.2019.11.066 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: S. Pan et al., Local hepcidin increased intracellular iron overload via the degradation of ferroportin in the kidney, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.066
2
S. Pan et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
of ferroportin (FPN1), the only known mammalian cellular iron exporter [19,20]. Hepcidin synthesis is regulated by body iron status [21,22], erythropoietic activity [23], hypoxia and inflammatory cytokines [24]. Hepcidin has been shown to be locally synthesized and secreted by renal proximal tubules [25]. A study showed that the degree of kidney iron overload may not correlates with the extent of systemic iron overload [26]. However, whether there is an autoregulatory mechanism for renal iron metabolism is not defined. In the present study, we explored the mechanism of iron accumulation in the kidney and the effect of local hepcidin. We used a unilateral ureteral obstruction (UUO) model, a defined model of kidney iron accumulation, and explored renal iron metabolism and hepcidin levels on days 1, 3, 5 and 7 of the UUO model. In the in vitro experiments, we overexpressed hepcidin in HK-2 cells and tested the expression of renal hepcidin, FPN1, DMT1. 2. Materials and methods 2.1. Materials Commercially available antibodies, including anti-hepcidin-25, hepcidin-25, anti-DMT1, anti-SLC40A1, anti-ferritin were purchased from Abcam (Cambridge, CA, UK); anti-GAPDH was purchased from Cell Signaling Technology (Beverly, MA, USA). 2.2. Animal preparation and procedure All experimental procedures were performed in strict accordance with the guidelines of the Animal Research Committee of the Chinese People’s Liberation Army General Hospital. Eight-week-old male C57BL/6J mice were obtained from HuafuKang Biotech Co., Ltd. (Beijing, China), and they were given free access to water and food. The surgical procedure to establish the UUO model was previously described in detail [9]. Briefly, under pentobarbital anesthesia, the left ureter was isolated and ligated with 3e0 silk sutures at 2 points at the proximal site. In the sham group mice, the left ureter was isolated but not ligated. The total operation period lasted approximately 10 min. After the operation, the mice were placed on a heating pad until they were completely awake. The mice in the UUO model group were sacrificed on days 1, 3, 5 and 7 after the surgery. The mice were perfused with saline, and the kidney was removed and stored at 80 C or fixed in 10% formalin at room temperature until use. 2.3. Cell culture HK-2 cells were obtained from frozen storage in our laboratory. The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; 1 g/L glucose; Corning; USA) supplemented with 5% fetal bovine serum (FBS, Cat. No. 0010). For each experiment, cells at passages 5e8 were used. The cells were grown to confluence and transferred to serum-free medium for 12 h before the start of the experiments. 2.4. The overexpression of human HAMP in HK-2 cells Human HAMP was amplified by RT-PCR, and the used primers were as follows: Forward: 5‘-CCGGAATTCGCCACCATGGCACTGA GCTCCCAGATCTGGGCCG-3’; Reverse: 5‘-CGCGGATCCCTACGTCTT GCAGCACATCCCACAC-3’. After that, human HAMP gene was cloned into a pIRES2-ZsGreen1 plasmid to generate pIRES2-ZsGreenhHAMP. The pIRES2-ZsGreen-hHAMP contained green fluorescent gene, which could be used as a marker for successful expression of the human HAMP gene. Then, the pIRES2-ZsGreen-hHAMP was
transfected into HK-2 cells by Lipofectamine 2000(Invitrogen). The pIRES2-ZsGreen plasmids were transfected as control. After 48 h, the transfected cells were collected and lysed in a RIPA buffer for PCR or supplemented with protease inhibitors for Western blot . Another group used hepcidin-25 200 nmol/l to stimulate HK-2 cells for 24 h. 2.5. Quantitative real-time PCR Total RNA extraction were performed using TRIzol reagent (Invitrogen). Then total RNA were digested with DNase (Beyotime, China). cDNA preparation used a First Strand cDNA synthesis kit (New England Biolabs, USA), respectively, according to the manufacturer’s instructions. Quantitative real-time (qRT)-PCR was carried out using SYBR Select Master Mix (Life Technologies, California, USA) and an RT-PCR detection system (ABI, Foster City, CA, USA). The expression levels of all target genes were normalized to that of 18S, an internal control. The primer sets used were as follows:50 TGGAGTACGTTCTGCTCTGGAAGG-30 and 50 - ACACAGGCTGGTTGT AGTAGGAGAC-30 for human FPN1; 50 -GTAACCCGTTGAACCCCATT-30 and 50 -CCATCCAACGGTAGTAGCG-30 for 18S. 2.6. Protein extraction and Western blotting analysis The tissue samples were lysed in RIPA buffer containing a protease inhibitor cocktail (1 lg/ml leupeptin, 1 lg/ml aprotinin, and 100 mM PMSF), homogenized and centrifuged (30 min, 4 C, and 15,000 rpm) [27]. The extracted proteins were separated using SDSPAGE and then transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked and incubated overnight at 4 C with each primary antibody: anti-hepcidin-25 (1:80), antiDMT1 (1:500), anti-SLC40A1 (1:500), anti-ferritin(1:1500) and anti-GAPDH (1:1000), followed by incubation for 2 h with the secondary antibody. Immunoreactive bands were detected using a ChemiDoc-It 600 Imaging System (UVP, Upland, USA). Densitometry analysis was performed using Image J. 2.7. Detection of labile ferrous iron Intracellular labile ferrous iron was detected using FeRhoNox-1 (Goryo Chemical Company, Japan) as previously described [7,28]. In brief, frozen sections were washed three times in Hank’s balanced salt solution (HBSS) and fixed in 4% paraformaldehyde for 15 min. After washing, the sections were incubated with the anti-hepcidin25 (1:40) primary antibody at 4 C overnight in a dark humidified container followed by incubation with FITC-conjugated anti-rabbit secondary antibody (green). After washing, these sections were incubated with RhoNox-1 in HBSS (5 mM) at room temperature for 30 min. The sections were counterstained with DAPI and covered with a small drop of mounting medium. RhoNox-1 was observed and quantified using confocal microscopy. 2.8. Intracellular labile ferrous iron detection Intracellular labile ferrous iron was detected using FeRhoNox-1 (Goryo Chemical Company, Japan) as the instructions described. HK-2 cells were stimulated with hepcidin-25 for 24 h or overexpressed human HAMP gene, and then incubated with RhoNox-1 in HBSS (5 mM) at 37 C for 30 min in the dark. After washing, ferrous iron production was observed using fluorescence microscopy. 2.9. Statistical analysis All statistical analyses were performed using GraphPad Prism 5.
Please cite this article as: S. Pan et al., Local hepcidin increased intracellular iron overload via the degradation of ferroportin in the kidney, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.066
S. Pan et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
3
Fig. 1. Iron accumulation and hepcidin synthesis in kidneys. (A) Representative immunofluorescence of DAPI (blue), hepcidin (green), and FeRhoNox (red) staining in the kidneys of sham operation group and the UUO model group at 1, 3, 5, and 7 days. (B) Semi-quantitative analysis of FeRhoNox fluorescence intensity. The values are expressed as means ± s.d. ***P＜0.001 vs sham operation group. (C) Western blot analysis showing the hepcidin expression in kidney. *P＜0.05; **P＜0.01 vs sham operation group.
Data are presented as the mean ± SEM. Differences between the groups were assessed by student t-test or one-way analysis of variance (ANOVA). A value of p < 0.05 was considered indicative of statistical significance.
3. Results 3.1. Changes in hepcidin expression and iron content in the kidney Ferrous ion production in the kidney was significantly increased on day 1 of the UUO model and remained higher than that in the sham operation group (Fig. 1B). Western blotting results showed that hepcidin protein expression was increased on the first day (Fig. 1C), and immunofluorescence supported this result. In addition, we observed that increased expression of hepcidin was associated with the degree of ferrous ions accumulation in the kidney (Fig. 1A).
3.2. Expression of DMT1 and FPN1 in the kidney Beginning on the first day of the UUO model, the protein expression of DMT1 gradually increased (Fig. 2A). However, the protein expression of FPN1 showed significant decreases on day 3 of the UUO model(Fig. 2B).
3.3. Expression of hepcidin, DMT1, FPN1, ferritin and iron content in HK-2 cells We found that hepcidin mRNA expression in HK-2 cells was increased after transfected pIRES2-ZsGreen-hHAMP plasmid into cells. In hepcidin overexpressed HK-2 cells, the mRNA and protein expression of FPN1 was decreased, while the expression of DMT1 has no significant change (Fig. 3). In addition, the expression of ferritin increased, and the production of intracellular ferrous ions augmented (Fig. 4). 4. Discussion Herein, we examined the endogenous formation of hepcidin in HK-2 cells, a human proximal tubular epithelial cell line, and in the kidney from a mouse UUO model and the association between local hepcidin and intracellular iron overload. To our knowledge, this study is the first to overexpress HAMP gene in HK-2 cell line and we have found that local renal hepcidin can regulate iron metabolism by adjusting the expression of FPN1. Our study showed that both HK-2 cells and kidneys from the mouse UUO model express significant levels of hepcidin, our study indicated that hepcidin was mainly synthesized by tubular cells in the kidney, which was consistent with the results published by Tania Veuthey [18] and H Kulaksiz [25], as they found that hepcidin was produced by the proximal tubules (S1 and S2), basolateral thick
Please cite this article as: S. Pan et al., Local hepcidin increased intracellular iron overload via the degradation of ferroportin in the kidney, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.066
4
S. Pan et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
Fig. 2. DMT1 and FPN1 expression in the kidney. (A) Western blot analysis showing the DMT1 expression in the kidney. *P＜0.05; ***P＜0.001 vs sham operation group. (B) Western blot analysis showing the FPN1 expression in the kidney. **P＜0.01; ***P＜0.001 vs sham operation group.
ascending limb and medullary collecting duct. Renal hepcidin protein expression was significantly increased on the first day of the UUO model, which was regulated by iron status, inflammatory stimuli and hypoxia [24,29,30]. In addition, studies showed that urinary iron content was increased in CKD patients and animal models of CKD [6,31]. Interestingly, we found that the site of renal hepcidin synthesis was highly coincident with the location of ferrous ion accumulation in the kidney. The relationship between kidney-derived hepcidin and renal ferrous ions overload was further confirmed by overexpressed HAMP gene HK-2 cells. We found that with upregulation of
endogenous hepcidin, the expression of FPN1 decreased and cellular ferrous ions increased. It was reported that administration of exogenous hepcidin reduced the expression of DMT1 instead of FPN1 in the kidney [32]. However, the degree of iron accumulation in the kidney is consistent with the expression of endogenous hepcidin mRNA [26], which was in accordance with our results. In addition, the study was also showed that in HAMP knockout (KO) mice, FPN was highly expressed in the basolateral and intracellular membrane in the epithelial cells of the thick ascending limbs. Moreover, a study showed that after specifically knocking out the FPN gene of the renal tubule, iron accumulation in the kidney was
Fig. 3. Hepcidin, DMT1 and FPN1 expression in HK-2 cells. (A) RT-PCR showing the hepcidin and FPN1 expression in cells. **P＜0.01; ***P＜0.001 vs control group. (B) Western blot analysis showing the FPN1 expression in cells. **P＜0.01; ***P＜0.001 vs control group.
Please cite this article as: S. Pan et al., Local hepcidin increased intracellular iron overload via the degradation of ferroportin in the kidney, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.066
S. Pan et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
5
Fig. 4. Iron accumulation and ferritin expression in HK-2 cells. (A) Representative immunofluorescence of FeRhoNox (red) staining in HK-2 cells. (B) Western blot analysis showing the ferritin expression in cells. The values are expressed as means ± s.d. ***P＜0.001 vs control group.
aggravated, and iron content in the liver and plasma was lowered [33]. In addition, studies showed that FPN expression in inflammatory monocytes is negatively affected by autocrine formation of hepcidin [34]. Iron overload was associated with renal tubular cell injury [35,36]. However, iron toxicity depends on the amount of free iron rather than the total iron load [37]. Renal iron was not altered, but protein overload enhanced ferrous iron levels in the kidney, resulting in tubulointerstitial injury that was ameliorated by iron restriction [7]. Moreover, although mice with diabetic nephropathy did not show an increase in renal iron, limiting iron intake prevented the progression of diabetic nephropathy [38]. And renal iron overload accelerated diabetes progression by activating the RAAS system [39]. UUO model mice have been reported to have elevated renal total iron content. Our study showed that renal ferrous iron levels, the main involvement of pathological renal conditions, were significantly increased on the first day of the UUO model. Funding This work was supported by grants from Programs of the National Natural Science Foundation of China [No. 81830019]. Declaration of competing interest There are no conflicts of interest.
Acknowledgements Sai Pan conducted experiments, acquired and analyzed the data, and wrote the manuscript; Zhong-Ming Qian; Shaoyuan Cui; Weiren Lan; Delong Zhao; Xu Wang analyzed and discussed the data; Xiangmei Chen designed the research, analyzed the data. References [1] M. El Nahas, The burden of chronic kidney disease on developing nations: a 21st century challenge in global health editorial comment, Nephron Clin. Pract. 118 (2011). C277-C277. [2] L. Zhang, F. Wang, L. Wang, et al., Prevalence of chronic kidney disease in China: a cross-sectional survey, Lancet 379 (2012) 815e822. [3] R. Lozano, M. Naghavi, K. Foreman, et al., Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010, Lancet 380 (2012) 2095e2128. [4] J.S. Duffield, Cellular and molecular mechanisms in kidney fibrosis, J. Clin. Investig. 124 (2014) 2299e2306. [5] H. Wang, K. Nishiya, H. Ito, et al., Iron deposition in renal biopsy specimens from patients with kidney diseases, Am. J. Kidney Dis. 38 (2001) 1038e1044. [6] D.A. Sears, P.R. Anderson, A.L. Foy, et al., Urinary iron excretion and renal metabolism of hemoglobin in hemolytic diseases, Blood 28 (1966) 708e725. [7] Y. Ikeda, Y. Horinouchi, H. Hamano, et al., Dietary iron restriction alleviates renal tubulointerstitial injury induced by protein overload in mice, Sci. Rep. 7 (2017) 10621. [8] Y. Naito, A. Fujii, H. Sawada, et al., Association between renal iron accumulation and renal interstitial fibrosis in a rat model of chronic kidney disease, Hypertens. Res. 38 (2015) 463e470. [9] Y. Ikeda, I. Ozono, S. Tajima, et al., Iron chelation by deferoxamine prevents renal interstitial fibrosis in mice with unilateral ureteral obstruction, PLoS One 9 (2014), e89355.
Please cite this article as: S. Pan et al., Local hepcidin increased intracellular iron overload via the degradation of ferroportin in the kidney, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.066
6
S. Pan et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
[10] M. Abouhamed, J. Gburek, W. Liu, et al., Divalent metal transporter 1 in the kidney proximal tubule is expressed in late endosomes/lysosomal membranes: implications for renal handling of protein-metal complexes, Am. J. Physiol. Renal. Physiol. 290 (2006) F1525eF1533. [11] M.L. Huang, D.J. Lane, D.R. Richardson, Mitochondrial mayhem: the mitochondrion as a modulator of iron metabolism and its role in disease, Antioxidants Redox Signal. 15 (2011) 3003e3019. [12] T.S. Koskenkorva-Frank, G. Weiss, W.H. Koppenol, et al., The complex interplay of iron metabolism, reactive oxygen species, and reactive nitrogen species: insights into the potential of various iron therapies to induce oxidative and nitrosative stress, Free Radic. Biol. Med. 65 (2013) 1174e1194. [13] C.P. Smith, F. Thevenod, Iron transport and the kidney, Biochim. Biophys. Acta 1790 (2009) 724e730. [14] J.A. Moreno, C. Martin-Cleary, E. Gutierrez, et al., AKI associated with macroscopic glomerular hematuria: clinical and pathophysiologic consequences, Clin. J. Am. Soc. Nephrol. 7 (2012) 175e184. [15] A.M.F. Martines, R. Masereeuw, H. Tjalsma, et al., Iron metabolism in the pathogenesis of iron-induced kidney injury, Nat. Rev. Nephrol. 9 (2013) 385e398. [16] J.J. Kroot, H. Tjalsma, R.E. Fleming, et al., Hepcidin in human iron disorders: diagnostic implications, Clin. Chem. 57 (2011) 1650e1669. [17] D.A. Vesey, Transport pathways for cadmium in the intestine and kidney proximal tubule: focus on the interaction with essential metals, Toxicol. Lett. 198 (2010) 13e19. [18] T. Veuthey, M.C. D’Anna, M.E. Roque, Role of the kidney in iron homeostasis: renal expression of Prohepcidin, Ferroportin, and DMT1 in anemic mice, Am. J. Physiol. Renal. Physiol. 295 (2008) F1213eF1221. [19] M.W. Hentze, M.U. Muckenthaler, B. Galy, et al., Two to tango: regulation of Mammalian iron metabolism, Cell 142 (2010) 24e38. [20] T. Ganz, E. Nemeth, Hepcidin and disorders of iron metabolism, Annu. Rev. Med. 62 (2011) 347e360. [21] D.A. Weinstein, C.N. Roy, M.D. Fleming, et al., Inappropriate expression of hepcidin is associated with iron refractory anemia: implications for the anemia of chronic disease, Blood 100 (2002) 3776e3781. [22] C. Pigeon, G. Ilyin, B. Courselaud, et al., A new mouse liver-specific gene, encoding a protein homologous to human antimicrobial peptide hepcidin, is overexpressed during iron overload, J. Biol. Chem. 276 (2001) 7811e7819. [23] L. Kautz, G. Jung, E.V. Valore, et al., Identification of erythroferrone as an erythroid regulator of iron metabolism, Nat. Genet. 46 (2014) 678e684. [24] G. Nicolas, C. Chauvet, L. Viatte, et al., The gene encoding the iron regulatory peptide hepcidin is regulated by anemia, hypoxia, and inflammation, J. Clin.
Investig. 110 (2002) 1037e1044. [25] H. Kulaksiz, F. Theilig, S. Bachmann, et al., The iron-regulatory peptide hormone hepcidin: expression and cellular localization in the mammalian kidney, J. Endocrinol. 184 (2005) 361e370. [26] B. Moulouel, D. Houamel, C. Delaby, et al., Hepcidin regulates intrarenal iron handling at the distal nephron, Kidney Int. 84 (2013) 756e766. [27] J. Gao, L. Wu, Y. Wang, et al., Knockdown of Cxcl10 inhibits mesangial cell proliferation in murine habu nephritis via ERK signaling, Cell. Physiol. Biochem. 42 (2017) 2118e2129. [28] K. Oshima, Y. Ikeda, Y. Horinouchi, et al., Iron suppresses erythropoietin expression via oxidative stress-dependent hypoxia-inducible factor-2 alpha inactivation, Lab. Investig. 97 (2017) 555e566. [29] C.Y. Wang, J.L. Babitt, Liver iron sensing and body iron homeostasis, Blood 133 (2019) 18e29. [30] I. Silval, V. Rausch, T. Peccerella, et al., Hypoxia enhances H2O2-mediated upregulation of hepcidin: evidence for NOX4-mediated iron regulation, Redox Biol. 16 (2018) 1e10. [31] D.C. Harris, Y.C. Tay, Mitochondrial function in rat renal cortex in response to proteinuria and iron, Clin. Exp. Pharmacol. Physiol. 24 (1997) 916e922. [32] R.P. van Swelm, J.F. Wetzels, V.G. Verweij, et al., Renal handling of circulating and renal-synthesized hepcidin and its protective effects against hemoglobinmediated kidney injury, J. Am. Soc. Nephrol. 27 (2016) 2720e2732. [33] X. Wang, X. Zheng, J. Zhang, et al., Physiological functions of ferroportin in the regulation of renal iron recycling and ischemic acute kidney injury, Am. J. Physiol. Renal. Physiol. 315 (2018) F1042eF1057. [34] I. Theurl, M. Theurl, M. Seifert, et al., Autocrine formation of hepcidin induces iron retention in human monocytes, Blood 111 (2008) 2392e2399. [35] K.A. Nath, Heme oxygenase-1: a provenance for cytoprotective pathways in the kidney and other tissues, Kidney Int. 70 (2006) 432e443. [36] H.T. Sponsel, A.C. Alfrey, W.S. Hammond, et al., Effect of iron on renal tubular epithelial cells, Kidney Int. 50 (1996) 436e444. [37] G.A. Ramm, R.G. Ruddell, Iron homeostasis, hepatocellular injury, and fibrogenesis in hemochromatosis: the role of inflammation in a noninflammatory liver disease, Semin. Liver Dis. 30 (2010) 271e287. [38] Y. Ikeda, H. Enomoto, S. Tajima, et al., Dietary iron restriction inhibits progression of diabetic nephropathy in db/db mice, Am. J. Physiol. Renal. Physiol. 304 (2013) F1028eF1036. [39] K. Chaudhary, A. Chilakala, S. Ananth, et al., Renal iron accelerates the progression of diabetic nephropathy in HFE (high Fe-iron) gene knockout mouse model of iron overload, Am. J. Physiol. Renal. Physiol. (2019), https://doi.org/ 10.1152/ajprenal.00184.2019.
Please cite this article as: S. Pan et al., Local hepcidin increased intracellular iron overload via the degradation of ferroportin in the kidney, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.066
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Local hepcidin increased intracellular iron overload via the degradation of ferroportin in the kidney Sai Pan a, Zhong-Ming Qian b, Shaoyuan Cui a, Delong Zhao a, Weiren Lan c, Xu Wang a, Xiangmei Chen a, * a
Department of Nephrology, Chinese PLA General Hospital, Chinese PLA Institute of Nephrology, Beijing Key Laboratory of Kidney Disease, State Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, Beijing, People’s Republic of China Laboratory of Neuropharmacology, Fudan University School of Pharmacy, Shanghai, 201203, People’s Republic of China c The Second Affiliated Hospital of Army Medical University, Chongqing, People’s Republic of China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 October 2019 Accepted 10 November 2019 Available online xxx
Background: Hepcidin is a key regulator of iron homeostasis. Some studies showed that exogenous hepcidin decreased the expression of divalent metal transporter (DMT1) rather than ferroportin(FPN1) to regulate renal iron metabolism. This study explored the effects of hepcidin synthesized by the kidney and its mechanism of iron regulation. Methods: In the in vivo experiments, mice were divided into a unilateral ureter obstruction (UUO) model group and a sham operation group, and mice in the UUO model group were sacrificed on days 1, 3, 5 and 7. The expression of renal hepcidin, FPN1, DMT1 and the retention of renal iron were studied. In the in vitro experiments, we overexpressed hepcidin in HK-2 cells. Then we tested the expression of renal hepcidin, FPN1, DMT1 and observed the production of intracellular ferrous ions. Results: Renal hepcidin expression was consistently higher in the UUO group than in the sham group from the first day. The expression of FPN1 gradually decreased, and the expression of DMT1 gradually increased in the UUO model. Intracellular ferrous ions significantly increased on the first day of the UUO model. In hepcidin overexpressed HK-2 cells, the expression of FPN1 was decreased, while the expression of DMT1 has no significant change. In addition, production of intracellular ferrous ions increased. Conclusion: local hepcidin can regulate iron metabolism in the kidney by adjusting the expression of FPN1. © 2019 Elsevier Inc. All rights reserved.
Keywords: Hepcidin Ferroportin Iron overload Kidney
1. Introduction The prevalence of chronic kidney disease (CKD) is high in both developing and developed countries [1,2]. CKD rose to 18th on the list of causes of the total number of global deaths in 2010 [3]. Notably, fibrosis is a pathological manifestation of all forms of CKD and irreversibly reduces kidney function [4]. Many studies have indicated that renal iron content is increased in patients with CKD [5,6] and animal models of CKD [7e9] and that dietary iron restriction or iron chelation reduces renal fibrosis. Excess iron in renal cells catalyzes the production of hydroxylation by the Fenton reaction, promoting lipid peroxidation, lysosomal damage, and
* Corresponding author. 28 Fuxing Road, Haidian District, Beijing, People’s Republic of China. E-mail address: [email protected] (X. Chen).
mitochondrial dysfunction, leading to apoptosis, ferroptosis and renal fibrosis [10e12]. Therefore, understanding the mechanism of kidney iron accumulation is important. Renal filtration and reabsorption of iron play an important role in the balance of iron homeostasis [13]. In patients with kidney diseases, renal tubules are exposed to a high concentration of iron owing to increased glomerular filtration of iron and iron-containing proteins, including hemoglobin, transferrin and neutrophil gelatinase-associated lipocalin (NGAL) [14,15]. Luminal proteinbound iron is endocytosed by transferrin receptor protein 1 (TFR1), transferrin receptor protein 2 (TFR2), cubilin and megalin [16]. Filtered free iron and iron released from proteins as a result of acidification of the filtrate as it passes along the nephron enter the tubular epithelial cells via the apical ion transporter DMT-1 (in both proximal and distal tubules) [17,18]. Hepcidin, which is mainly synthesized by hepatocytes, is a central iron regulator and induces internalization and degradation
https://doi.org/10.1016/j.bbrc.2019.11.066 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: S. Pan et al., Local hepcidin increased intracellular iron overload via the degradation of ferroportin in the kidney, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.066
2
S. Pan et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
of ferroportin (FPN1), the only known mammalian cellular iron exporter [19,20]. Hepcidin synthesis is regulated by body iron status [21,22], erythropoietic activity [23], hypoxia and inflammatory cytokines [24]. Hepcidin has been shown to be locally synthesized and secreted by renal proximal tubules [25]. A study showed that the degree of kidney iron overload may not correlates with the extent of systemic iron overload [26]. However, whether there is an autoregulatory mechanism for renal iron metabolism is not defined. In the present study, we explored the mechanism of iron accumulation in the kidney and the effect of local hepcidin. We used a unilateral ureteral obstruction (UUO) model, a defined model of kidney iron accumulation, and explored renal iron metabolism and hepcidin levels on days 1, 3, 5 and 7 of the UUO model. In the in vitro experiments, we overexpressed hepcidin in HK-2 cells and tested the expression of renal hepcidin, FPN1, DMT1. 2. Materials and methods 2.1. Materials Commercially available antibodies, including anti-hepcidin-25, hepcidin-25, anti-DMT1, anti-SLC40A1, anti-ferritin were purchased from Abcam (Cambridge, CA, UK); anti-GAPDH was purchased from Cell Signaling Technology (Beverly, MA, USA). 2.2. Animal preparation and procedure All experimental procedures were performed in strict accordance with the guidelines of the Animal Research Committee of the Chinese People’s Liberation Army General Hospital. Eight-week-old male C57BL/6J mice were obtained from HuafuKang Biotech Co., Ltd. (Beijing, China), and they were given free access to water and food. The surgical procedure to establish the UUO model was previously described in detail [9]. Briefly, under pentobarbital anesthesia, the left ureter was isolated and ligated with 3e0 silk sutures at 2 points at the proximal site. In the sham group mice, the left ureter was isolated but not ligated. The total operation period lasted approximately 10 min. After the operation, the mice were placed on a heating pad until they were completely awake. The mice in the UUO model group were sacrificed on days 1, 3, 5 and 7 after the surgery. The mice were perfused with saline, and the kidney was removed and stored at 80 C or fixed in 10% formalin at room temperature until use. 2.3. Cell culture HK-2 cells were obtained from frozen storage in our laboratory. The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; 1 g/L glucose; Corning; USA) supplemented with 5% fetal bovine serum (FBS, Cat. No. 0010). For each experiment, cells at passages 5e8 were used. The cells were grown to confluence and transferred to serum-free medium for 12 h before the start of the experiments. 2.4. The overexpression of human HAMP in HK-2 cells Human HAMP was amplified by RT-PCR, and the used primers were as follows: Forward: 5‘-CCGGAATTCGCCACCATGGCACTGA GCTCCCAGATCTGGGCCG-3’; Reverse: 5‘-CGCGGATCCCTACGTCTT GCAGCACATCCCACAC-3’. After that, human HAMP gene was cloned into a pIRES2-ZsGreen1 plasmid to generate pIRES2-ZsGreenhHAMP. The pIRES2-ZsGreen-hHAMP contained green fluorescent gene, which could be used as a marker for successful expression of the human HAMP gene. Then, the pIRES2-ZsGreen-hHAMP was
transfected into HK-2 cells by Lipofectamine 2000(Invitrogen). The pIRES2-ZsGreen plasmids were transfected as control. After 48 h, the transfected cells were collected and lysed in a RIPA buffer for PCR or supplemented with protease inhibitors for Western blot . Another group used hepcidin-25 200 nmol/l to stimulate HK-2 cells for 24 h. 2.5. Quantitative real-time PCR Total RNA extraction were performed using TRIzol reagent (Invitrogen). Then total RNA were digested with DNase (Beyotime, China). cDNA preparation used a First Strand cDNA synthesis kit (New England Biolabs, USA), respectively, according to the manufacturer’s instructions. Quantitative real-time (qRT)-PCR was carried out using SYBR Select Master Mix (Life Technologies, California, USA) and an RT-PCR detection system (ABI, Foster City, CA, USA). The expression levels of all target genes were normalized to that of 18S, an internal control. The primer sets used were as follows:50 TGGAGTACGTTCTGCTCTGGAAGG-30 and 50 - ACACAGGCTGGTTGT AGTAGGAGAC-30 for human FPN1; 50 -GTAACCCGTTGAACCCCATT-30 and 50 -CCATCCAACGGTAGTAGCG-30 for 18S. 2.6. Protein extraction and Western blotting analysis The tissue samples were lysed in RIPA buffer containing a protease inhibitor cocktail (1 lg/ml leupeptin, 1 lg/ml aprotinin, and 100 mM PMSF), homogenized and centrifuged (30 min, 4 C, and 15,000 rpm) [27]. The extracted proteins were separated using SDSPAGE and then transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked and incubated overnight at 4 C with each primary antibody: anti-hepcidin-25 (1:80), antiDMT1 (1:500), anti-SLC40A1 (1:500), anti-ferritin(1:1500) and anti-GAPDH (1:1000), followed by incubation for 2 h with the secondary antibody. Immunoreactive bands were detected using a ChemiDoc-It 600 Imaging System (UVP, Upland, USA). Densitometry analysis was performed using Image J. 2.7. Detection of labile ferrous iron Intracellular labile ferrous iron was detected using FeRhoNox-1 (Goryo Chemical Company, Japan) as previously described [7,28]. In brief, frozen sections were washed three times in Hank’s balanced salt solution (HBSS) and fixed in 4% paraformaldehyde for 15 min. After washing, the sections were incubated with the anti-hepcidin25 (1:40) primary antibody at 4 C overnight in a dark humidified container followed by incubation with FITC-conjugated anti-rabbit secondary antibody (green). After washing, these sections were incubated with RhoNox-1 in HBSS (5 mM) at room temperature for 30 min. The sections were counterstained with DAPI and covered with a small drop of mounting medium. RhoNox-1 was observed and quantified using confocal microscopy. 2.8. Intracellular labile ferrous iron detection Intracellular labile ferrous iron was detected using FeRhoNox-1 (Goryo Chemical Company, Japan) as the instructions described. HK-2 cells were stimulated with hepcidin-25 for 24 h or overexpressed human HAMP gene, and then incubated with RhoNox-1 in HBSS (5 mM) at 37 C for 30 min in the dark. After washing, ferrous iron production was observed using fluorescence microscopy. 2.9. Statistical analysis All statistical analyses were performed using GraphPad Prism 5.
Please cite this article as: S. Pan et al., Local hepcidin increased intracellular iron overload via the degradation of ferroportin in the kidney, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.066
S. Pan et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
3
Fig. 1. Iron accumulation and hepcidin synthesis in kidneys. (A) Representative immunofluorescence of DAPI (blue), hepcidin (green), and FeRhoNox (red) staining in the kidneys of sham operation group and the UUO model group at 1, 3, 5, and 7 days. (B) Semi-quantitative analysis of FeRhoNox fluorescence intensity. The values are expressed as means ± s.d. ***P＜0.001 vs sham operation group. (C) Western blot analysis showing the hepcidin expression in kidney. *P＜0.05; **P＜0.01 vs sham operation group.
Data are presented as the mean ± SEM. Differences between the groups were assessed by student t-test or one-way analysis of variance (ANOVA). A value of p < 0.05 was considered indicative of statistical significance.
3. Results 3.1. Changes in hepcidin expression and iron content in the kidney Ferrous ion production in the kidney was significantly increased on day 1 of the UUO model and remained higher than that in the sham operation group (Fig. 1B). Western blotting results showed that hepcidin protein expression was increased on the first day (Fig. 1C), and immunofluorescence supported this result. In addition, we observed that increased expression of hepcidin was associated with the degree of ferrous ions accumulation in the kidney (Fig. 1A).
3.2. Expression of DMT1 and FPN1 in the kidney Beginning on the first day of the UUO model, the protein expression of DMT1 gradually increased (Fig. 2A). However, the protein expression of FPN1 showed significant decreases on day 3 of the UUO model(Fig. 2B).
3.3. Expression of hepcidin, DMT1, FPN1, ferritin and iron content in HK-2 cells We found that hepcidin mRNA expression in HK-2 cells was increased after transfected pIRES2-ZsGreen-hHAMP plasmid into cells. In hepcidin overexpressed HK-2 cells, the mRNA and protein expression of FPN1 was decreased, while the expression of DMT1 has no significant change (Fig. 3). In addition, the expression of ferritin increased, and the production of intracellular ferrous ions augmented (Fig. 4). 4. Discussion Herein, we examined the endogenous formation of hepcidin in HK-2 cells, a human proximal tubular epithelial cell line, and in the kidney from a mouse UUO model and the association between local hepcidin and intracellular iron overload. To our knowledge, this study is the first to overexpress HAMP gene in HK-2 cell line and we have found that local renal hepcidin can regulate iron metabolism by adjusting the expression of FPN1. Our study showed that both HK-2 cells and kidneys from the mouse UUO model express significant levels of hepcidin, our study indicated that hepcidin was mainly synthesized by tubular cells in the kidney, which was consistent with the results published by Tania Veuthey [18] and H Kulaksiz [25], as they found that hepcidin was produced by the proximal tubules (S1 and S2), basolateral thick
Please cite this article as: S. Pan et al., Local hepcidin increased intracellular iron overload via the degradation of ferroportin in the kidney, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.066
4
S. Pan et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
Fig. 2. DMT1 and FPN1 expression in the kidney. (A) Western blot analysis showing the DMT1 expression in the kidney. *P＜0.05; ***P＜0.001 vs sham operation group. (B) Western blot analysis showing the FPN1 expression in the kidney. **P＜0.01; ***P＜0.001 vs sham operation group.
ascending limb and medullary collecting duct. Renal hepcidin protein expression was significantly increased on the first day of the UUO model, which was regulated by iron status, inflammatory stimuli and hypoxia [24,29,30]. In addition, studies showed that urinary iron content was increased in CKD patients and animal models of CKD [6,31]. Interestingly, we found that the site of renal hepcidin synthesis was highly coincident with the location of ferrous ion accumulation in the kidney. The relationship between kidney-derived hepcidin and renal ferrous ions overload was further confirmed by overexpressed HAMP gene HK-2 cells. We found that with upregulation of
endogenous hepcidin, the expression of FPN1 decreased and cellular ferrous ions increased. It was reported that administration of exogenous hepcidin reduced the expression of DMT1 instead of FPN1 in the kidney [32]. However, the degree of iron accumulation in the kidney is consistent with the expression of endogenous hepcidin mRNA [26], which was in accordance with our results. In addition, the study was also showed that in HAMP knockout (KO) mice, FPN was highly expressed in the basolateral and intracellular membrane in the epithelial cells of the thick ascending limbs. Moreover, a study showed that after specifically knocking out the FPN gene of the renal tubule, iron accumulation in the kidney was
Fig. 3. Hepcidin, DMT1 and FPN1 expression in HK-2 cells. (A) RT-PCR showing the hepcidin and FPN1 expression in cells. **P＜0.01; ***P＜0.001 vs control group. (B) Western blot analysis showing the FPN1 expression in cells. **P＜0.01; ***P＜0.001 vs control group.
Please cite this article as: S. Pan et al., Local hepcidin increased intracellular iron overload via the degradation of ferroportin in the kidney, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.066
S. Pan et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
5
Fig. 4. Iron accumulation and ferritin expression in HK-2 cells. (A) Representative immunofluorescence of FeRhoNox (red) staining in HK-2 cells. (B) Western blot analysis showing the ferritin expression in cells. The values are expressed as means ± s.d. ***P＜0.001 vs control group.
aggravated, and iron content in the liver and plasma was lowered [33]. In addition, studies showed that FPN expression in inflammatory monocytes is negatively affected by autocrine formation of hepcidin [34]. Iron overload was associated with renal tubular cell injury [35,36]. However, iron toxicity depends on the amount of free iron rather than the total iron load [37]. Renal iron was not altered, but protein overload enhanced ferrous iron levels in the kidney, resulting in tubulointerstitial injury that was ameliorated by iron restriction [7]. Moreover, although mice with diabetic nephropathy did not show an increase in renal iron, limiting iron intake prevented the progression of diabetic nephropathy [38]. And renal iron overload accelerated diabetes progression by activating the RAAS system [39]. UUO model mice have been reported to have elevated renal total iron content. Our study showed that renal ferrous iron levels, the main involvement of pathological renal conditions, were significantly increased on the first day of the UUO model. Funding This work was supported by grants from Programs of the National Natural Science Foundation of China [No. 81830019]. Declaration of competing interest There are no conflicts of interest.
Acknowledgements Sai Pan conducted experiments, acquired and analyzed the data, and wrote the manuscript; Zhong-Ming Qian; Shaoyuan Cui; Weiren Lan; Delong Zhao; Xu Wang analyzed and discussed the data; Xiangmei Chen designed the research, analyzed the data. References [1] M. El Nahas, The burden of chronic kidney disease on developing nations: a 21st century challenge in global health editorial comment, Nephron Clin. Pract. 118 (2011). C277-C277. [2] L. Zhang, F. Wang, L. Wang, et al., Prevalence of chronic kidney disease in China: a cross-sectional survey, Lancet 379 (2012) 815e822. [3] R. Lozano, M. Naghavi, K. Foreman, et al., Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010, Lancet 380 (2012) 2095e2128. [4] J.S. Duffield, Cellular and molecular mechanisms in kidney fibrosis, J. Clin. Investig. 124 (2014) 2299e2306. [5] H. Wang, K. Nishiya, H. Ito, et al., Iron deposition in renal biopsy specimens from patients with kidney diseases, Am. J. Kidney Dis. 38 (2001) 1038e1044. [6] D.A. Sears, P.R. Anderson, A.L. Foy, et al., Urinary iron excretion and renal metabolism of hemoglobin in hemolytic diseases, Blood 28 (1966) 708e725. [7] Y. Ikeda, Y. Horinouchi, H. Hamano, et al., Dietary iron restriction alleviates renal tubulointerstitial injury induced by protein overload in mice, Sci. Rep. 7 (2017) 10621. [8] Y. Naito, A. Fujii, H. Sawada, et al., Association between renal iron accumulation and renal interstitial fibrosis in a rat model of chronic kidney disease, Hypertens. Res. 38 (2015) 463e470. [9] Y. Ikeda, I. Ozono, S. Tajima, et al., Iron chelation by deferoxamine prevents renal interstitial fibrosis in mice with unilateral ureteral obstruction, PLoS One 9 (2014), e89355.
Please cite this article as: S. Pan et al., Local hepcidin increased intracellular iron overload via the degradation of ferroportin in the kidney, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.066
6
S. Pan et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
[10] M. Abouhamed, J. Gburek, W. Liu, et al., Divalent metal transporter 1 in the kidney proximal tubule is expressed in late endosomes/lysosomal membranes: implications for renal handling of protein-metal complexes, Am. J. Physiol. Renal. Physiol. 290 (2006) F1525eF1533. [11] M.L. Huang, D.J. Lane, D.R. Richardson, Mitochondrial mayhem: the mitochondrion as a modulator of iron metabolism and its role in disease, Antioxidants Redox Signal. 15 (2011) 3003e3019. [12] T.S. Koskenkorva-Frank, G. Weiss, W.H. Koppenol, et al., The complex interplay of iron metabolism, reactive oxygen species, and reactive nitrogen species: insights into the potential of various iron therapies to induce oxidative and nitrosative stress, Free Radic. Biol. Med. 65 (2013) 1174e1194. [13] C.P. Smith, F. Thevenod, Iron transport and the kidney, Biochim. Biophys. Acta 1790 (2009) 724e730. [14] J.A. Moreno, C. Martin-Cleary, E. Gutierrez, et al., AKI associated with macroscopic glomerular hematuria: clinical and pathophysiologic consequences, Clin. J. Am. Soc. Nephrol. 7 (2012) 175e184. [15] A.M.F. Martines, R. Masereeuw, H. Tjalsma, et al., Iron metabolism in the pathogenesis of iron-induced kidney injury, Nat. Rev. Nephrol. 9 (2013) 385e398. [16] J.J. Kroot, H. Tjalsma, R.E. Fleming, et al., Hepcidin in human iron disorders: diagnostic implications, Clin. Chem. 57 (2011) 1650e1669. [17] D.A. Vesey, Transport pathways for cadmium in the intestine and kidney proximal tubule: focus on the interaction with essential metals, Toxicol. Lett. 198 (2010) 13e19. [18] T. Veuthey, M.C. D’Anna, M.E. Roque, Role of the kidney in iron homeostasis: renal expression of Prohepcidin, Ferroportin, and DMT1 in anemic mice, Am. J. Physiol. Renal. Physiol. 295 (2008) F1213eF1221. [19] M.W. Hentze, M.U. Muckenthaler, B. Galy, et al., Two to tango: regulation of Mammalian iron metabolism, Cell 142 (2010) 24e38. [20] T. Ganz, E. Nemeth, Hepcidin and disorders of iron metabolism, Annu. Rev. Med. 62 (2011) 347e360. [21] D.A. Weinstein, C.N. Roy, M.D. Fleming, et al., Inappropriate expression of hepcidin is associated with iron refractory anemia: implications for the anemia of chronic disease, Blood 100 (2002) 3776e3781. [22] C. Pigeon, G. Ilyin, B. Courselaud, et al., A new mouse liver-specific gene, encoding a protein homologous to human antimicrobial peptide hepcidin, is overexpressed during iron overload, J. Biol. Chem. 276 (2001) 7811e7819. [23] L. Kautz, G. Jung, E.V. Valore, et al., Identification of erythroferrone as an erythroid regulator of iron metabolism, Nat. Genet. 46 (2014) 678e684. [24] G. Nicolas, C. Chauvet, L. Viatte, et al., The gene encoding the iron regulatory peptide hepcidin is regulated by anemia, hypoxia, and inflammation, J. Clin.
Investig. 110 (2002) 1037e1044. [25] H. Kulaksiz, F. Theilig, S. Bachmann, et al., The iron-regulatory peptide hormone hepcidin: expression and cellular localization in the mammalian kidney, J. Endocrinol. 184 (2005) 361e370. [26] B. Moulouel, D. Houamel, C. Delaby, et al., Hepcidin regulates intrarenal iron handling at the distal nephron, Kidney Int. 84 (2013) 756e766. [27] J. Gao, L. Wu, Y. Wang, et al., Knockdown of Cxcl10 inhibits mesangial cell proliferation in murine habu nephritis via ERK signaling, Cell. Physiol. Biochem. 42 (2017) 2118e2129. [28] K. Oshima, Y. Ikeda, Y. Horinouchi, et al., Iron suppresses erythropoietin expression via oxidative stress-dependent hypoxia-inducible factor-2 alpha inactivation, Lab. Investig. 97 (2017) 555e566. [29] C.Y. Wang, J.L. Babitt, Liver iron sensing and body iron homeostasis, Blood 133 (2019) 18e29. [30] I. Silval, V. Rausch, T. Peccerella, et al., Hypoxia enhances H2O2-mediated upregulation of hepcidin: evidence for NOX4-mediated iron regulation, Redox Biol. 16 (2018) 1e10. [31] D.C. Harris, Y.C. Tay, Mitochondrial function in rat renal cortex in response to proteinuria and iron, Clin. Exp. Pharmacol. Physiol. 24 (1997) 916e922. [32] R.P. van Swelm, J.F. Wetzels, V.G. Verweij, et al., Renal handling of circulating and renal-synthesized hepcidin and its protective effects against hemoglobinmediated kidney injury, J. Am. Soc. Nephrol. 27 (2016) 2720e2732. [33] X. Wang, X. Zheng, J. Zhang, et al., Physiological functions of ferroportin in the regulation of renal iron recycling and ischemic acute kidney injury, Am. J. Physiol. Renal. Physiol. 315 (2018) F1042eF1057. [34] I. Theurl, M. Theurl, M. Seifert, et al., Autocrine formation of hepcidin induces iron retention in human monocytes, Blood 111 (2008) 2392e2399. [35] K.A. Nath, Heme oxygenase-1: a provenance for cytoprotective pathways in the kidney and other tissues, Kidney Int. 70 (2006) 432e443. [36] H.T. Sponsel, A.C. Alfrey, W.S. Hammond, et al., Effect of iron on renal tubular epithelial cells, Kidney Int. 50 (1996) 436e444. [37] G.A. Ramm, R.G. Ruddell, Iron homeostasis, hepatocellular injury, and fibrogenesis in hemochromatosis: the role of inflammation in a noninflammatory liver disease, Semin. Liver Dis. 30 (2010) 271e287. [38] Y. Ikeda, H. Enomoto, S. Tajima, et al., Dietary iron restriction inhibits progression of diabetic nephropathy in db/db mice, Am. J. Physiol. Renal. Physiol. 304 (2013) F1028eF1036. [39] K. Chaudhary, A. Chilakala, S. Ananth, et al., Renal iron accelerates the progression of diabetic nephropathy in HFE (high Fe-iron) gene knockout mouse model of iron overload, Am. J. Physiol. Renal. Physiol. (2019), https://doi.org/ 10.1152/ajprenal.00184.2019.
Please cite this article as: S. Pan et al., Local hepcidin increased intracellular iron overload via the degradation of ferroportin in the kidney, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.066