Supplementary Data. References

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1 Supplementary Data Materials and Methods Sample collection from acute kidney injury patients Urine samples were obtained from patients who had cardiac surgery at one academic medical center and two local community teaching hospitals in Taiwan and were enrolled in the National Taiwan University Study Group on Acute Renal Failure (NSARF) (1 5). Informed consent was obtained in accordance with the Institutional Review Board approved protocol at each institution. Samples were collected and stored using a standard operating procedure, included centrifugation, storage at - 80 C, and thawing immediately prior to analysis. Clinical data including patient demographics, prior health history, serum creatinine, and hourly urine output as available in the hospital record were collected. Samples were analyzed at Astute Medical by technicians blinded to clinical data. Inclusion criteria were surgery of the heart or ascending aorta and development of acute kidney injury (AKI) within 2 days of surgery according to the KDIGO AKI classification. Results We tried to find the best cutoff values by Youden index and it showed the best cutoff values of urine hemojuvelin adjusted by urine creatinine is 0.82 (ng/ml). References 1. Huang TM, Wu VC, Young GH, Lin YF, Shiao CC, Wu PC, et al. Preoperative proteinuria predicts adverse renal outcomes after coronary artery bypass grafting. J Am Soc Nephrol 22: , ShiaoCC,WuVC,LiWY,LinYF,HuFC,YoungGH,et al. Late initiation of renal replacement therapy is associated with worse outcomes in acute kidney injury after major abdominal surgery. Critical care (London, England)13: R171, Wu VC, Ko WJ, Chang HW, Chen YS, Chen YW, Chen YM, et al. Early renal replacement therapy in patients with postoperative acute liver failure associated with acute renal failure: effect on postoperative outcomes. J Am Coll Surg 205: , Wu VC, Ko WJ, Chang HW, Chen YW, Lin YF, Shiao CC, et al. Risk factors of early redialysis after weaning from postoperative acute renal replacement therapy. Intensive Care Med 34: , Wu VC, Wang CH, Wang WJ, Lin YF, Hu FC, Chen YW, et al. Sustained low-efficiency dialysis versus continuous veno-venous hemofiltration for postsurgical acute renal failure. Am J Surg 199: , 2009.

2 Supplementary Table S1. Characteristic Protein Identified by Proteomic Methods Gene name Protein name Score pi MW AACT Alpha-1-antichymotrypsin ALBU Serum albumin precursor AMBP Protein AMBP APOA1 Apolipoprotein A-I precursor BMP4 Bone morphogenetic protein DCD Dermcidin precursor FIBA Fibrinogen alpha chain GSTO Glutathione transferase omega GSTO1 Glutathione transferase omega HEMO Hemopexin HJV Hemojuvelin HPT Haptoglobin HPTR Haptoglobin-related protein HRNR Hornerin HV305 Ig heavy chain V-III region BRO HV305 Ig heavy chain V-III region BRO HYOU1 Hypoxia upregulated protein IGHG1 Ig gamma-1 chain C region IGHG2 Ig gamma-2 chain C region IGHG3 Ig gamma-3 chain C region IGHG4 Ig gamma-4 chain C region IGKC Ig kappa chain C region IREB2 Iron-responsive element-binding protein KV101 Ig kappa chain V-I region AG KV101 Ig kappa chain V-I region AG KV104 Ig kappa chain V-I region CAR KV105 Ig kappa chain V-I region DEE KV107 Ig kappa chain V-I region Gal KV114 Ig kappa chain V-I region OU KV114 Ig kappa chain V-I region OU KV115 Ig kappa chain V-I region Rei KV116 Ig kappa chain V-I region Roy KV118 Ig kappa chain V-I region WEA KV119 Ig kappa chain V-I region Wes KV119 Ig kappa chain V-I region Wes KV120 Ig kappa chain V-I region Mev KV121 Ig kappa chain V-I region Ni KV301 Ig kappa chain V-III region B KV302 Ig kappa chain V-III region SIE KV302 Ig kappa chain V-III region SIE KV307 Ig kappa chain V-III region GOL KV308 Ig kappa chain V-III region CLL LAC Ig lambda chain C regions LAC6 Ig lambda-6 chain C region LDHA L-lactate dehydrogenase A chain LDHB L-lactate dehydrogenase B chain LDHC L-lactate dehydrogenase C chain LV102 Ig lambda chain V-I region HA LV302 Ig lambda chain V-III region LOI NEO1 Neogenin PGRP2 N-acetylmuramoyl-L-alanine amidase PRDS1 Peroxiredoxin PRDX2 Peroxiredoxin SMAD7 Mothers against decapentaplegic homolog SODC Superoxide dismutase TRFE Serotransferrin

3 SUPPLEMENTARY FIG. S1. Kidney periodic acid-schiff (PAS) sections from acute kidney injury (AKI) mice. Shown are 40 fields of representative kidney sections from the cortex and medulla of C57BL6 wild-type mice. Injury scores were defined based on cast formation, dilation, and necrosis as detailed in Materials and Methods section. Scale bars: 20 lm. *p < 0.05 versus control (n = 10).

4 SUPPLEMENTARY FIG. S2. Temporal expression of membrane-bound hemojuvelin/soluble hemojuvelin (mhjv/ shjv) ratios in glycerol-induced acute tubular necrosis mice. (A) A representative western blot (30 lg/well) of the total kidney lysate and total liver lysate (B) for mhjv/shjv ratios. Typical results of 10 independent experiments are shown. Each experiment was normalized with the sham group as previously described. (C) The same kidney lysates were analyzed at each time point using anti-hepcidin and anti-ngal antibodies. (D) Total kidney and liver iron concentration measured after mice received glycerol injection. Total iron was measured using an ICP-AES analysis (*p < 0.05).

5 SUPPLEMENTARY FIG. S3. The effect of HJV on kidney iron store after AKI. A representative western blot (30 lg/ well) for mhjv/shjv ratios from the total kidney. Total kidney iron concentration was measured using inductively coupled plasma atomic emission spectroscopy (ICP-AES) analysis. After Ln transformation, the total kidney iron content after AKI significantly correlated to the ratio of mhjv/shjv. (Spearman correlation, r =-0.851, p < 0.001).

6 SUPPLEMENTARY FIG. S4. The uptake and release of iron in HK2 cells after free iron stimulation. Human renal proximal renal cell lines (HK2) were pretreated with 50 lm furin inhibitor (FI), 50 lm deferoxamine (DFO), or 0.5 lg/ml recombinant human HJV protein (rhhjv) for 1 h. Then, 100 lm ferric chloride was added to the culture media for 24 h as indicated. A representative western blot (30 lg/well) of (A) the cell lysate for mhjv and immunoprecipitation of (B) cultural media for shjv. FI and DFO suppressed shjv in the media. Typical results of five independent experiments are shown. Each experiment was normalized with the control group and 150 ng rhhjv proteins, which produced bands at 37 and 32 kda. (C) Confocal microscopy was used to determine the localization of HJV (green labels) in iron-induced HK2 cells. (D) The cellular iron deposition of HK2 cells were examined using Prussian blue staining. Scale bars: 50 lm. *p < 0.05 versus control (n = 5).

7 SUPPLEMENTARY FIG. S5. Temporal expression of HJV from mice that received renal ischemia/reperfusion (I/ R) injury. Representative western blots (30 lg/well) of the (A) kidney, (B) liver, and (C) muscle lysates. The antibody against b-actin or GAPDH was used as a loading control.

8 SUPPLEMENTARY FIG. S6. Temporal expression of TGF-b from mice that received renal I/R injury. A representative western blot (30 lg/well) of the kidney lysate showed intrarenal TGF-b augment after 96 h. The antibody against b-actin was used as a loading control.

9 SUPPLEMENTARY FIG. S7. Kidney PAS sections from AKI mice with or without FI injection. Shown are 40 fields of representative kidney sections from the cortex and medulla of C57BL6 mice. Scale bars: 20 lm.

10 SUPPLEMENTARY FIG. S8. The cell toxicity of FI on human HK2 cells cells were treated with various concentrations of FI (D6R, 0 to 100 lm) till 48 h. Cytotoxicity assays were determined by cell count kit-8 (CCK8) according to manufacturer s protocol (Dojindo Molecular Technologies). The experiment was repeated thrice and the results represent the means standard deviations of all three experiments.