Hemojuvelin modulates iron stress during acute kidney injury: improved by furin inhibitor

Abstract

Aims: Free iron plays an important role in the pathogenesis of acute kidney injury (AKI) via the formation of hydroxyl radicals. Systemic iron homeostasis is controlled by the hemojuvelin-hepcidin-ferroportin axis in the liver, but less is known about this role in AKI.

Results: By proteomics, we identified a 42 kDa soluble hemojuvelin (sHJV), processed by furin protease from membrane-bound hemojuvelin (mHJV), in the urine during AKI after cardiac surgery. Biopsies from human and mouse specimens with AKI confirm that HJV is extensively increased in renal tubules. Iron overload enhanced the expression of hemojuvelin-hepcidin signaling pathway. The furin inhibitor (FI) decreases furin-mediated proteolytic cleavage of mHJV into sHJV and augments the mHJV/sHJV ratio after iron overload with hypoxia condition. The FI could reduce renal tubule apoptosis, stabilize hypoxic induced factor-1, prevent the accumulation of iron in the kidney, and further ameliorate ischemic-reperfusion injury. mHJV is associated with decreasing total kidney iron, secreting hepcidin, and promoting the degradation of ferroportin at AKI, whereas sHJV does the opposite.

Innovation: This study suggests the ratio of mHJV/sHJV affects the iron deposition during acute kidney injury and sHJV could be an early biomarker of AKI.

Conclusion: Our findings link endogenous HJV inextricably with renal iron homeostasis for the first time, add new significance to early predict AKI, and identify novel therapeutic targets to reduce the severity of AKI using the FI.

FIG. 1.

The identification of urinary hemojuvelin by proteomic methods. (A) The comparison of urinary proteins from pooled urine samples from healthy volunteers, post-cardiac surgery patients with and without AKI using 2-DE maps, respectively. Protein spot identified as HJV is labeled in a dotted circle. (B) 2-DE maps of pooled urinary proteins from pre- and post-ischemia/reperfusion (I/R) injury rats. The gels were stained with Coomassie blue R250, transferred into a PVDF membrane, and developed using a polyclonal antibody against HJV (arrow). Significant changes in spots were processed and analyzed using LC-MS/MS as described. 2-DE, two-dimensional electrophoresis.

FIG. 2.

Temporal change of HJV in renal I/R injury. (A) Change in serum creatinine and blood urea nitrogen (BUN) at different time points in mice. (n=10 at each time point). (B) Disease severity after I/R injury was shown by tubular injury score according to periodic acid–Schiff (PAS)-stained renal section at each time point. (C) Kidney sections stained with Prussian blue in terms of iron content. (D) Immunohistochemical (IHC) analysis using anti-HJV antibody (scale bars: 50 μM). (E) A representative western blot (30 μg/well) for membrane-bound hemojuvelin/soluble hemojuvelin (mHJV/sHJV) ratios from the total kidney and (F) liver lysate **p<0.01 vs. sham (n=10). Typical results of 10 independent experiments are shown. Each experiment was normalized with the sham group and 300 ng recombinant mouse HJV (rmHJV) proteins, which produced bands at 57 and 38 kDa. (G) Kidney lysates at each time point were analyzed using anti-hepcidin, anti-ferroportin, and anti- neutrophil gelatinase-associated lipocalin (NGAL) antibodies. (H) Total kidney and liver iron concentration measured after renal I/R injury. The total iron was measured using inductively coupled plasma atomic emission spectroscopy (ICP-AES) analysis. (I) Temporal excretion of plasma HJV measured using ELISA and (J) Trichloroacetic acid/acetone precipitated urinary protein for western analysis using anti-HJV antibody. Urinary HJV was observed predominantly as sHJV (42 kDa), with lower amounts of mHJV (50 kDa) (*p<0.05). To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 3.

Increase in expression of HJV in the cortical renal tubules in mice and humans after AKI. (A) In mice, increased HJV after renal I/R injury was predominantly in the renal tubules. (B) The staining of HJV in the normal human kidney cortex was less apparent; however, HJV was prominent in the renal tubules after hypovolemic shock-related AKI. The staining was homogenous and predominant in the proximal renal tubules. The histological images display five patients with acute tubular necrosis (ATN, patients 1–5), including fuzzy brush border, dilated renal tubule, and relative denuded nucleus. Scale bars: 50 μM. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 4.

Expression of urinary HJV and NGAL in different kidney diseases of patients. Human urine samples from patients after cardiac surgery and rhabdomyolysis-related AKI exhibited high levels of HJV, whereas samples from patients with chronic kidney disease (CKD), glomerulonephropathy, and urinary tract infection exhibited a low level of HJV, as detected by an ELISA. These results were similar to those obtained for urine NGAL expression.

FIG. 5.

The uptake and release of iron in HK2 cells after free iron stimulation under hypoxic condition. Human renal proximal renal cell lines (HK2) were pretreated with 50 μM furin inhibitor (FI), 50 μM deferoxamine (DFO), or 0.5 μg/ml recombinant human HJV protein (rhHJV) for 1 h. Then, 100 μM ferric chloride was added to the culture media under hypoxia chamber (1% oxygen) for 24 h as indicated. (A) Representative western blot (30 μg/well) of the cell lysate was analyzed. 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. (B) Confocal microscopy was used to determine the localization of HJV (green labels) in iron-induced HK2 cells. (C) The cellular iron deposition of HK2 cells were examined using Prussian blue staining. Scale bars: 10 μM. *p≤0.05 versus control (n=5). To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 6.

Amelioration of renal I/R (AKI) injury, iron deposition, and tubular apoptosis by the FI. C57BL/6 mice were injected with 10 μg rmHJV protein, 100 μM FI, or PBS prior to unilateral renal I/R injury and sham-operated mice were used as a control. (A) The total kidney and liver iron concentration were measured after the mice received a kidney I/R injury. Organic iron was quantified using ICP-AES analysis. (B) Disease severity after I/R, evaluated by kidney injury score, improved by FI injection and aggravated by rmHJV injection. (C) Immunostaining of the whole kidney lysates (30 μg/well) and the ratio of mHJV/sHJV after treatment. The antibody against β-actin was used as a loading control. (D) Tubular apoptosis. The kidney tubules of TUNEL⁺ apoptotic cells (green fluorescence in I/R injury) were reduced by pretreatment with an FI, as determined by fluorescent staining. The same field was merged with a DAPI-stained image, showing the nucleus as a counter stain (blue). Scale bars: 50 μM. (E) In situ localization of reactive oxygen species (ROS). Dihydroethidium (DHE) fluorescent analysis was used for evaluation of ROS. The same field was merged with an FITC-labeled lotus tetragonolobus lectin (LTL), which is a proximal tubular marker for histological co-localization. Scale bars: 50 μM. *p≤0.05 versus control (n=10). To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 7.

FI ameliorated renal I/R (AKI) injury via HJV-mediated mechanism. (A) Iron accumulated in hemojuvelin knockout (HJV^−/−) mice. Kidney iron was detected by Prussian blue staining. (B) HJV^+/+ or HJV^−/− mice received FI or PBS prior to unilateral renal I/R injury and sham-operation as control. Mice were sacrificed at 24 h after I/R injury. The expression levels of HJV, hepcidin, ferroportin, NGAL, and β-actin in kidney were determined by western analysis. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars