Change in iron metabolism in rats after renal ischemia/reperfusion injury

Abstract

Previous studies have indicated that hepcidin, which can regulate iron efflux by binding to ferroportin-1 (FPN1) and inducing its internalization and degradation, acts as the critical factor in the regulation of iron metabolism. However, it is unknown whether hepcidin is involved in acute renal ischemia/reperfusion injury (IRI). In this study, an IRI rat model was established via right renal excision and blood interruption for 45 min in the left kidney, and iron metabolism indexes were examined to investigate the change in iron metabolism and to analyze the role of hepcidin during IRI. From 1 to 24 h after renal reperfusion, serum creatinine and blood urea nitrogen were found to be time-dependently increased with different degrees of kidney injury. Regular variations in iron metabolism indexes in the blood and kidneys were observed in renal IRI. Renal iron content, serum iron and serum ferritin increased early after reperfusion and then declined. Hepcidin expression in the liver significantly increased early after reperfusion, and its serum concentration increased beginning at 8 h after reperfusion. The splenic iron content decreased significantly in the early stage after reperfusion and then increased time-dependently with increasing reperfusion time, and the hepatic iron content showed a decrease in the early stage after reperfusion. The early decrease of the splenic iron content and hepatic iron content might indicate their contribution to the increase in serum iron in renal IRI. In addition, the duodenal iron content showed time-dependently decreased since 12 h after reperfusion in the IRI groups compared to the control group. Along with the spleen, the duodenum might contribute to the decrease in serum iron in the later stage after reperfusion. The changes in iron metabolism indexes observed in our study demonstrate an iron metabolism disorder in renal IRI, and hepcidin might be involved in maintaining iron homeostasis in renal IRI. These findings might suggest a self-protection mechanism regulating iron homeostasis in IRI and provide a new perspective on iron metabolism in attenuating renal IRI.

Fig 1. Renal pathological changes and changes in renal function after renal IRI.

(A) Varying degrees of renal pathological changes from 1 h to 24 h after reperfusion in the IRI groups, including loss of brush borders, vacuolar degeneration and necrosis in epithelial cells, dilatation, cast formation, and cell debris in the kidney tubules. Arrow denotes pathological change of the renal tissue. The control group displayed normal histology. (B) SCr and (C) BUN levels were significantly elevated in the IRI groups compared with levels in the control group, and both SCr and BUN were time-dependently elevated from 1 h to 24 h after reperfusion in the IRI groups. Kidney sections were stained with hematoxylin and eosin. Original magnification, X200. The data are presented as the means ± standard deviation. * P <0.05, compared to control group; n = 6 in each group. IRI: ischemia/reperfusion injury.

Fig 2. Evaluation of iron metabolism indexes in the serum and kidney after renal IRI.

(A) SI increased significantly early after reperfusion, and it time-dependently decreased after reperfusion in the IRI groups. (B) The level of SF began to rise at 4 h after reperfusion and started to decline after reaching a maximum value at 16 h after reperfusion in the IRI groups. (C) Serum hepcidin increased significantly beginning at 8 h after reperfusion and started to decline after reaching a maximum value at 16 h after reperfusion in the IRI groups. (D) Renal iron content was time-dependently increased from 1 h to 8 h after reperfusion and then declined gradually in the IRI groups. The data are presented as the means ± standard deviation. * P <0.05, compared to control group; n = 6 in each group. IRI: ischemia/reperfusion injury; SI: serum iron; SF: serum ferritin.

Fig 3. Iron analysis in the spleen, liver and duodenum after IRI.

(A) The splenic iron content was significantly decreased in the early stage after IR and then increased time-dependently with increasing reperfusion time. (B) The hepatic iron content declined in the early stage of IRI but was not significantly different than the hepatic iron content in the control group. (C) The duodenal iron content showed no obvious change in the early stage after reperfusion, but it time-dependently decreased since 16 h after reperfusion in the IRI groups. The data are presented as the means ± standard deviation. * P <0.05, compared to control group; n = 6 in each group. IRI: ischemia/reperfusion injury.

Fig 4. Liver hepcidin expression and kidney FPN1 expression after IRI.

(A) In the IRI groups, hepcidin mRNA levels increased rapidly after reperfusion and then declined gradually beginning at 8 h after reperfusion. (B) FPN1 mRNA levels in the kidney decreased gradually after reperfusion in the IRI groups. (C) Hepcidin protein expression was significantly increased beginning at 4 h after reperfusion and started to decline after reaching a maximum value at 12 h after reperfusion in the IRI groups. (D) FPN1 protein expression in the kidney clearly decreased beginning at 4 h after reperfusion in the IRI groups compared with expression in the control group. The data are presented as the means ± standard deviation. * P <0.05, compared to control group; n = 6 in each group. FPN1: ferroportin-1; IRI: ischemia/reperfusion injury.

Fig 5. Immunohistochemical analyses of hepcidin and FPN1 after renal IRI.

(A) Hepcidin protein expression in the liver increased gradually after renal reperfusion. Arrow denotes hepcidin in the hepatocytes. (B) FPN1 protein expression in the kidney clearly decreased after reperfusion. Original magnification of liver tissues, X400. Original magnification of kidney tissues, X200. The data are presented as the means ± standard deviation. * P <0.05, compared to control group; n = 6 in each group. FPN1: ferroportin-1; IRI: ischemia/reperfusion injury; IOD: integrated optical density.