Investigating the Molecular Mechanisms of Renal Hepcidin Induction and Protection upon Hemoglobin-Induced Acute Kidney Injury.

Laura E. Diepeveen,Rachel P. L. van Swelm,Erwin T. Wiegerinck,Rian Roelofs,Gaby Stegemann,Myrthe Naber,Dorine W. Swinkels,Olivier Lóreal,Bart Smeets,Frank Thévenod

doi:10.3390/ijms23031352

Abstract

Hemolysis is known to cause acute kidney injury (AKI). The iron regulatory hormone hepcidin, produced by renal distal tubules, is suggested to exert a renoprotective role during this pathology. We aimed to elucidate the molecular mechanisms of renal hepcidin synthesis and its protection against hemoglobin-induced AKI. In contrast to known hepatic hepcidin induction, incubation of mouse cortical collecting duct (mCCDcl1) cells with IL-6 or LPS did not induce Hamp1 mRNA expression, whereas iron (FeS) and hemin significantly induced hepcidin synthesis (p < 0.05). Moreover, iron/heme-mediated hepcidin induction in mCCDcl1 cells was caused by the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, as indicated by increased nuclear Nrf2 translocation and induced expression of Nrf2 downstream targets GCLM (p < 0.001), NQO1 (p < 0.001), and TXNRD1 (p < 0.005), which could be prevented by the known Nrf2 inhibitor trigonelline. Newly created inducible kidney-specific hepcidin KO mice demonstrated a significant reduction in renal Hamp1 mRNA expression. Phenylhydrazine (PHZ)-induced hemolysis caused renal iron loading and oxidative stress in both wildtype (Wt) and KO mice. PHZ treatment in Wt induced inflammatory markers (IL-6, TNFα) but not Hamp1. However, since PHZ treatment also significantly reduced systemic hepcidin levels in both Wt and KO mice (both p < 0.001), a dissection between the roles of systemic and renal hepcidin could not be made. Combined, the results of our study indicate that there are kidney-specific mechanisms in hepcidin regulation, as indicated by the dominant role of iron and not inflammation as an inducer of renal hepcidin, but also emphasize the complex interplay of various iron regulatory mechanisms during AKI on a local and systemic level.

Highlights

Major surgery with cardiopulmonary bypass (CPB) is the second most common cause of life-threatening acute kidney injury (AKI), after sepsis [1]
Incubation of mCCDcl1 cells with hemin previously resulted in increased intracellular iron and inflammation [8], both of which can induce systemic hepcidin synthesis in the liver [18,19]
Hepcidin synthesis was not increased upon IL-6 incubation as indicated by both the Hamp1 mRNA expression levels and luciferase activity in cells transfected with a luciferase construct coupled to a Hamp promotor (Figure 1A)

Summary

Introduction

Major surgery with cardiopulmonary bypass (CPB) is the second most common cause of life-threatening acute kidney injury (AKI), after sepsis [1]. AKI as a postoperative complication affects up to one-third of patients and is associated with increased mortality and morbidity. It has been recognized that an important contribution to postoperative AKI is made by hemolysis as a result of the mechanical forces of both the blood pump and suction system during CPB [2,3]. Upon saturation of the systemic hemoglobin scavenger haptoglobin, hemoglobin and its toxic reactive breakdown products heme and free iron are filtered by the glomerulus [5,6]. During severe hemolysis, hemoglobin, heme, and free iron may accumulate in the kidney, promoting renal cell damage [11]. The exact mechanisms underlying hemoglobin-induced AKI have not been completely elucidated but appear to be multi-factorial and intertwined. There are no adequate measures to prevent or treat hemoglobin-induced AKI

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Results