Effect Of Exogenous Hepcidin On Sepsis-induced Acute Kidney Injury And Its Mechanism

Background and ObjectiveAcute kidney injury(AKI)predicts worse prognosis following sepsis,with limited available interventions.Hepcidin is a peptide hormone secreted by the liver in response to iron loading and inflammation.Studies have found that hepatic hepcidin synthesis is increased during sepsis,which may play an important role in the immune defense and prognosis of sepsis.However,it remains unclear about the effect of exogenous hepcidin on sepsis-induced AKI and its pathophysiologic mechanism.The objective of the current investigation was to study the effect of exogenous hepcidin in sepsis-induced AKI,and to preliminarily study its mechanism through in vitro and in vivo experiments.We also try to determine whether iron homeostasis and dynamic regulation of iron homeostasis are beneficial in the treatment of septic AKI.It may be provide a novel theoretical basis for the clinical prevention and treatment of sepsis AKI,and also provides a basis for the clinical application of hepcidin.MethodsFirst,a prospective cohort study was conducted.A total of 133 eligible adult patients with sepsis and septic AKI were selected and divided into survival and death groups according to 60-day all-cause mortality.Healthy volunteers(n=20)were enrolled as a control group.The differences in baseline clinical characteristics and serum hepcidin,serum iron and ferritin levels were compared between the two groups.Univariate and multivariate Cox proportional hazards regression was used to analyze the association of serum hepcidin,serum iron and ferritin with all-cause mortality in patients.The differences in mortality between different levels of iron metabolism-related parameters were further analyzed by Kaplan-Meier curve.Secondly,a septic AKI cell model was constructed by stimulating mouse tubular epithelial cell(TCMK-1)cells with LPS,and a co-culture system of TCMK-1-J774A.1 macrophage was established.TCMK-1 cells were treated directly or indirectly,the gene expression of kidney injury markers(NGAL,KIM-1)in the cell lysate was detected by Realtime-PCR,the levels of inflammatory cytokines in the supernatant were determined by ELISA,and Fe2+ in living cells was detected by fluorescent probe.Confocal microscopy and flow cytometry were used to observe the phagocytic function of macrophages by the method of phagocytosing fluorescent particles,Western Blot was used to detect the expression of ferroportin(FPN)and heavy chain-ferritin(FTH)in macrophages,etc.The direct and indirect effects of exogenous hepcidin on sepsis-induced AKI were initially investigated,and the possible mechanisms were explored in vitro.Finally,cecal ligation and puncture(CLP)surgery was performed to induce an animal model of sepsis-induced AKI in mice.Using wild-type mice and Hamp knockout mice as tool mice,the effects of exogenous hepcidin and vehicle intervention on the survival status,degree of bacterial infection,serum inflammatory cytokines,renal function,renal histopathological changes,renal oxidative stress and inflammatory cell infiltrate were compared in mice with sepsis-induced AKI.In addition,the role of exogenous hepcidin in sepsis-induced AKI and its possible mechanism were further explored by detecting the changes of serum iron,non-heme iron levels in liver,spleen,and kidney as well as the ferroportin(PFN)and H-ferritin(FTH)protein expressions in spleen.Results(1)The 60-day all-cause deaths in the sepsis and sepsis-induced AKI cohorts were 61(45.8%)and 26(60.5%),respectively.Patients with sepsis or septic-induced AKI showed significant increases in serum hepcidin,ferritin and significant decreases in serum iron on ICU admission compared with healthy volunteers.Patients in the death group were more likely than those in the survival group to have lower concentrations of serum hepcidin,but higher serum iron levels(all P<0.05).Multivariate cox regression and Kaplan-Meier analysis showed that only serum iron(cutoff 9.50 μmol/l)was still a risk factor for all-cause mortality in patients with sepsis after adjusting for confounders such as age,sex,previous history of congestive heart failure,and disease severity(APACHE Ⅱ score,SOFA score,shock,and mechanical ventilation)(HR=2.359,95%CI 1.349-4.127,P=0.003).For the septic AKI cohort,both lower concentrations of hepcidin(cutoff 62.03 ng/ml)and higher serum iron concentrations(cutoff 9.50 μmol/1)were independently associated with increased risk of allcause mortality in patients(HR=0.520,95%CI 0.319-0.847,P=0.009 and HR=2.162,95%CI 1.044-4.477,P=0.038,respectively).(2)Compared with the control group or the TCMK-1 cells treated with hepcidin alone,the cell viability was significantly reduced after 24 hours of the LPS treatment,and The percentage of lactate dehydrogenase(LDH)released from cells into the media was significantly increased,and the differences were statistically significant(both P<0.0001);however,cytotoxicity and NGAL and KIM-1 mRNA levels were comparably increased by LPS-treated cells and hepcidin pretreated TCMK-1 cells(all P>0.05).However,in the coculture model of TCMK-1 and J774A.1 macrophage,pretreatment of macrophages with hepcidin can significantly reduce the inflammatory cytokines TNF-α,IL-6 and IL-β levels,LDH release from TCMK-1 cells,KIM-1 and NGAL gene expression were also significantly decreased.Compared with PBS,the red fluorescence emitted by Fe2+in J774A.1 macrophage treated with hepcidin was significantly weaker,the fluorescence intensity was lower quantitatively,and the number and fluorescence intensity of phagocytosed E.coli particles were significantly increase.At the same time,the expression of FPN protein in macrophages pretreated with hepcidin was significantly decreased,and the expression level of anti-inflammatory protein FTH was significantly increased.(3)CLP-induced AKI led to significant changes in systemic iron homeostasis.In WT mice,it is mainly manifested as significantly elevated hepcidin,induced liver and spleen iron retention,decreased serum iron,and the body initiates hypoferremia to fight pathogenic microorganisms,and exogenous hepcidin treatment can further strengthen this defense mechanism.However,in Hamp knockout mice,deficiency of hepcidin resulted in systemic iron mobilization in liver and spleen,increased serum iron,and iron accumulation in kidneys,further aggravating CLP-induced AKI.Hepcidin reconstitution restores iron homeostasis by degrading splenic FPN(an iron export protein),inducing FTH production and effective liver and spleen iron sequestration,reducing systemic inflammatory response,reducing serum iron,increasing the number of macrophages and phagocytic function,inhibiting bacteremia,and regressing renal iron deposition(Hamp-/-mice),thereby improving local inflammation in the kidney,Oxidative stress,mitochondrial damage,and effective protection against sepsis-induced AKI.Conclusion1.Iron dyshomeostasis is an important factor affecting the prognosis of critically ill patients with sepsis and septic AKI.The decreased level of hepcidin and elevated serum iron on admission are independently associated with all-cause mortality,and may be usefully prognostic markers in patients with septic AKI.It may provide therapeutic benefit in this patient population to supplement exogenous hepcidin or reduce serum iron concentrations.2.Exogenous hepcidin cannot directly act on renal tubular epithelial cells to improve LPS-induced AKI,but it can indirectly protect sepsis-induced AKI and improves its survival mainly by regulating systemic and intracellular iron homeostasis.3.Mechanistically,hepcidin mainly targets the FPN-FTH signaling pathway of the reticuloendothelial system,regulates iron metabolism,restores iron homeostasis,reduces systemic inflammatory response,increases the number of macrophages and phagocytic function,and inhibits bacteria;thereby exerting an indirect protective effect on sepsisinduced AKI.However,in hepcidin-deficient mice,hepcidin reconstitution not only restores systemic iron homeostasis,but also helps reduce renal iron deposition and exerts a direct protective effect on the kidney.