Use of hepcidin in preparation of drug for blocking ferroptosis of collecting tubule in acute kidney injuries and protecting kidney function

Abstract

The present invention belongs to the technical field of medicine. Disclosed in the present invention is a new pharmaceutical use of hepcidin, specifically the use of hepcidin in the preparation of a drug for blocking ferroptosis of a collecting tubule in acute kidney injuries and protecting kidney function. It has been demonstrated in the present invention that both renal endogenous and exogenous hepcidin can alleviate tubular injury and improve renal function in ischemia-reperfusion-induced acute kidney injury by inhibiting ferroptosis in intercalated cells of the collecting tubule. Disclosed in the present invention is a drug for alleviating ferroptosis of a collecting tubule and protecting kidney function in ischemic-reperfusion-induced acute kidney injury. The drug is an intraperitoneal injection prepared by dissolving hepcidin at a therapeutic concentration in PBS.

Description

Application of hepcidin in the preparation of drugs for blocking ferroptosis in collecting ducts of acute kidney injury and protecting renal function Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a new pharmaceutical use of hepcidin, specifically the application of hepcidin in the preparation of a drug to reduce iron death in the collecting ducts of mice induced by ischemia-reperfusion injury and to protect renal function. Background Technology

[0002] Acute kidney injury (AKI) is a significant public health burden, with high morbidity and mortality rates worldwide, reaching up to 50% in intensive care units (ICUs). Cardiac surgery-related AKI is the second leading cause of AKI in ICUs, primarily due to intraoperative ischemia-reperfusion injury. Severe cardiac surgery-related AKI is independently associated with a 3-8 times increase in perioperative mortality. However, early diagnosis remains challenging, and specific treatment strategies beyond mitigating further damage and providing supportive care are lacking. Therefore, a deeper understanding of the underlying mechanisms of ischemia-reperfusion-induced AKI is essential.

[0003] Recent clinical studies have shown that elevated urinary hepcidin concentrations 24 hours after cardiac surgery are generally associated with a reduced incidence of acute kidney injury (AKI). However, notably, serum hepcidin did not show any predictive power, suggesting that endogenous renal hepcidin may be involved in the development of AKI in these patients. Therefore, the exact role and mechanism of endogenous renal hepcidin in regulating ischemia-reperfusion injury-induced AKI warrants further investigation. Summary of the Invention

[0004] To address the aforementioned problems, this invention discloses the application of hepcidin in the preparation of drugs for blocking ferroptosis in collecting ducts of acute kidney injury and protecting renal function. This invention unexpectedly discovered that hepcidin protein levels were significantly elevated in the renal tissue of mice with ischemia-reperfusion-induced acute kidney injury, primarily located in the collecting ducts. Further transcriptome sequencing revealed that knocking down hepcidin significantly exacerbated hypoxia-induced ferroptosis in the collecting ducts, suggesting that the anti-ferroptosis function of hepcidin may improve ischemia-reperfusion-induced acute kidney injury. This hypothesis was further verified experimentally.

[0005] This invention includes the following technical solutions:

[0006] This invention discloses for the first time the application of hepcidin in the preparation of a drug for blocking ferroptosis in collecting ducts of acute kidney injury and protecting renal function.

[0007] Furthermore, in the above applications, the drug is a drug that inhibits the degree of ferroptosis in renal collecting duct cells under hypoxia-reoxygenation injury.

[0008] Furthermore, in the above application, the inhibition is achieved by hepcidin in the renal collecting duct through direct binding to ltf and upregulation of its protein, revealing the role of ferroportin independent of the hepcidin receptor.

[0009] Furthermore, in the above applications, the drug is a drug that inhibits intercalated cell ferroptosis, thereby improving renal tubular damage and renal function.

[0010] Furthermore, in the above application, the acute kidney injury occurred in mice.

[0011] Furthermore, in the above applications, the drug is administered via intraperitoneal injection, using PBS as a solvent, at a concentration of 2 mg / kg, with a maximum of 50 μg per mouse.

[0012] The present invention also discloses a drug that blocks ferroptosis in the collecting ducts of acute kidney injury and protects kidney function. The drug is an intraperitoneal injection prepared by dissolving therapeutic concentrations of hepcidin in PBS.

[0013] The present invention has the following beneficial effects:

[0014] This invention unexpectedly discovered that the level of hepcidin protein was significantly increased in the kidney tissue of mice with ischemia-reperfusion-induced acute kidney injury, and it was mainly located in the collecting duct. Transcriptome sequencing revealed that hepcidin could reduce the degree of ferroptosis in the collecting duct, suggesting that the anti-ferroptosis function of hepcidin may improve ischemia-reperfusion-induced acute kidney injury. The above hypothesis was further verified by experiments.

[0015] Specifically, this invention proposes that hepcidin expressed in collecting ducts and exogenous hepcidin can directly bind to lactoferrin and stabilize its expression, thereby inhibiting intercalated cell ferroptosis and alleviating ischemia-reperfusion-induced acute kidney injury.

[0016] This invention investigates the role of hepcidin in alleviating ischemia-reperfusion-induced acute kidney injury (AKI) using in vitro cell models of hypoxia-reoxygenation and mouse models of AKI. Results showed that hepcidin significantly improved tubular damage and renal function, intercalated cell ferroptosis, and reduced tubulointerstitial inflammation in AKI in both the ischemia-reperfusion mouse model and in vitro cell models. Our findings have the potential to provide effective clinical drugs for the prevention and treatment of AKI. Attached Figure Description

[0017] Figure 1: Showing that hepcidin in collecting ducts inhibits ferroptosis in collecting duct cells under hypoxia-reoxygenation injury by directly binding to ltf and upregulating its protein expression.

[0018] The following tests were performed on the following four groups: normoxic siNC group, normoxic siHepcidin group, hypoxia-reoxygenation siNC group, and hypoxia-reoxygenation siHepcidin group: (A) Ptgs2 protein (B) Ptgs2 protein quantification (C) PI staining (D) PI staining quantification (E) C11 bodipy staining (F) FerroOrange staining (G) FerroOrange staining quantification.

[0019] (H)IP experiments were used to verify the interaction between Hepcidin and Ltf proteins;

[0020] (I) Ltf protein and quantification in four groups: normoxic Flag-Control group, normoxic Flag-Hepcidin group, hypoxia-reoxygenation Flag-Control group, and hypoxia-reoxygenation Flag-Hepcidin group;

[0021] (J) Gpx4 protein and quantification in six groups: normoxic Flag-Control+siNC group, normoxic Flag-Hepcidin+siNC group, normoxic Flag-Hepcidin+siLtf group, hypoxia-reoxygenation Flag-Control+siNC group, hypoxia-reoxygenation Flag-Hepcidin+siNC group and hypoxia-reoxygenation Flag-Hepcidin+siLtf group;

[0022] Figure 2: This shows that hepcidin in the collecting duct significantly improved renal tubular injury and renal function under ischemia-reperfusion injury by inhibiting intercalated cell ferroptosis.

[0023] Hepcidin flox / flox sham group, Hepcidin KspKI sham group, Hepcidin flox / flox bIRI 32min group and Hepcidin KspKI In the bIRI32min group, among the four groups, (A) kidney PAS staining, (B) kidney injury score, (C) Scr, (D) BUN, (E) 4HNE, Gpx4, Acsl4, and Ptgs2 proteins, (F) 4HNE protein quantification, (G) Gpx4 protein quantification, (H) Acsl4 protein quantification, (I) Ptgs2 protein quantification, and (J) Acsl4 staining co-localized with ATP6V1B1.

[0024] Figure 3: Exogenous hepcidin significantly improved renal tubular injury and renal function under ischemia-reperfusion injury by inhibiting intercalated cell ferroptosis; (A) renal PAS staining (B) renal injury score (C) Scr (D) BUN (E) Acsl4, Gpx4, Ptgs2 protein (F) Acsl4 protein quantification (G) Gpx4 protein quantification (H) Ptgs2 protein quantification (I) Acsl4 staining co-localized with ATP6V1B1. Detailed Implementation

[0025] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Unless otherwise specified, all reagents or instruments used in the embodiments of this invention are commercially available conventional reagent products.

[0027] The materials and methods used in the following embodiments are as follows:

[0028] (I) Materials and Reagents

[0029] Hepcidin flox / + Heterozygous mice were constructed by Nanjing Jicui Pharmaceutical Co., Ltd. (https: / / cn.gempharmatech.com / shop / productDetails / 64668). The CAG-LSL-mHamp-flag-WPRE-PolyA gene fragment was specifically inserted into the H11 site of the mouse using CRISPR / Cas9 technology. Hepcidin... flox / + Heterozygous mice and Hepcidin flox / + Hepcidin was obtained after breeding heterozygous mice. flox / flox Homozygous mice. Hepcidin was then administered. flox / flox After crossing homozygous mice with Cdh16-Cre tool mice, heterozygous mice with collecting duct-specific hepcidin knock-in were obtained. KspKI(Overexpression of hepcidin). C57bL / 6J wild-type mice were purchased from Beijing Vital River Biotechnology Co., Ltd. Mouse cortical collecting duct cell line (M-1) was purchased from Guangzhou Saiku Biotechnology Co., Ltd. Hepcidin small interfering RNA and overexpression plasmid, and Ltf small interfering RNA were purchased from Shanghai Hanheng Biotechnology Co., Ltd. PI kit was purchased from Nanjing Beyotime Biotechnology Co., Ltd. C11bodipy and FerroOrange probes were purchased from Dojindo. Scr and BUN detection kits were purchased from Nanjing Jiancheng Biotechnology Co., Ltd. Hepcidin peptide.

[0030] (H-Asp-Thr-Asn-Phe-Pro-Ile-Cys-Ile-Phe-Cys-Cys-Lys-Cys-Cys-Asn-Asn-Ser-Gln-Cys-Gly-Ile-Cys-Cys-Lys-Thr-OH)SEQ ID NO: 1, purchased from Peptide International. Ptgs2 and Gpx4 antibodies were purchased from Abcam, Ltf from Boster, 4HNE antibody from Bioss, and Acsl4, ATP6V1B1, and β-actin antibodies from Proteintech.

[0031] Cell culture and processing

[0032] M-1 cells were cultured in DMEM / F12 medium containing 10% fetal bovine serum, 1% penicillin, streptomycin, and amphotericin B at 37°C, 5% carbon dioxide, and 95% aerobic air. To investigate the effect of hepcidin on hypoxia / reoxygenation-induced damage to collecting duct cells, cells were transfected with siNC or siHepcidin at 70-80% confluency, then transferred to serum-free and triple-antibiotic medium. After 24 hours, the H / R group was replaced with serum-free and glucose-free medium and placed in a tri-gas incubator under hypoxic conditions (37°C, 5% carbon dioxide, 1% oxygen, and 94% nitrogen). After 24 hours, the medium was replaced with complete medium and cultured normally for 2 hours. Finally, cells were collected for Western blotting and PI, C11bodipy, and FerroOrange staining. For cells transfected with Hepcidin overexpression plasmids, transfection was performed at 50-60% confluency, followed by H / R treatment under the same conditions after 48 hours.

[0033] (III) Animal Grouping

[0034] Select 10 Hepcidin KspKI Hepcidin in male mice and 10 littermates flox / flox Male mice, 6-8 weeks old, weighing 22-25g, were divided into the following four groups, containing Hepcidin.flox / flox Four animals were in the sham surgery group; Hepcidin KspKI 4 birds in group sham; Hepcidin flox / flox Six animals were in the bilateral renal ischemia-reperfusion (bIRI) group; Hepcidin KspKI Six mice were selected from 15 male C57BL / 6J mice aged 6-8 weeks and weighing 22-25g. They were randomly divided into three groups of five mice each: the sham group, the bIRI 32min + Vehicle (PBS) group, and the bIRI 32min + rHepcidin group.

[0035] Hepcidin KspKI Mouse genes that specifically overexpress hepcidin in collecting ducts; Hepcidin flox / flox In control mice, there was no overexpression of hepcidin in the collecting ducts.

[0036] (iv) Animal handling

[0037] An acute kidney injury model in mice was induced by renal ischemia-reperfusion. Mice were anesthetized with isoflurane gas, and the renal pedicles were clamped bilaterally through a lateral spinal incision for 32 minutes. The arterial clamps were then removed to restore renal blood supply, and the mice's body temperature was maintained at 36-37°C throughout the process using a thermostat. One day after reperfusion, blood samples were collected from the retro-orbital venous plexus for renal function tests, and the kidneys were harvested for subsequent PAS staining and immunofluorescence staining.

[0038] (V) Kidney function testing

[0039] After centrifuging the blood samples, the supernatant was collected, and the serum creatinine and blood urea nitrogen of each group of mice were measured according to the kit instructions.

[0040] (vi) Renal tubular injury score

[0041] Ten regions were randomly selected from each kidney PAS slice. Brush border loss, tubular dilation, cast formation, and tubular necrosis were used as evaluation criteria. The percentage of tubular damage was calculated using a semi-quantitative scoring method: 0, no damage; 1, <25%; 2, 25-50%; 3, 50-75%; 4, <75%.

[0042] Immunofluorescence staining

[0043] After fixation, embedding, and sectioning, the kidney tissue was stained with Acsl4 (1:200) and ATP6V1B1 (1:600) antibodies, and finally stained with DAPI and mounted according to the instructions.

[0044] Western blot of proteins

[0045] After extraction of total protein from the kidneys, the procedure was performed according to the instructions. The antibody ratios were Ptgs2 (1:1000), Ltf (1:1000), and β-actin (1:20000). The bands were analyzed using ImageJ for grayscale analysis.

[0046] PI, C11bodipy and FerroOrange staining

[0047] Follow the instructions in the kit.

[0048] Statistical analysis

[0049] Data are expressed as mean ± SD. For non-normally distributed data, the Mann-Whitney test was used. For normally distributed data with two or more groups, one-way ANOVA was used. For data with more than one variable, two-way ANOVA was used. A p-value < 0.05 was considered statistically significant.

[0050] Example 1

[0051] Hepcidin in collecting duct cells inhibits ferroptosis in collecting duct cells under hypoxia-reoxygenation injury by directly binding to LTF and upregulating its protein expression.

[0052] To evaluate the effect of hepcidin on collecting duct cells under hypoxia / reoxygenation (H / R) injury, we examined ferroptosis-related markers. After H / R, the expression of the ferroptosis marker Ptgs2 protein increased in the collecting ducts (Fig. 1A and B), cell death increased (Fig. 1C and D), and the production of ferroptosis indicators such as lipid peroxides (Fig. 1E) and ferrous ions (Fig. 1F and G) increased. Knockdown of hepcidin exacerbated the increase in these markers. Therefore, knockdown of hepcidin intensified ferroptosis in collecting duct cells under H / R.

[0053] To investigate the specific mechanism by which Hepcidin inhibits ferroptosis in collecting tube cells after hematopoiesis (H / R), we found that Hepcidin can directly interact with Ltf (Fig. 1H). It is known that Ltf can alleviate ferroptosis by chelating ferric ions. Furthermore, Ltf protein expression in collecting tubes decreases after H / R, while overexpression of Hepcidin can upregulate Ltf protein expression after H / R (Fig. 1I). Gpx4 protein expression, a negative marker of ferroptosis, decreases in collecting tubes after H / R, and overexpression of Hepcidin can rescue Gpx4 protein expression, but this rescue effect is inhibited by knockdown of Ltf (Fig. 1J). Therefore, Hepcidin in collecting tubes exerts an anti-ferroptosis effect in collecting tube cells after H / R by covalently binding to Ltf.

[0054] Example 2

[0055] In an ischemia-reperfusion-induced acute kidney injury model, collecting duct hepcidin significantly improved renal tubular injury and renal function by inhibiting intercalated cell ferroptosis.

[0056] To investigate the role of collecting duct hepcidin in ischemia-reperfusion-induced acute kidney injury, we first evaluated renal pathology. One day after ischemia-reperfusion, Hepcidin KspKI mice showed less renal tubular injury compared to Hepcidin flux / flox mice (Figures 2A and 2B). Next, we assessed relevant biochemical parameters in mouse serum; one day after ischemia-reperfusion, Hepcidin... flox / flox Rat serum creatinine and blood urea nitrogen were significantly elevated, while hepcidin... KspKI Compared with blood urea nitrogen, mouse serum creatinine and blood urea nitrogen were treated with Hepcidin. flox / flox The rats had lower levels of hepcidin (Fig. 2C and D). One day after ischemia-reperfusion, hepcidin levels were lower. flox / flox In rat kidneys, the expression of 4HNE, Acsl4, and Ptgs2 proteins was increased, while the expression of Gpx4 protein was decreased, but Hepcidin... KspKI Mice exhibited lower levels of 4HNE, Acsl4, and Ptgs2 proteins and higher levels of Gpx4 protein (Figure 2E-I). Finally, we found that Hepcidin levels decreased one day after ischemia-reperfusion. flox / flox Iron death in intercalated cells of the collecting duct of mice was exacerbated, while Hepcidin... KspKI Mice showed milder symptoms (Figure 2J). These results suggest that collecting duct hepcidin can protect against ischemia-reperfusion-induced acute kidney injury by inhibiting intercalated cell ferroptosis.

[0057] Example 3

[0058] In an ischemia-reperfusion-induced acute kidney injury model, exogenous hepcidin significantly improved renal tubular injury and renal function by inhibiting intercalated cell ferroptosis.

[0059] We pretreated mice with exogenous hepcidin (2 mg / kg) via intraperitoneal injection 24 h before ischemia-reperfusion surgery, followed by 32 min of ischemia and reperfusion for 1 day. We assessed the pathological changes in the kidneys and relevant biochemical parameters in the serum of mice. After ischemia-reperfusion, mice treated with exogenous hepcidin showed a reduction in renal casts (Fig. 3A and B) and improved renal function compared to mice injected with PBS (Fig. 3C and D). We then compared the degree of renal ferroptosis. Mice injected with PBS showed increased expression of Acsl4 and Ptgs2 proteins and decreased expression of Gpx4 protein after ischemia-reperfusion, while mice treated with exogenous hepcidin showed decreased expression of Acsl4 and Ptgs2 proteins and increased expression of Gpx4 protein, suggesting that the degree of renal ferroptosis was reduced after treatment (Fig. 3E-H), and the degree of intercalated cell ferroptosis was also reduced after treatment (Fig. 3I). These results indicate that exogenous hepcidin can protect against ischemia-reperfusion-induced acute kidney injury by inhibiting intercalated cell ferroptosis.

[0060] In summary, this invention provides the use of hepcidin in the preparation of a drug for alleviating ischemia-reperfusion-induced acute kidney injury. The drug is administered via intraperitoneal injection (using PBS as a solvent, at a concentration of 2 mg / kg, with a maximum of 50 μg per mouse). It improves acute kidney injury by directly binding to lactoferrin and upregulating its protein expression, thereby inhibiting intercalated cell ferroptosis.

[0061] The above embodiments illustrate and describe the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. Application of hepcidin in the preparation of drugs for blocking ferroptosis in collecting ducts of acute kidney injury and protecting renal function.

2. The application according to claim 1, characterized in that, The drug is used to inhibit ferroptosis in renal collecting duct cells under hypoxia-reoxygenation injury.

3. The application according to claim 2, characterized in that, The inhibition is achieved by hepcidin in the renal collecting duct directly binding to ltf and upregulating its protein.

4. The application according to claim 1, characterized in that, The drug is used to inhibit ferroptosis of intercalated cells, thereby improving renal tubular damage and renal function.

5. The application according to claim 4, characterized in that, The inhibition is achieved by suppressing intercalated cell ferroptosis through hepcidin in the renal collecting duct.

6. The application according to claim 1, characterized in that, The acute kidney injury occurred in mice.

7. The application according to claim 6, characterized in that, The mice were collecting duct-specific hepcidin knock-in heterozygous mice. KspKI .

8. The application according to claim 6, characterized in that, The heterozygous mouse Hepcidin KspKI Hepcidin purchased from Nanjing Jicui Yaokang Biotechnology Co., Ltd. flox / + Hepcidin obtained by hybridization of heterozygous mice flox / flox The mice were obtained by crossbreeding homozygous mice with Cdh16-Cre mice.

9. The application according to claim 6, characterized in that, The drug is administered via intraperitoneal injection, using PBS as a solvent, at a concentration of 2 mg / kg, with a maximum dose of 50 μg per mouse.

10. A drug that blocks ferroptosis in the collecting ducts of acute kidney injury and protects renal function, characterized in that, The drug is an intraperitoneal injection prepared by dissolving therapeutic concentrations of hepcidin in PBS.

Images