Application of hepcidin in preparation of medicine for blocking acute kidney injury collecting tube ferroptosis and protecting kidney function

Abstract

The invention discloses a novel pharmaceutical application of hepcidin, particularly relates to an application of hepcidin in preparation of drugs for blocking acute kidney injury collecting tube ferroptosis and protecting renal functions, and belongs to the technical field of medicines. It is proved for the first time that both the kidney endogenous hepcidin and the exogenous hepcidin can inhibit the ferreath of the collecting tube cells, so that the tubule injury caused when the acute kidney injury is induced by ischemia-reperfusion is relieved, and the kidney function is improved. The invention opens up a new medical approach for the treatment of acute kidney injury; furthermore, the invention discloses a medicine for relieving ischemia reperfusion induced acute kidney injury collecting tube ferroptosis and protecting renal function, and the medicine is an intraperitoneal injection and is prepared by dissolving hepcidin with therapeutic concentration by using PBS (Phosphate Buffer Solution).

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to a new use of hepcidin as a medicine, and specifically relates to the use of hepcidin in the preparation of a medicine for alleviating ferroptosis of collecting ducts in acute renal injury of mice caused by ischemia-reperfusion and protecting renal function. Background Art

[0002] Acute kidney injury is a serious public health burden with high morbidity and mortality worldwide, with an incidence of up to 50% in intensive care units. Cardiac surgery-related acute kidney injury is the second most common cause of acute kidney injury in intensive care units, mainly due to intraoperative ischemia-reperfusion injury. Severe cardiac surgery-related acute kidney injury is independently associated with a 3- to 8-fold increase in perioperative mortality. However, to date, early diagnosis is challenging, and specific treatment strategies are lacking in addition to mitigating further damage and supportive care, so it is necessary to have a deeper understanding of the potential mechanisms of ischemia-reperfusion-induced acute kidney injury.

[0003] Recent clinical studies have shown that high urinary hepcidin concentrations 24 hours after cardiac surgery are generally associated with a reduced incidence of acute kidney injury. However, it is worth noting that serum hepcidin did not show any predictive efficacy, suggesting that endogenous hepcidin in the kidney may be involved in the development of acute kidney injury in these patients. Therefore, the exact role and mechanism of endogenous hepcidin in the kidney regulating ischemia-reperfusion injury-induced acute kidney injury deserves further study. Summary of the invention

[0004] To solve the above problems, the present invention discloses the use of hepcidin in the preparation of a drug for blocking acute kidney injury collecting duct ferroptosis and protecting renal function. The present invention unexpectedly found that the level of hepcidin protein in the renal tissue of mice with acute kidney injury induced by ischemia-reperfusion was significantly increased and mainly localized in the collecting duct. Further transcriptome sequencing revealed that knocking down hepcidin can significantly aggravate hypoxia-induced collecting duct ferroptosis, suggesting that the anti-ferroptosis function of hepcidin may improve ischemia-reperfusion-induced acute kidney injury, and further verified the above speculation through experiments.

[0005] The present invention includes the following technical solutions:

[0006] The present invention discloses for the first time the use of hepcidin in preparing a medicine for blocking collecting duct ferroptosis in acute renal injury and protecting renal function.

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

[0008] Furthermore, in the above application, the inhibition is achieved by renal collecting duct hepcidin directly binding to LTF and upregulating its protein, revealing the role of hepcidin receptor ferroportin that is independent of it.

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

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

[0011] Furthermore, in the above application, the drug is in the form of an intraperitoneal injection, using PBS as a solvent, with a drug concentration of 2 mg / kg and a maximum of 50 ug per mouse.

[0012] The invention also discloses a medicine for blocking ferroptosis of collecting ducts in acute renal injury and protecting renal function. The medicine is an intraperitoneal injection prepared by dissolving hepcidin at a therapeutic concentration in PBS.

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

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

[0015] Specifically, the present invention proposes that hepcidin expressed in the collecting duct and exogenous hepcidin inhibit intercalated cell ferroptosis by directly binding to lactoferrin and stabilizing its expression, thereby alleviating ischemia-reperfusion-induced acute renal injury.

[0016] The present invention studies collecting duct hepcidin and exogenous hepcidin in an in vitro cell model of hypoxia and reoxygenation and an ischemia-reperfusion-induced acute kidney injury mouse model, and explores the use of hepcidin in drugs for alleviating ischemia-reperfusion-induced acute kidney injury. The results showed that hepcidin can significantly improve tubular damage and renal function, intercalated cell ferroptosis, and reduce tubulointerstitial inflammation in ischemia-reperfusion mouse models and in vitro cells during acute kidney injury. Our discovery is very likely to provide effective clinical drugs for the prevention and treatment of acute kidney injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 : It shows that collecting duct hepcidin inhibits the degree of ferroptosis of collecting duct cells under hypoxia-reoxygenation injury by directly binding to LTF and upregulating its protein expression;

[0018] (A) Ptgs2 protein in the normoxic siNC group, normoxic siHepcidin group, hypoxia / reoxygenation siNC group, and hypoxia / reoxygenation siHepcidin group (B) Ptgs2 protein quantification (C) PI staining (D) PI staining quantification (E) C11bodipy staining (F) FerroOrange staining (G) FerroOrange staining quantification;

[0019] (H) IP experiments verified the interaction between Hepcidin protein and Ltf protein;

[0020] (I) Ltf protein and quantification in 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 : It showed that collecting duct hepcidin significantly improved renal tubular damage 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 bIRI32min group and Hepcidin KspKI bIRI32min group in four groups (A) PAS staining of kidney (B) Kidney injury score (C) Scr (D) BUN (E) 4HNE, Gpx4, Acsl4, Ptgs2 proteins (F) 4HNE protein quantification (G) Gpx4 protein quantification (H) Acsl4 protein quantification (I) Ptgs2 protein quantification (J) Acsl4 staining co-localized with ATP6V1B1;

[0024] Figure 3: It showed that exogenous hepcidin significantly improved renal tubular injury and renal function under ischemia-reperfusion injury by inhibiting intercalated cell ferroptosis; in the sham+Vehicle group, bIRI32min+Vehicle group and bIRI32min+rHepcidin group, (A) renal PAS staining (B) renal injury score (C) Scr (D) BUN (E) Acsl4, Gpx4, Ptgs2 proteins (F) Acsl4 protein quantification (G) Gpx4 protein quantification (H) Ptgs2 protein quantification (I) Acsl4 staining co-localized with ATP6V1B1. DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] The reagents or instruments used in the examples of the present invention without indicating the manufacturer are all conventional reagent products that can be obtained through commercial purchase.

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

[0028] (I) Materials and reagents

[0029] Hepcidin flox / + Heterozygous mice were commissioned to Nanjing Gemharmatech Biotechnology Co., Ltd. (https: / / cn.gempharmatech.com / shop / productDetails / 64668). The CAG-LSL-mHamp-flag-WPRE-PolyA gene fragment was inserted into the mouse H11 locus using CRISPR / Cas9 technology. flox / + Heterozygous mice and Hepcidin flox / + Hepcidin is obtained after breeding heterozygous mice flox / flox Homozygous mice. flox / flox Homozygous mice were crossed with Cdh16-Cre mice to obtain collecting duct-specific hepcidin knock-in heterozygous mice (Hepcidin KspKI(overexpressing hepcidin)). C57bL / 6J background wild-type mice were purchased from Beijing Weitong Lihua 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, Ltf small interfering RNA were purchased from Shanghai Hanheng Biotechnology Co., Ltd. PI kit was purchased from Nanjing Biyuntian 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 was purchased from Boster, 4HNE antibody was purchased from Bioss, Acsl4, ATP6V1B1, β-actin antibodies were purchased from Proteintech.

[0031] Cell culture and treatment

[0032] M-1 cells were cultured in DMEM / F12 medium containing 10% fetal bovine serum, 1% penicillin, streptomycin and amphotericin at 37°C, 5% carbon dioxide and 95% air. To study the role of Hepcidin in collecting duct cell damage caused by hypoxia / reoxygenation, when the cells grew to 70-80% density, the medium without serum and triple antibody was changed to transfect siNC or siHepcidin. After 24 hours, the H / R group was changed to medium without serum and glucose and placed in a three-gas incubator for hypoxia. The culture conditions were 37°C, 5% carbon dioxide, 1% oxygen and 94% nitrogen. After 24 hours, the medium was changed to complete medium and cultured normally for 2 hours. Finally, the cells were collected for Western Blot detection and PI, C11bodipy and FerroOrange staining. For transfection of Hepcidin overexpression plasmid, the cells were transfected when they grew to 50-60% density, and H / R treatment was performed 48 hours later without changing the conditions.

[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 22-25 g, were divided into the following four groups: Hepcidinflox / flox 4 rats in the sham group; KspKI 4 in the sham group; flox / flox Bilateral renal ischemia-reperfusion (bIRI) group (n=6); Hepcidin KspKI bIRI group: 6. Fifteen 6-8 week old male mice with C57BL / 6J background and weighing 22-25 g were randomly divided into 3 groups, 5 mice in each group, namely sham group, bIRI32min+Vehicle(PBS) group, and bIRI32min+rHepcidin group.

[0035] Hepcidin KspKI :Genetic mice with collecting duct-specific overexpression of hepcidin, Hepcidin flox / flox : Control mice, collecting ducts do not overexpress hepcidin.

[0036] (IV) Animal handling

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

[0038] (V) Renal function test

[0039] After centrifugation of the blood samples, the supernatant was taken and the blood creatinine and urea nitrogen of each group of mice were detected according to the instructions of the kit.

[0040] 6. Renal tubular injury score

[0041] Ten areas were randomly selected from each renal PAS section, and the percentage of tubular damage was calculated using a semiquantitative scoring method using brush border loss, tubular dilatation, tubular cast formation, and tubular necrosis as evaluation criteria: 0, no damage; 1, <25%; 2, 25-50%; 3, 50-75%; 4, <75%.

[0042] Immunofluorescence staining

[0043] The kidney tissue was fixed, embedded, and sectioned, then stained with Acsl4 (1:200) and ATP6V1B1 (1:600) antibodies, and then the nuclei were stained with DAPI and sealed according to the instructions.

[0044] Western blotting

[0045] After total kidney protein extraction, follow the instructions. The antibody ratio is Ptgs2 (1:1000), Ltf (1:1000), β-actin (1:20000). The bands were analyzed by grayscale using Image J.

[0046] PI, C11bodipy and FerroOrange staining

[0047] Follow the instructions of the kit respectively.

[0048] Statistical analysis

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

[0050] Example 1

[0051] Hepcidin in the collecting duct inhibits the degree of ferroptosis of 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 detected ferroptosis-related markers. The expression of Ptgs2 protein, a ferroptosis marker, was increased in the collecting duct after H / R ( Figure 1 A and B), increased cell death ( Figure 1 C and D), lipid peroxides, an indicator of ferroptosis ( Figure 1 E) and ferrous ions ( Figure 1 F and G) were increased. Knockdown of Hepcidin could aggravate the increase of these indicators. Therefore, knockdown of Hepcidin aggravated the ferroptosis of collecting duct cells under H / R.

[0053] To explore the specific mechanism by which Hepcidin inhibits ferroptosis of collecting duct cells after H / R, we found that Hepcidin can directly interact with Ltf ( Figure 1 H). It is known that ltf can reduce ferroptosis by chelating trivalent iron ions. In addition, the expression of Ltf protein in the collecting duct is reduced after H / R, while overexpression of Hepcidin can upregulate the expression of Ltf protein after H / R ( Figure 1 I). After H / R, the expression of Gpx4 protein, a negative indicator of ferroptosis, decreased in the collecting ducts. Overexpression of Hepcidin could rescue the expression of Gpx4 protein, but this rescue effect was inhibited by knocking down Ltf at the same time ( Figure 1J). Therefore, collecting duct hepcidin exerts an anti-ferroptosis effect in collecting duct cells after H / R by covalently binding to Ltf.

[0054] Example 2

[0055] In the ischemia-reperfusion-induced acute renal injury model, collecting duct hepcidin significantly improved 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. After 1 day of ischemia-reperfusion, renal tubular injury was milder in HepcidinKspKI mice than in Hepcidinflox / flox mice ( Figure 2 A and B). Then, the relevant biochemical indexes in the serum of mice were evaluated. After 1 day of ischemia-reperfusion, Hepcidin flox / flox Serum creatinine and urea nitrogen increased significantly, while Hepcidin KspKI Compared with Hepcidin flox / flox Mouse lower ( Figure 2 C and D). Hepcidin flox / flox The expression of 4HNE, Acsl4 and Ptgs2 proteins in the kidney of mice was increased, while the expression of Gpx4 protein was decreased. However, Hepcidin KspKI Mice have lower 4HNE, Acsl4, and Ptgs2 protein levels and higher Gpx4 protein levels ( Figure 2 Finally, we found that Hepcidin flox / flox Ferroptosis of intercalated cells in the collecting duct of mice is aggravated, while Hepcidin KspKI Mice are lighter ( Figure 2 J). 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 acute renal injury model induced by ischemia-reperfusion, exogenous hepcidin significantly improved tubular injury and renal function by inhibiting intercalated cell ferroptosis

[0059] We injected exogenous hepcidin (2 mg / kg) intraperitoneally 24 h before ischemia-reperfusion surgery for pretreatment, followed by ischemia for 32 min and reperfusion for 1 day. We evaluated the renal pathology and related biochemical parameters in the serum of mice. After ischemia-reperfusion, the number of casts in the kidneys of mice treated with exogenous hepcidin was reduced compared with that of mice injected with PBS ( Figure 3 A and B), kidney function improved ( Figure 3 C and D). We then compared the extent of renal ferroptosis. After ischemia-reperfusion, the expression of Acsl4 and Ptgs2 proteins in mice injected with PBS increased, while the expression of Gpx4 protein decreased. However, after exogenous hepcidin treatment, the expression of Acls4 and Ptgs2 proteins decreased, while the expression of Gpx4 protein increased, indicating that the extent of renal ferroptosis in mice was alleviated after treatment ( Figure 3 EH), and the degree of intercalated cell ferroptosis was also reduced after treatment ( Figure 3 I). These results suggest that exogenous hepcidin can protect against ischemia-reperfusion-induced acute kidney injury by inhibiting intercalated cell ferroptosis.

[0060] In summary, the present invention provides a use of hepcidin in the preparation of a drug for alleviating ischemia-reperfusion-induced acute kidney injury, which is administered by intraperitoneal injection (using PBS as a solvent, a drug concentration of 2 mg / kg, and a maximum of 50 ug per mouse), directly binds to lactoferrin and upregulates its protein expression, thereby inhibiting intercalated cell ferroptosis and improving acute kidney injury.

[0061] The above embodiments show 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 form, and any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

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

2. The use according to claim 1, characterized in that: The drug is a drug for inhibiting the degree of ferroptosis of renal collecting duct cells under hypoxia-reoxygenation injury.

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

4. The use according to claim 1, characterized in that: The drug is a drug that inhibits intercalated cell ferroptosis and improves renal tubular damage and renal function.

5. The use according to claim 4, characterized in that: The inhibition is achieved by inhibiting intercalated cell ferroptosis through renal collecting duct hepcidin.

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

7. The use according to claim 6, characterized in that: The mouse is a collecting duct-specific hepcidin knock-in heterozygous mouse Hepcidin KspKI .

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

9. The use according to claim 6, characterized in that: The drug is dosed for intraperitoneal injection, using PBS as the solvent, at a concentration of 2 mg / kg, with a maximum of 50 ug per mouse.

10. A drug for blocking collecting duct ferroptosis in acute renal injury and protecting renal function, characterized in that: The drug is an intraperitoneal injection, which is prepared by dissolving hepcidin at a therapeutic concentration in PBS.

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