Application of FXR protein as target spot in preparation of medicine for relieving and / or treating renal toxicity of tacrolimus

Abstract

The invention provides application of FXR protein as a target spot in preparation of a medicine for relieving and / or treating renal toxicity of tacrolimus, and belongs to the technical field of biological medicine. The invention proves that the activity or expression level of the FXR protein is regulated in a targeted manner, so that the Tacrolimus-induced renal tubule epithelial cell vacuolation and renal tubule injury can be effectively prevented or relieved, and the abnormal rise of early renal injury markers such as urine NAG enzyme and the like is reduced, thereby directly improving the renal toxicity problem of Tacrolimus. The invention provides a new drug action target and a treatment strategy for clinic, and by developing an FXR agonist or a gene intervention means, the long-term renal injury risk of tacrolimus can be reduced while the anti-rejection curative effect is maintained, the transplanted kidney function and the survival time of a patient are prolonged, and the clinical application prospect is wide. And an effective means is provided for solving the unsatisfied clinical demand of the renal toxicity of the tacrolimus after the transplantation operation.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and in particular to the use of FXR protein as a target in the preparation of a drug for alleviating and / or treating tacrolimus nephrotoxicity. Background Art

[0002] The calcineurin inhibitor tacrolimus (also known as FK506), as the cornerstone of the anti-rejection regimen for solid organ transplantation, is the first-choice drug after organ transplantation, and transplant patients must take it for life. Although the clinical application of tacrolimus has increased the short-term survival rate of transplantation by 80%, there is no significant difference in the long-term survival rate. The incidence of tacrolimus nephrotoxicity is as high as 44% one year after surgery, and none of all patients are spared after 10 years. This nephrotoxicity is irreversible and severely restricts the survival time of the transplanted kidney and the recipient. As is well known, tubular vacuolization is the pathological feature of tacrolimus nephrotoxicity, but its injury mechanism is still unclear. Moreover, there is currently a lack of drugs in clinical practice to reduce tacrolimus nephrotoxicity. Clinicians can only reduce the dosage to reduce its toxicity by jointly using other second-line anti-rejection drugs or the CYP3A4 inhibitor of tacrolimus metabolism enzyme, Wuzi Capsule, etc.

[0003] Renal FXR is mainly expressed in the proximal tubules, especially in the initial segment of the proximal tubules and the proximal tubules of the cortical medullary rays. As a nuclear transcription factor, after activation, FXR can initiate and activate a series of target genes, play functions in regulating urine volume, osmotic pressure and lipid metabolism, and participate in the occurrence and development of acute and chronic kidney injuries, diabetic nephropathy, renal vascular diseases, etc. The urine volume of FXR knockout mice increases, and the osmotic pressure is lower than that of the control group. Under hypertonic stress, TonEBP and CRYZ-dependent apoptosis occurs in the medullary collecting duct cells (MCD) of FXR-deficient mice. Specific deletion of Fxr flox-Kap in the renal proximal tubules inhibits the transcription of Pparγ and Cpt1 and improves cisplatin-induced acute kidney injury. The novel natural FXR agonist Alisol B 23-acetate (ABA) can reduce acute kidney injury induced by ischemia-reperfusion in mice. However, regarding the molecular mechanism of tacrolimus-induced kidney structure damage and tubular vacuolization, there is still insufficient understanding, and there is no research report on targeted drug targets. Therefore, clarifying the molecular mechanism of tacrolimus nephrotoxicity and exploring potential action targets have important theoretical value and practical significance for reducing drug adverse reactions and delaying graft failure. Summary of the Invention

[0004] The purpose of the present invention is to provide the use of FXR protein as a target in the preparation of a drug for alleviating and / or treating tacrolimus nephrotoxicity, providing an effective means for anti-rejection and reducing side effects after transplantation.

[0005] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0006] The present invention provides the use of FXR protein as a target in the preparation of a medicament for treating kidney injury.

[0007] The present invention provides the use of FXR protein as a target in the preparation of a medicament for alleviating and / or treating tacrolimus nephrotoxicity.

[0008] The present invention provides the use of FXR protein as a target in the preparation of a drug screening model for alleviating and / or treating tacrolimus nephrotoxicity.

[0009] The present invention provides a drug screening model, which is based on the expression or activity level of FXR protein for screening candidate compounds for preventing and treating tacrolimus nephrotoxicity.

[0010] The present invention provides a kit for detecting tacrolimus nephrotoxicity, which kit contains reagents for detecting the expression level or activity of FXR protein in kidney tissues.

[0011] The present invention provides the use of an FXR agonist in the preparation of a medicament for alleviating and / or treating tacrolimus nephrotoxicity.

[0012] Preferably, the FXR agonist is GW4064.

[0013] The present invention provides the use of a pharmaceutical composition in the preparation of a medicament for alleviating and / or treating tacrolimus nephrotoxicity, which pharmaceutical composition contains an FXR agonist and a pharmaceutically acceptable carrier.

[0014] The present invention provides the use of a gene-edited animal model in simulating tacrolimus nephrotoxicity, which gene-edited animal model is an animal with specific knockout or overexpression of FXR gene in renal tubular epithelial cells.

[0015] The present invention provides an anti-rejection pharmaceutical composition for organ transplantation, which anti-rejection pharmaceutical composition for organ transplantation includes tacrolimus and an FXR agonist.

[0016] Advantages of the present invention:

[0017] The present invention proves that by targeting and regulating the activity or expression level of FXR protein, it is possible to effectively prevent or reduce vacuolization of renal tubular epithelial cells and renal tubular injury induced by tacrolimus, and reduce the abnormal increase of early kidney injury markers such as urinary NAG enzyme, thereby directly improving the problem of tacrolimus nephrotoxicity. The present invention provides a new drug action target and treatment strategy for clinical practice. By developing FXR agonists or gene intervention means, it is possible to reduce the long-term renal injury risk of tacrolimus while maintaining the anti-rejection efficacy, extend the renal function of transplantation and the survival time of patients, and provide an effective means for solving the unmet clinical need of tacrolimus nephrotoxicity after transplantation. Description of the Drawings

[0018] Figure 1 For the normal donor control group, the HE staining results of renal tubular epithelial cells in the FK506 treatment group after kidney transplantation;

[0019] Figure 2 For the HE staining results of renal tubular epithelial cells in mice of the normal saline control group and the FK506 experimental group;

[0020] Figure 3 For the expression of FXR in the kidneys of mice in the normal saline control group and the FK506 experimental group detected by Western blot and qPCR;

[0021] Figure 4 For FXR fl / fl Control group mice and FXR △RTEC HE staining results of renal tubular epithelial cells in experimental group mice;

[0022] Figure 5 For FXR fl / fl Control group mice and FXR △RTEC HE staining results of renal tubular epithelial cells in experimental group mice after FK506-induced tacrolimus nephrotoxicity;

[0023] Figure 6 For the HE staining results of renal tubular epithelial cells in mice of the AAV9-Control control group and the AAV9-FXR experimental group after FK506-induced tacrolimus nephrotoxicity;

[0024] Figure 7 For the HE staining results of renal tubular epithelial cells in mice of the normal saline control group and the GW4064 experimental group after FK506-induced tacrolimus nephrotoxicity;

[0025] Figure 8 For the levels of urinary NAG enzyme, an early renal tubular injury marker, in mice of the normal saline control group and the FK506 experimental group;

[0026] Figure 9 For FXR fl / fl Control group mice and FXR △RTEC Levels of urinary NAG enzyme, an early renal tubular injury marker, in experimental group mice;

[0027] Figure 10 For FXR fl / fl Control group mice and FXR △RTEC Levels of urinary NAG enzyme, an early renal tubular injury marker, in experimental group mice after FK506-induced tacrolimus nephrotoxicity;

[0028] Figure 11To determine the levels of urinary NAG enzyme, an early renal tubular injury marker, in mice of the AAV9-Control group and the AAV9-FXR experimental group after induction of tacrolimus nephrotoxicity with FK506.

[0029] Figure 12 To determine the levels of urinary NAG enzyme, an early renal tubular injury marker, in mice of the normal saline control group and the GW4064 experimental group after induction of tacrolimus nephrotoxicity with FK506. Detailed implementation mode

[0030] The technical solutions provided by the present invention will be described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0031] Embodiment

[0032] By collecting clinical specimens from normal organ donors and patients treated with FK506 after kidney transplantation, and observing the kidney morphology by HE staining, it was found that vacuolization of renal tubular epithelial cells occurred in the FK506 treatment group after kidney transplantation.

[0033] By constructing a mouse model of tacrolimus nephrotoxicity, observing the kidney morphology of mice by HE staining and detecting the level of urinary NAG enzyme, an early renal tubular injury marker, with a kit, it was found that vacuolization of renal tubular epithelial cells and an increase in urinary NAG enzyme occurred in mice with tacrolimus nephrotoxicity. Western blot and qPCR were used to detect the expression of FXR in the kidneys, and it was found that the expression of FXR in the kidneys of mice with tacrolimus nephrotoxicity decreased; indicating that tacrolimus can induce vacuolization of renal tubular epithelial cells and early renal tubular injury, and reduce the expression of FXR in the kidneys.

[0034] By constructing a mouse model with specific knockout of the FXR gene in renal tubular epithelial cells, observing the kidney morphology of mice by HE staining and detecting the level of urinary NAG enzyme, an early renal tubular injury marker, with a kit, it was found that compared with FXR fl / fl mice, vacuolization of renal tubular epithelial cells and an increase in urinary NAG enzyme occurred in FXR △RTEC mice, indicating that specific knockout of the FXR gene in renal tubular epithelial cells can induce vacuolization of renal tubular epithelial cells and early renal injury.

[0035] To verify the role of FXR in tacrolimus nephrotoxicity, by inducing tacrolimus nephrotoxicity with FK506 in FXR fl / fl mice and FXR △RTEC mice, observing the kidney morphology of mice by HE staining and detecting the level of urinary NAG enzyme, an early renal tubular injury marker, with a kit, it was found that under the same treatment with FK506, compared with FXR fl / fl mice, FXR △RTECVacuolization and tubular damage were more pronounced in renal tubular epithelial cells of mice, indicating that tubular epithelial cell-specific FXR gene knockout could aggravate tacrolimus-induced nephrotoxicity;

[0036] To verify the role of FXR in tacrolimus nephrotoxicity, we induced tacrolimus nephrotoxicity in AAV9-Control mice and AAV9-FXR mice using FK506. Renal morphology was observed using HE staining, and urinary NAGase levels, a marker of early tubular injury, were measured using a kit. We found that FK506 treatment reduced renal tubular epithelial cell vacuolation and tubular injury in AAV9-FXR mice compared with AAV9-Control mice, suggesting that overexpression of the FXR gene in renal tubular epithelial cells can mitigate tacrolimus-induced nephrotoxicity.

[0037] To validate the role of FXR in tacrolimus nephrotoxicity, we induced tacrolimus nephrotoxicity in saline-treated and GW4064-treated mice using FK506. Renal morphology was observed using HE staining, and urinary NAGase levels, a marker of early tubular injury, were measured using a kit. We found that, compared with saline-treated mice, FXR agonist GW4064-treated mice exhibited less vacuolization and tubular injury in renal tubular epithelial cells under FK506 treatment, suggesting that FXR agonist GW4064 treatment can alleviate tacrolimus-induced nephrotoxicity.

[0038] In the above experiment, the method for constructing the tacrolimus nephrotoxicity model includes the following steps:

[0039] Wild-type mice were used as experimental subjects. Male mice of appropriate age were randomly divided into two groups (A and B). Group A served as the control group and received normal saline by gavage, while Group B served as the experimental group and received 2 mg / kg / d of FK506 by gavage at the same time every day for four weeks. Samples were then collected from the experimental mice for analysis.

[0040] Get FXR fl / fl Mice (mice with loxP sites in the FXR gene) were crossed with Ggt1-Cre mice (renal tubular tissue-specific Cre tool mice) and FXR was screened in the same littermates. fl / fl Mice served as a control group and their littermates were screened for FXR fl / fl .Ggt1-Cre mice, namely FXR △RTEC Mice (renal tubular epithelial cell-specific FXR gene knockout mice) were used as experimental subjects and were gavaged with 2 mg / kg / d of FK506 at the same time every day for four weeks. Samples were then analyzed.

[0041] Wild-type mice were used as experimental subjects, and male mice of appropriate age were randomly divided into two groups (A and B). Group A was injected with AAV9-Control adenovirus via tail vein as a control group of empty virus mice, while Group B was injected with AAV9-FXR adenovirus via tail vein as an experimental group of mice with overexpression of the FXR gene in renal tubular epithelial cells. Two weeks after injection, the mice were gavaged with 2 mg / kg / d of FK506 at the same time every day for four weeks, and then samples were analyzed.

[0042] Wild-type mice were used as experimental subjects. Male mice of appropriate age were randomly divided into two groups (A and B). Group A served as the control group and received normal saline and 2 mg / kg / d of FK506 by oral gavage. Group B served as the experimental group and received 30 mg / kg / d of the FXR agonist GW4064 and 2 mg / kg / d of FK506 by oral gavage. After four weeks of oral gavage at the same time every day, samples were collected and analyzed.

[0043] The results are shown in the attached figure. Figure 1 HE staining results of renal tubular epithelial cells in the normal donor control group and the FK506 treatment group after kidney transplantation;

[0044] Figure 2 The HE staining results of renal tubular epithelial cells in the normal saline control group and FK506 experimental group mice;

[0045] Figure 3 Western blot and qPCR were used to detect the expression of FXR in the kidneys of mice in the normal saline control group and the FK506 experimental group;

[0046] Figure 4 For FXR fl / fl Control mice and FXR △RTEC HE staining results of renal tubular epithelial cells in experimental group mice;

[0047] Figure 5 For FXR fl / fl Control mice and FXR △RTEC HE staining results of renal tubular epithelial cells in the experimental group mice after tacrolimus nephrotoxicity was induced by FK506;

[0048] Figure 6 The results of HE staining of renal tubular epithelial cells in mice after tacrolimus nephrotoxicity was induced by FK506 in AAV9-Control mice and AAV9-FXR experimental mice.

[0049] Figure 7 The results of HE staining of renal tubular epithelial cells in mice treated with normal saline control group and GW4064 experimental group after tacrolimus nephrotoxicity was induced by FK506;

[0050] Figure 8 For the levels of urinary NAG enzyme, an early renal tubular injury marker, in mice of the normal saline control group and the FK506 experimental group;

[0051] Figure 9 For FXR fl / fl In control group mice and FXR △RTEC For the levels of urinary NAG enzyme, an early renal tubular injury marker, in mice of the experimental group;

[0052] Figure 10 For FXR fl / fl In control group mice and FXR △RTEC For the levels of urinary NAG enzyme, an early renal tubular injury marker, in mice of the experimental group after inducing tacrolimus nephrotoxicity with FK506 in control group mice and FXR experimental group mice;

[0053] Figure 11 For the levels of urinary NAG enzyme, an early renal tubular injury marker, in mice of the AAV9-Control control group mice and the AAV9-FXR experimental group mice after inducing tacrolimus nephrotoxicity with FK506;

[0054] Figure 12 For the levels of urinary NAG enzyme, an early renal tubular injury marker, in mice of the normal saline control group and the GW4064 experimental group after inducing tacrolimus nephrotoxicity with FK506.

[0055] The results show that the construction of a tacrolimus nephrotoxicity mouse model with specific knockout of the FXR gene in renal tubular epithelial cells provides a relatively mature in vivo model for the study of tacrolimus nephrotoxicity, which is conducive to further in-depth research in the field of tacrolimus nephrotoxicity.

[0056] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. Use of FXR protein as a target in the preparation of a drug for treating kidney injury.

2. Use of FXR protein as a target in the preparation of a drug for alleviating and / or treating tacrolimus nephrotoxicity.

3. Use of FXR protein as a target in the preparation of a drug screening model for alleviating and / or treating tacrolimus nephrotoxicity.

4. A drug screening model, characterized in that, The drug screening model is used to screen candidate compounds for preventing and treating tacrolimus nephrotoxicity based on the expression or activity level of FXR protein.

5. A kit for detecting tacrolimus nephrotoxicity, characterized in that, The kit contains reagents for detecting the expression level or activity of FXR protein in kidney tissue.

6. Use of an FXR agonist in the preparation of a drug for alleviating and / or treating tacrolimus nephrotoxicity.

7. The application according to claim 6, characterized in that, The FXR agonist is GW4064.

8. Use of a pharmaceutical composition in the preparation of a medicament for relieving and / or treating tacrolimus nephrotoxicity, characterized in that, The pharmaceutical composition contains an FXR agonist and a pharmaceutically acceptable carrier.

9. Use of a gene-edited animal model in simulating tacrolimus nephrotoxicity, characterized in that, The gene-edited animal model is an animal with specific knockout or overexpression of FXR gene in renal tubular epithelial cells.

10. An anti-rejection drug composition for organ transplantation, characterized in that, The anti-rejection drug composition for organ transplantation includes tacrolimus and an FXR agonist.

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