Application of disulfiram in preparation of medicine for treating UMOD gene mutant autosomal dominant renal tubular interstitial nephropathy

Abstract

The invention discloses application of disulfiram in preparation of a medicine for treating UMOD gene mutation type autosomal dominant renal tubulointerstitial nephropathy, and belongs to the field of biomedicine. The application comprises the application of the disulfiram in preparation of the medicine for treating the hereditary tubulointerstitial nephropathy. According to the invention, a new pathogenic mutation UMOD c.106Cgt is identified; according to the invention, the pathogenicity of the gene is verified through a UMODH36Y mouse model constructed by CRISPR / Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR associated 9). DSF intervention is adopted, the serum creatinine and glomerular filtration rate (tGFR) of mice can be remarkably improved, and renal interstitial inflammation and fibrosis are relieved. The discovery prompts that the DSF realizes the therapeutic effect of renal function protection-inflammation inhibition-fibrosis relief in an ADTKD-UMOD animal model through targeted regulation of a pyroptosis pathway, a novel therapeutic strategy is provided for ADTKD-UMOD, and the clinical transformation period can be remarkably shortened due to the characteristic of new use of old medicines. The discovery lays an experimental foundation for developing a precise treatment scheme for the hereditary renal tubulointerstitial disease.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the use of disulfiram in the preparation of drugs for treating UMOD gene-mutant autosomal dominant tubulointerstitial nephropathy. Background Technology

[0002] Autosomal dominant tubulointerstitial kidney disease (ADTKD) is a group of inherited kidney diseases characterized by renal tubular damage, renal interstitial fibrosis, and slow progression of renal function. ADTKD-UMOD, caused by mutations in the UMOD gene encoding uromodulin, is the most common subtype of ADTKD, with a prevalence of approximately 1.67 to 9 cases per million people. The mutated UMOD protein abnormally accumulates in the renal tubules (loops of Henry and distal tubules), triggering endoplasmic reticulum stress (ER stress) and the unfolded protein response (UPR), ultimately leading to progressive deterioration of renal function and progression to end-stage renal disease (ESRD), for which there is currently no effective treatment strategy.

[0003] Disulfiram (DSF) is a FDA-approved drug for the treatment of alcohol dependence, and its safety has been well-established in long-term clinical use. Recent studies have shown that DSF can effectively inhibit pyroptosis, a key pathological process, through multiple target mechanisms, including blocking GSDMD pore formation, inhibiting NLRP3 inflammasome oligomerization, and regulating copper metabolism. Currently, DSF has demonstrated potential therapeutic value in phase II clinical trials for various malignant tumors. Its pharmacokinetic characteristics and existing formulation processes provide a solid foundation for clinical translation. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention proposes the application of disulfiram in the preparation of drugs for treating UMOD gene-mutant autosomal dominant tubulointerstitial nephropathy.

[0005] The objective of this invention can be achieved through the following technical solutions:

[0006] A first aspect of the present invention relates to the use of disulfiram in the preparation of a medicament for treating hereditary tubulointerstitial nephropathy.

[0007] Optionally, the hereditary tubulointerstitial nephropathy is hereditary tubulointerstitial nephropathy caused by UMOD gene mutation.

[0008] Optionally, the UMOD gene mutation type is UMOD c.106C>T.

[0009] Optionally, the treatment of hereditary tubulointerstitial nephropathy includes alleviating renal function impairment.

[0010] Optionally, the treatment of hereditary tubulointerstitial nephropathy includes: inhibiting macrophage pyroptosis in kidney tissue.

[0011] Optionally, the treatment of hereditary tubulointerstitial nephropathy includes: reducing the infiltration of inflammatory cells and the degree of fibrosis in the renal interstitium.

[0012] Optionally, the dosage of the drug is configured as follows: 50-75 mg per kilogram of body weight.

[0013] Optionally, the chemical formula of the disulfiram is:

[0014]

[0015] A second aspect of the invention relates to the use of a macrophage clearance agent in the preparation of a medicament for treating hereditary tubulointerstitial nephropathy, said macrophage clearance agent comprising liposome-clophosphate sodium.

[0016] The beneficial effects of this invention are:

[0017] This invention identified a family carrying a novel pathogenic mutation UMOD c.106C>T (NM_003361.3 transcript, p.His36Tyr) and constructed a Umod PCR system using CRISPR / Cas9. H36Y / + The pathogenicity was verified in mouse models. Key findings included: Compared with wild-type mice, mutant mice (1) showed enhanced macrophage infiltration: Both patients and mouse models showed renal interstitial inflammation, especially characterized by macrophage aggregation in the medullary region; (2) Pyroptosis drove disease progression: Single-cell RNA sequencing (scRNA-seq) and functional experiments confirmed that macrophage pyroptosis is a key mechanism for renal interstitial inflammation activation and fibrosis; (3) Further verification: Clearing macrophages or inhibiting pyroptosis can significantly improve renal function, suggesting the clinical value of targeted intervention of the pyroptosis pathway.

[0018] Based on the crucial role of pyroptosis in the pathogenesis of ADTKD-UMOD, this invention employs medium-dose (50 mg / kg) and high-dose (75 mg / kg) DSF to treat Umod. H36Y / +Mice underwent an 8-week intervention. Results showed that DSF treatment significantly improved serum creatinine (Scr) and glomerular filtration rate (tGFR) in mice, and reduced renal interstitial inflammation and fibrosis. These findings suggest that DSF achieves a therapeutic effect of renal function protection, inflammation suppression, and fibrosis relief in the ADTKD-Umod animal model by targeting and regulating the pyroptosis pathway. Given that DSF is an FDA-approved drug with well-defined pharmacokinetic characteristics and clinical safety data, this study is highly recommended.

[0019] This invention not only provides a novel treatment strategy for ADTKD-UMOD, but also significantly shortens the clinical translation cycle due to its repurposing of existing drugs. This discovery lays the experimental foundation for developing precision treatments for hereditary tubulointerstitial diseases. Attached Figure Description

[0020] The invention will now be further described with reference to the accompanying drawings.

[0021] Figure 1 Umod, constructed using CRISPR / Cas9 technology in the embodiments of this application. H36Y / + Methodological procedures for mouse models;

[0022] Figure 2 Umod in the embodiments of this application H36Y / + Mouse model characterization;

[0023] Figure 3 Single-cell sequencing in the embodiments of this application indicates that macrophages exhibit the classic pyroptosis phenotype;

[0024] Figure 4 This is an in vivo characterization of macrophage pyroptosis in the embodiments of this application;

[0025] Figure 5 This application's embodiments demonstrate the crucial role of macrophages in pyroptosis through macrophage clearance.

[0026] Figure 6 The disulfiram in the embodiments of this application significantly improved renal function and kidney damage in mutant mice. Detailed Implementation

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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.

[0028] In some embodiments of the present invention, an experimental method for constructing a Umod mutant mouse and intervening in or treating its tubulointerstitial nephropathy is disclosed, specifically including the following steps:

[0029] 1. Construction of disease models

[0030] Using CRISPR / Cas9 gene editing technology, an H36Y point mutation (CAC→TAC) was introduced into exon 3 of the UMOD gene (transcript UMOD-201, ENSMUST0000033263.5) in C57BL / 6 background mice. F0 mutants were obtained by microinjection of sgRNA (5′-CACACCATCCACCGTGCAGG-3′, SEQ ID NO.3), Cas9 mRNA, and homologous recombination donor vector into fertilized eggs. After verification by PCR and sequencing, these mutants were mated with wild-type C57BL / 6J mice to establish a stably inherited Umod mutant. H36Y / + Heterozygous model (F1 generation). All experiments used male mice aged 8-70 weeks, housed in an SPF-grade environment (22±2℃, 50%±10% humidity, 12h light / dark cycle). Genotyping was performed using TaqMan SNP detection (primers: forward 5′-ctggggaggattcgctaaactctag-3′ (SEQ ID NO.1); reverse 5′-agcactcatccacatcagtgcag-3′ (SEQ ID NO.2)).

[0031] 2. UMOD H36Y Validation of point mutation mouse models

[0032] 2.1 Immunofluorescence of kidney tissue

[0033] Kidney tissue was fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned (4 μm). H&E, PAS, and Masson's trichrome staining were performed sequentially. The immunofluorescence detection procedure was as follows: after dewaxing, antigen retrieval (EDTA / citric acid), and serum blocking, sections were treated with species-specific primary antibody (incubated overnight at 4°C) and corresponding fluorescent secondary antibody (protected from light at room temperature for 1 hour). After nuclear counterstaining with DAPI, the sections were mounted with glycerol and stored at 4°C protected from light until analysis.

[0034] like Figure 2 As shown in B, the mutation Umod can be seen to be aggregated in the tubule endoplasmic reticulum by observing the tissue staining.

[0035] 2.2 Western blot

[0036] Tissue proteins were extracted by lysis with RIPA buffer, quantified using the BCA method, and then separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane. The membrane was blocked with blocking buffer for 1 hour, then reacted sequentially with primary antibody (incubated overnight at 4°C) and HRP-labeled secondary antibody (incubated at room temperature for 1 hour). After washing with TBST, the membrane was developed using ECL substrate, and target protein analysis was performed based on band position and intensity. The detection results are as follows: Figure 2 As shown in D, Umod H36Y Point mutant mice showed increased immature Umod protein in kidney tissue and decreased urinary Umod excretion.

[0037] 2.3 Biochemical Measurement

[0038] Blood samples (200 μL) were collected from mice before and after drug intervention via glass capillaries through the posterior orbital venous plexus. Hemostasis was achieved by gentle intraocular pressure control. Samples were centrifuged at 3,000 rpm for 15 min at room temperature to separate serum, which was then stored at -80°C. Serum creatinine levels were measured using a fully automated biochemical analyzer (BS360S, Mindray Bio-Medical Electronics Co., Ltd., Shenzhen, China).

[0039] 2.4tGFR determination

[0040] FITC-myoglobin clearance was dynamically measured using a transcutaneous GFR monitor (Medibeacon GmbH) to assess renal function. The specific steps included: shaving the back hair 24 hours prior to the experiment to expose the testing area; fixing the monitoring device under isoflurane anesthesia; injecting FITC-myoglobin (7 mg / 100 g body weight) via the tail vein; continuously recording the fluorescence clearance rate for 60 minutes; and collecting and processing the clearance rate data using dedicated software (Sensor_ctrl_app.exe) to calculate the GFR value. Figure 2 As shown in A, Umod H36Y Point mutant mice showed significantly reduced GFR values ​​and impaired kidney function.

[0041] 3. Single-cell RNA sequencing and data analysis

[0042] For Umod + / + Mice and 24-week-old Umod H36Y / +Mouse kidneys were used for cellular RNA sequencing and data analysis. Kidney samples (3 mice per group) were digested to prepare single-cell suspensions, and GEMs were constructed using a 10x Genomics Chromium system. Cells were resuspended in PBS and sorted, with approximately 16,000 cells loaded per channel. After cell lysis, RNA was barcoded and reverse transcribed within GEMs, and cDNA was amplified and quality-checked using an Agilent 4200. Library construction was followed by sequencing on an Illumina platform. Data analysis was performed using Seurat 3.0: low-quality cells were filtered, and principal component analysis (PCA) was performed followed by dimensionality reduction and visualization using t-SNE / UMAP. Differentially expressed genes were used to annotate cell clusters. The set of genes related to programmed cell death (apoptosis, necrosis, pyroptosis) was derived from GeneOntology, KEGG, FerrDb, and other literature.

[0043] Test results as follows Figure 3 As shown, this specifically includes cell clustering based on marker genes. Figure 3 A) in Umod H36Y / + The number (113 vs. 454) and proportion (1.13% vs. 2.81%) of renal macrophages in the group were significantly increased. Figure 3 In B), the cell death score showed Umod H36Y / + The macrophage pyroptosis score of the group was significantly increased ( Figure 3 C), accompanied by upregulation of typical pyrolysis factors such as Casp1, Gsdmd, and Nlrp3. Figure 3 (D in the middle).

[0044] 4. In vivo wet test verification

[0045] 4.1 Immunohistochemical staining

[0046] Kidney tissue was fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned (4 μm). The sections underwent dewaxing, hydration, and citrate-buffered antigen retrieval. After blocking endogenous peroxidase, non-specific binding sites were blocked with 10% goat serum. The tissue was then incubated overnight at 4°C with primary antibody (IL-18 / Caspase 1 / F4 / 80). After washing with PBS, the target antigen was bound with HRP-labeled secondary antibody, followed by DAB staining and hematoxylin counterstaining. Figure 4 As shown in CD, immunohistochemistry of consecutive paraffin-embedded kidney sections suggests Umod. H36Y / + The relationship between group F4 / 80 and the presentation and expression of Casp1 and IL-18. Figure 5 B shows that, compared with the lipo-PBS group, the F4 / 80 expression in the lipo-Clo group was significantly reduced.

[0047] 5. In vivo drug intervention studies

[0048] 5.1 Macrophage clearance

[0049] To clarify the role of macrophages in pyroptosis in ADTKD-Umod mouse tissues ( Figure 5 In section A), we used liposome-based sodium clodronate (a targeted drug that specifically eliminates macrophages, Liposoma Cat#CP-010-010) to consume Umod. H36Y / + Mouse renal macrophages. Specifically, 30-week-old mice were randomly divided into a clophosphamide liposome group (treatment group, n=5) and a PBS liposome group (control group, n=5). Both groups of mice received intraperitoneal injections (10 μL / g body weight) every 7 days for 8 weeks. Results showed that after clearance of renal macrophages, the infiltration of inflammatory cells and the degree of fibrosis in the renal interstitium were significantly reduced. Figure 5 In the B group, the levels of various pro-inflammatory factors and pyroptosis factors were also significantly reduced. Figure 5 The C in the figure suggests the key role of macrophages in pro-inflammatory and pyroptosis processes.

[0050] 5.2 DSF in vivo intervention

[0051] In this embodiment, disulfiram (DSF) (T0054, TargetMol, USA) was used to modify the Umod constructed in the above embodiment. H36Y In vivo intervention was performed on point mutant mice. Specifically, DSF was administered via intraperitoneal injection every other day for 8 consecutive weeks, prepared in a solvent containing 5% DMSO (T0341, TargetMol, USA), 40% PEG300 (T7022, TargetMol, USA), 5% Tween 80 (T13947, TargetMol, USA), and 50% sterile PBS. Experimental groups included: standard-dose DSF (50 mg / kg, n=6), high-dose DSF (75 mg / kg, n=6), and a solvent control group receiving an equal volume of physiological saline (6.7 ml / kg, n=6), with solvent concentrations matched. All experimental groups had similar baseline characteristics before intervention, including age, body weight, tGFR, and serum creatinine levels (p>0.05). Throughout the treatment period, animals maintained normal body weight and physiological status (coat condition and locomotor activity). After 8 weeks of intervention, mice underwent renal function assessment (tGFR), blood collection, and kidney tissue sampling to systematically evaluate the therapeutic effects of DSF on renal function, degree of renal interstitial inflammation activation, degree of renal interstitial fibrosis, and pyroptosis.

[0052] Kidney pathological section staining results showed that the degree of interstitial inflammatory cell infiltration and fibrosis in the disulfiram-treated group was significantly reduced (e.g., Figure 6(As shown in E in the figure). Western blot analysis further confirmed that the levels of α-SMA and pyroptosis factor, which are associated with renal fibrosis, were significantly decreased in the disulfiram-treated group, and this decrease was disulfiram dose-dependent (e.g., as shown in E in the figure). Figure 6 (As shown in F in the diagram). Therefore, this invention reveals that disulfiram improves renal function and delays the progression of end-stage renal disease (ESRD) by inhibiting pyroptosis in renal tissue, thereby suppressing renal inflammation and reducing renal fibrosis. This discovery provides strong experimental evidence for new applications of disulfiram in the treatment of kidney diseases.

[0053] The embodiments of this application successfully constructed Umod based on CRISPR / Cas9 technology. H36Y A point-mutant mouse model fully reproduced the typical disease characteristics of ADTKD-UMOD patients. Through single-cell transcriptome sequencing (scRNA-seq) combined with in vivo functional validation, it was revealed for the first time that macrophage pyroptosis is a key mechanism mediating renal interstitial inflammation activation and renal function impairment. Based on this, targeted intervention with the FDA-approved drug disulfiram demonstrated that it significantly inhibited pyroptosis-related signaling pathways in the kidney tissue of mutant mice, effectively reduced renal interstitial inflammatory cell infiltration, improved renal function indicators, and delayed disease progression. In the clinical treatment gap of ADTKD-UMOD, this study not only elucidated a novel mechanism of disease development but also provided a treatment strategy with clear molecular targets and clinical translational potential, providing experimental evidence for drug repositioning in this rare disease.

[0054] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0055] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. Use of disulfiram in the preparation of drugs for the treatment of hereditary tubulointerstitial nephropathy.

2. Use of disulfiram according to claim 1 in the preparation of a medicament for treating hereditary tubulointerstitial nephropathy, characterized in that: The hereditary tubulointerstitial nephropathy is a hereditary tubulointerstitial nephropathy caused by UMOD gene mutation.

3. Use of disulfiram according to claim 2 in the preparation of a medicament for treating hereditary tubulointerstitial nephropathy, characterized in that: The UMOD gene mutation type is UMOD c.106C>T.

4. Use of disulfiram according to claim 1 in preparing a medicament for treating hereditary tubulointerstitial nephropathy, characterized in that: The treatment of hereditary tubulointerstitial nephropathy includes: alleviating renal function damage.

5. Use of disulfiram according to claim 1 in preparing a medicament for treating hereditary tubulointerstitial nephropathy, characterized in that: The treatment of hereditary tubulointerstitial nephropathy includes: inhibiting macrophage pyroptosis in kidney tissue.

6. Use of disulfiram according to claim 1 in the preparation of a medicament for treating hereditary tubulointerstitial nephropathy, characterized in that: The treatment of hereditary tubulointerstitial nephropathy includes: alleviating the infiltration of inflammatory cells and the degree of fibrosis in the renal interstitium.

7. Use of disulfiram according to claim 1 in preparing a medicament for treating hereditary tubulointerstitial nephropathy, characterized in that: The dosage of the drug is: 50 to 75 mg per kilogram of body weight.

8. Use of disulfiram according to claim 1 in preparing a medicament for treating hereditary tubulointerstitial nephropathy, characterized in that: The chemical formula of disulfiram is:

9. Use of a macrophage scavenging agent in the preparation of a drug for treating hereditary tubulointerstitial nephropathy, characterized in that: The macrophage depletion agent includes liposome-sodium clodronate.

10. Use of the macrophage clearing reagent according to claim 9 in preparing a medicament for treating hereditary tubulointerstitial nephropathy, characterized in that: The hereditary tubulointerstitial nephropathy is hereditary tubulointerstitial nephropathy caused by UMOD gene mutation; the UMOD gene mutation type is UMOD c.106C>T.

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