Use of ring1b inhibitors in the protection of acute kidney injury

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of Ring1B inhibitor in protection of acute kidney injury. Ring1B inhibitor Ms147 is found and verified for the first time, which can effectively prevent cisplatin-induced renal tubular epithelial cell injury. Experimental results show that Ms147 pretreatment can significantly reduce expression levels of kidney injury markers KIM-1 and NGAL, reduce cisplatin-induced renal tubular epithelial cell injury and maintain normal morphology of renal tubules. Meanwhile, Ms147 can significantly inhibit Ring1B protein expression and reduce histone H2AK119 ubiquitination modification level. The application provides a brand-new drug selection and strategy for preventing cisplatin chemotherapy-induced acute kidney injury in clinic.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of Ring1B inhibitors in protecting against acute kidney injury. Background Technology

[0002] Acute kidney injury (AKI) is a clinical syndrome characterized by sudden loss of kidney function within a short period of time. Its core pathological manifestation is renal tubular damage, accompanied by microcirculatory, inflammatory and interstitial changes. Cisplatin is one of the most commonly used basic chemotherapy drugs in clinical practice. It has a broad spectrum of anticancer activity and is effective against various solid tumors such as lung cancer, ovarian cancer, gastric cancer, head and neck cancer, and bladder cancer. It is clinically mature and inexpensive. However, it has significant toxic side effects, especially damage to the renal tubules. Cisplatin mainly damages the proximal renal tubular epithelial cells, inducing oxidative stress, inflammatory response, and DNA damage, leading to degeneration, apoptosis, and necrosis of renal tubular epithelial cells. This results in impaired renal tubular reabsorption function, causing proteinuria, casts in the urine, and electrolyte disturbances such as hypomagnesemia and hypokalemia. It can also cause a decrease in glomerular filtration function, leading to an increase in serum creatinine and blood urea nitrogen. Mild cases can partially recover after drug withdrawal and hydration. However, excessive doses or repeated use can cause persistent and irreversible kidney damage. In severe cases, it can progress to acute kidney injury or even develop into chronic renal failure.

[0003] Currently, there is no specific drug to treat renal tubular damage. Treatment for AKI mainly focuses on helping to maintain renal blood supply, maintain electrolyte balance, and using dialysis to replace renal function to alleviate symptoms. However, these measures have limited protective effects and cannot completely prevent kidney damage.

[0004] Ring1B is an important member of the RING family of E3 ubiquitin ligases. Currently, a variety of inhibitors targeting Ring1B have been reported, including small molecule inhibitors that directly target the catalytic activity of Ring1B and Ring1B protein degraders. Research on these inhibitors has mainly focused on the field of oncology, and their role in renal protection has not yet been reported. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide the application of Ring1B inhibitors in protecting against acute kidney injury.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A first aspect of the invention provides the use of Ring1B inhibitors in the preparation of drugs that protect against cisplatin-induced acute kidney injury.

[0007] A second aspect of the invention provides the use of a composition of a Ring1B inhibitor and cisplatin in the preparation of a medicament to protect against cisplatin-induced acute kidney injury.

[0008] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: This invention discovers and validates for the first time the novel use of the Ring1B inhibitor Ms147 in the preparation of drugs to protect against cisplatin-induced acute kidney injury. Experimental results show that in cisplatin-induced renal tubular epithelial cell and kidney organoid injury models, Ms147 pretreatment significantly reduces the expression levels of kidney injury markers KIM-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL), effectively alleviating cisplatin-induced renal tubular epithelial cell damage and maintaining the normal morphology and structure of renal tubules. Immunofluorescence staining results show that Ms147 significantly inhibits Ring1B protein expression and effectively reduces the ubiquitination modification level of downstream histone H2AK119.

[0009] The technical solution provided by this invention can intervene in patients before cisplatin administration, with smooth clinical operation and strong feasibility of administration method. It does not require changes to the existing chemotherapy regimen and has good prospects for clinical translation. It provides a brand-new drug option and strategy for the prevention of acute kidney injury caused by cisplatin chemotherapy in clinical practice. Attached Figure Description

[0010] Figure 1 Immunofluorescence identification image of kidney organoids derived from induced pluripotent stem cells. In the image, ECAD (gray) marks the distal renal tubules, LTL (green) marks the proximal renal tubules, CD31 (red) marks the endothelial cells, and PODXL (gray) marks the podocytes. The superimposed images represent the superimposed images of each channel. Scale bar: 200 µm.

[0011] Figure 2 The structure is Ms147.

[0012] Figure 3The figures show the experimental results of the protective effect of Ms147 against cisplatin-induced acute kidney injury. A shows the expression level of KIM-1 (red) in each group detected by immunofluorescence staining, with LTL (green) labeling renal tubules and DAPI (blue) counterstaining cell nuclei. The superimposed images represent the overlay of each channel. Scale bar: 40 µm. B shows the expression level of NGAL (red) in each group detected by immunofluorescence staining, with LTL (green) labeling proximal renal tubules, ECAD (gray) labeling distal renal tubules, and DAPI (blue) counterstaining cell nuclei. The superimposed images represent the overlay of each channel. Scale bar: 40 µm. C shows the band diagram of KIM-1 and NGAL protein expression levels in each group detected by Western blotting, with GAPDH as an internal control. GAPDH is glyceraldehyde-3-phosphate dehydrogenase. D shows the bar chart of KIM-1 mRNA expression levels in cells of each group detected by RT-qPCR. This indicates a significant difference (P<0.05). This indicates a highly significant difference (P<0.01).

[0013] Figure 4 Representative images for detecting Ring1B protein expression levels in cells of each group using immunofluorescence staining are shown. In this image, A is treated with Ms147, and B is treated with PTC-209. Ring1B is displayed as red fluorescence, LTL (green) is used to label the proximal renal tubules, ECAD (gray) is used to label the distal renal tubules, and DAPI (blue) is used to counterstain the cell nuclei. The images are superimposed to represent the superimposed images of each channel. Scale bar: 40 µm.

[0014] Figure 5 Representative images for detecting the level of histone H2AK119 ubiquitination (H2AK119ub) modification in each group by immunofluorescence staining. In the images, A is Ms147 treated and B is PTC-209 treated. H2AK119ub is shown as red fluorescence, LTL (green) is used to label the proximal renal tubules, and ECAD (gray) is used to label the distal renal tubules. The superimposed images represent the superimposed images of each channel. Scale bar: 40 µm. Detailed Implementation

[0015] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0016] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0017] A typical embodiment of the present invention provides the use of Ring1B inhibitors in the preparation of drugs that protect against cisplatin-induced acute kidney injury.

[0018] In some embodiments, the protection includes preventing and / or mitigating damage to renal tubular epithelial cells.

[0019] In some embodiments, the prevention and / or mitigation of renal tubular epithelial cell damage includes: (a1) Reduce the expression levels of KIM-1 and / or NGAL proteins; (a2) Reduce histone H2AK119 ubiquitination modification level.

[0020] In some embodiments, the Ring1B inhibitor includes Ms147.

[0021] In this invention, after treating a cisplatin-induced acute kidney injury (AKI) in vitro model with Ms147, kidney injury markers such as KIM-1 were significantly downregulated, renal tubular epithelial cell apoptosis was reduced, and no obvious tubular injury phenotype was observed. This indicates that Ms147 has a protective effect on renal tubules in cisplatin-induced AKI.

[0022] In some embodiments, the concentration of Ms147 is 10~100 nM, and the treatment time is 12~96 h.

[0023] In some embodiments, the concentration of Ms147 is 20-60 nM, and the treatment time is 24-72 h.

[0024] In some embodiments, the drug is administered before, simultaneously with, and / or after cisplatin administration, preferably before cisplatin administration.

[0025] In some embodiments, the drug includes pharmaceutically acceptable excipients.

[0026] In this invention, the pharmaceutically acceptable excipients, which can be determined by those skilled in the art to meet clinical standards, include at least one of the following: solvents, propellants, cosolvents, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, penetration enhancers, pH adjusters, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculators, filter aids, and release inhibitors.

[0027] In some embodiments, the dosage form of the drug is a variety of dosage forms conventional in the art, preferably in the form of a solid, semi-solid or liquid, and may be an aqueous solution, a non-aqueous solution or a suspension, more preferably a tablet, capsule, soft capsule, granule, pill, oral liquid, dry suspension, drop pill, dry extract or injection.

[0028] In some embodiments, the medicament of the present invention can be administered into the body by known means, such as intravenous systemic delivery. Alternatively, it can be administered via intravenous, percutaneous, intranasal, mucosal, or other delivery methods. Such administration can be performed via a single dose or multiple doses. Those skilled in the art will understand that the actual dose to be administered in the present invention can vary considerably depending on a variety of factors, such as biological type or tissue, the general condition of the subject to be treated, the route of administration, the manner of administration, etc.

[0029] In some embodiments, the drug can be administered to humans and non-human mammals, including mice, rats, guinea pigs, cattle, sheep, cats, dogs, horses, monkeys, and orangutans, etc., without specific limitations.

[0030] Another typical embodiment of the present invention provides the use of a composition of a Ring1B inhibitor and cisplatin in the preparation of a medicament to protect against cisplatin-induced acute kidney injury.

[0031] In some embodiments, the Ring1B inhibitor includes Ms147.

[0032] In some embodiments, the drug is administered before, simultaneously with, and / or after cisplatin administration, preferably before cisplatin administration.

[0033] In some embodiments, the concentration of Ms147 is 10-100 nM and the concentration of cisplatin is 1-50 μM; preferably, the concentration of Ms147 is 20-60 nM and the concentration of cisplatin is 10-25 μM; more preferably, the concentration of Ms147 is 50 nM and the concentration of cisplatin is 15 μM.

[0034] In some embodiments, the protection includes preventing and / or mitigating damage to renal tubular epithelial cells.

[0035] In some embodiments, the prevention and / or mitigation of renal tubular epithelial cell damage includes: (a1) Reduce the expression levels of KIM-1 and / or NGAL proteins; (a2) Reduce histone H2AK119 ubiquitination modification level.

[0036] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0037] Example 1 (1) Human induced pluripotent stem cells (iPSCs) induced to differentiate into renal tubular epithelial cells iPSCs were cultured using mTeSR plus (STEMCELL Technologies, 100-0276) until cell confluence reached 70% or higher. The iPSCs were then dissociated using Accutase (STEMCELL Technologies, 07920) and seeded at a density of 35,000 cells per well onto 12-well transwell inserts (Corning, CLS3460) coated with Matrigel (Corning, 354277). iPSC differentiation was as follows: 1) 4 days of basal differentiation medium supplemented with 10 mM CHIR99021 (Tocris, 4423) (Advanced RPMI 1640, 1x L-GlutaMAX and 1x NEAA, Life Technologies); 2) 3 days of basal differentiation medium; 3) 2 days of basal differentiation medium supplemented with 3 μM CHIR99021 and 50 ng / mL recombinant human FGF9 (Peprotech, 100-23-1000); 4) 2 days of basal differentiation medium containing 50 ng / mL FGF9 until day 20 of differentiation.

[0038] (2) iPSCs induce differentiation of kidney organoids To generate 3D kidney organoids, differentiated cells from days 11–14 were dissociated into single-cell suspensions using Accutase. Approximately 35,000 cells were aggregated in each well of a Nunclon Sphera ultra-low attachment 96-well U-bottom microplate (Thermo Scientific, 174925) and cultured for 24 hours in basal differentiation medium supplemented with 50 ng / mL FGF9 and 10 mM Y-27632. Subsequently, a follow-up 6-day culture was performed using basal differentiation medium containing 50 ng / mL FGF9. The organoids were then transferred to the upper chamber of a Corning Transwell chamber. From day 24 of differentiation, only basal differentiation medium was used.

[0039] After obtaining mature cells, cell types were identified by immunofluorescence staining. The results showed that the 3D kidney organoids contained ECAD, LTL, PODXL, and CD31 positive cells, indicating that the kidney organoids were successfully constructed. Figure 1 In this study, ECAD represented distal renal tubular epithelial cells, LTL represented proximal renal tubular epithelial cells, PODXL positivity indicated podocytes, and CD31 positivity indicated the presence of endothelial cells, demonstrating the establishment of a stable renal tubular epithelial cell and kidney organoid induction culture system.

[0040] Example 2 The cisplatin-induced acute kidney injury (AKI) in vitro model constructed in Example 1 was randomly divided into a normal control group, a cisplatin-treated group (DDP), and a Ms147-treated group (C). 54 H 70 N 12 O6S, MCE, catalog number: HY-160924) processing group (DDP+Ms147), where the structure of Ms147 is as follows Figure 2 As shown.

[0041] The treatment methods for each group were as follows: the normal control group received an equal volume of solvent (PBS) as a control; the cisplatin treatment group received only 15 μM cisplatin for 24 h; the Ms147 treatment group received 50 nM Ms147 for 48 h pretreatment, followed by 15 μM cisplatin for another 24 h. The treatment time for cisplatin in all cisplatin-treated groups was 24 h.

[0042] The expression levels of kidney injury markers, such as kidney injury molecule-1 (KIM-1), were detected by immunofluorescence staining. The results are as follows: Figure 3 As shown, after Ms147 treatment, KIM-1 expression was significantly reduced, renal tubular morphology was good, and no signs of damage were observed. Figure 3In the cisplatin-treated group, the fluorescence signal of neutrophil gelatinase-associated lipotransferase (NGAL) was significantly increased, while after Ms147 treatment, the expression level of NGAL was significantly decreased. Figure 3 B in the text). Western blot experiments further confirmed that KIM-1 and NGAL were significantly reduced after Ms147 treatment compared to the DDP group. Figure 3 (C in the text); RT-qPCR experiments showed that KIM-1 levels were significantly lower in the Ms147 group compared to the DDP group (C). Figure 3 (D in the text). The above results indicate that Ms147 pretreatment can effectively alleviate cisplatin-induced renal tubular epithelial cell damage.

[0043] Example 3 This embodiment examines the effects of Ms147 and PTC-209 on RING1B protein expression and the ubiquitination level of PRC1 downstream histone H2AK119.

[0044] The cisplatin-induced acute kidney injury (AKI) in vitro model constructed in Example 1 was randomly divided into a normal control group, a cisplatin treatment group (DDP), a Ms147 treatment group (DDP+Ms147), and a PTC-209 (CAS No:315704-66-6) treatment group (DDP+PTC-209).

[0045] The treatment methods for each group are as follows: the normal control group was treated with an equal volume of solvent (PBS); the cisplatin treatment group was treated with only 15 μM cisplatin for 24 h; the Ms147 treatment group was pretreated with 50 nM Ms147 for 48 h, and then treated with 15 μM cisplatin for another 24 h; the PTC-209 treatment group was pretreated with 10 μM PTC-209 for 48 h, and then treated with 15 μM cisplatin for another 24 h. The treatment time for cisplatin in all cisplatin-treated groups was 24 h.

[0046] After treatment, the expression level of RING1B protein in each group of cells was detected by immunofluorescence staining. The results are as follows: Figure 4 As shown, Ring1B fluorescence signal was strong in the cisplatin-damaged group; Ring1B fluorescence signal was significantly weakened and almost invisible in the Ms147-treated group; Ring1B fluorescence signal was also weakened in the PTC-209-treated group, but the degree of weakening was not as significant as in the Ms147-treated group. Meanwhile, co-localization staining with LTL and ECAD showed that Ring1B was mainly expressed in the renal tubular epithelial cell region. These results indicate that both Ms147 and PTC-209 can inhibit Ring1B expression, but Ms147's inhibitory effect on Ring1B expression is significantly better than that of PTC-209.

[0047] Further immunofluorescence staining was used to detect the level of histone H2AK119 ubiquitination (H2AK119ub) in cells of each group, and the results are as follows: Figure 5 As shown in the figure, the fluorescence signal of H2AK119ub was weak in the normal control group; the fluorescence signal of H2AK119ub was significantly enhanced in the cisplatin-damaged group (DDP), indicating that the ubiquitination modification level of histone H2AK119 by the PRC1 complex was significantly increased after cisplatin treatment; the fluorescence signal of H2AK119ub in the Ms147-treated group (DDP+Ms147) was significantly lower than that in the cisplatin-damaged group, almost returning to the normal level; the fluorescence signal of H2AK119ub in the PTC-209-treated group (DDP+PTC-209) was also reduced, but the reduction effect was minimal. These results indicate that Ms147 effectively inhibits the ubiquitination modification of histone H2AK119 by degrading RING1B protein, and this inhibitory effect is significantly better than that of PTC-209.

[0048] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of Ring1B inhibitors in the preparation of drugs to protect against cisplatin-induced acute kidney injury.

2. The application as described in claim 1, characterized in that, The protection includes preventing and / or mitigating damage to renal tubular epithelial cells.

3. The application as described in claim 1, characterized in that, The Ring1B inhibitor includes Ms147.

4. The application as described in claim 3, characterized in that, The concentration of Ms147 was 10~100 nM, and the treatment time was 12~96 h.

5. The application as described in claim 4, characterized in that, The concentration of Ms147 was 20-60 nM, and the treatment time was 24-72 h.

6. The application as described in claim 1, characterized in that, The drug includes pharmaceutically acceptable excipients.

7. The application as described in claim 6, characterized in that, The pharmaceutically acceptable excipients include at least one of the following: solvents, propellants, solubilizers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, penetration enhancers, pH adjusters, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, filter aids, and release inhibitors.

8. The application as described in claim 1, characterized in that, The dosage forms of the drugs include tablets, capsules, granules, pills, oral liquids, dry suspensions, dry extracts, and lyophilized powder injections.

9. Use of a composition of a Ring1B inhibitor and cisplatin in the preparation of a medicament for protecting against cisplatin-induced acute kidney injury.

10. The application as described in claim 9, characterized in that, The Ring1B inhibitor includes Ms147.

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