Application of CTRP13 in the preparation of drugs for treating renal fibrosis
By using CTRP13 to inhibit the Smad3 signaling pathway, the problem of difficulty in effectively treating renal tissue fibrosis in existing technologies is solved, and a safe and efficient renal fibrosis inhibition effect is achieved.
Patent Information
- Application Number
- CN202211120327.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-09-15
- Publication Date
- 2025-09-09
- Estimated Expiration
- 2042-09-15
AI Technical Summary
Existing technologies are unable to effectively inhibit renal tissue fibrosis, especially in fibrosis caused by aging, infection, drugs, hypertension and other diseases, and there is a lack of safe and effective treatment options.
By applying complement C1q tumor necrosis factor-related protein 13 (CTRP13), as an inhibitor of Smad3 phosphorylation and downstream signaling pathways, the transdifferentiation of renal tubular epithelial cells is inhibited, thereby preventing and treating renal tissue fibrosis.
CTRP13 can effectively inhibit the Smad3 downstream signaling pathway, prevent the transdifferentiation of renal tubular epithelial cells, prevent and treat renal tissue fibrosis, and show safety without obvious liver or kidney toxicity.
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Figure CN115607652B_ABST
Abstract
Description
Technical field:
[0001] The present invention relates to the field of biomedicine, and in particular to the use of CTRP13 in preparing a drug for treating renal tissue fibrosis. Background technology:
[0002] With the aging of the population, the incidence of age-related diseases is showing a significant upward trend. According to data from large-scale studies, the prevalence of chronic kidney disease has reached 10% over the past decade. The kidneys are one of the body's most important metabolic organs, playing a vital role in maintaining normal life. Aging-related glomerulosclerosis, tubular atrophy, and renal interstitial fibrosis alter kidney structure and hemodynamics, leading to progressive deterioration and even loss of renal function. Renal fibrosis is a terminal manifestation of chronic kidney disease. Once it develops, it severely impacts a patient's quality of life and survival. Therefore, the search for effective treatments for age-related fibrosis has significant medical and social significance. Actively improving chronic renal tissue damage and fibrosis is a key component of the prevention and treatment of chronic kidney disease.
[0003] Transforming growth factor (TGF) β1 is a recently discovered member of the TGF-β superfamily that regulates cell growth and differentiation. It is the most potent signaling factor currently discovered to promote renal fibrosis, and its signaling is achieved through the expression of Smad proteins. Smads play a key role in transmitting TGF-β signals from cell surface receptors to the nucleus, with different Smads mediating signaling by different TGF-β family members. TGF-β, as a ligand, forms a receptor complex that enters the nucleus by activating Smads, thereby co-activating or repressing target gene transcription. Prolonged ligation time increases the severity of renal fibrosis, suggesting that the TGF-β / Smad3 signaling pathway plays a crucial role in the development and progression of renal interstitial fibrosis.
[0004] Complement C1q tumor necrosis factor-related protein 13, also known as CTRP13, also known as C1q And Tumor Necrosis Factor-Related Protein 13, contains 255 amino acids with a molecular weight of 26,719 Da. NCBI reference number: NP_001010908.1. The amino acid sequence is as follows:
[0005] MVLLLVILIPVLVSSAGTSAHYEMLGTCRMVCDPYGGTKAPSTAATPDRGLMQSLPTFIQGPKGEAGRPGKAGPRGPPGEPGPPGPMGPPGEKGEPGRQGLPGPPGAPGLNAAGAISAATYSTVPKI AFYAGLKRQHEGYEVLKFDDVVTNLGNHYDPTTGKFTCSIPGIYFFTYHVLMRGGDGTSMWADLCKNNQVRASAIAQDADQNYDYASNSVVLHLEPGDEVYIKLDGGKAHGGNNNKYSTFSGFIIYAD
[0006] In recent years, research on members of the complement C1q / TNF-related protein (CTRP) family in metabolic disorders, including metabolic syndrome and diabetes, has attracted significant attention. CTRP13, a highly conserved novel adipokine within the CTRP family, is implicated in the regulation of cardiovascular and metabolic diseases. Studies have shown that blood glucose levels influence CTRP13 expression, and multiple lines of evidence from various groups have demonstrated that serum CTRP13 expression is significantly reduced in metabolic diseases, including fatty liver disease and diabetes. Furthermore, studies have found that CTRP13 can significantly inhibit foam cell formation, delay the progression of atherosclerosis, and reduce the incidence of vascular calcification in renal failure and hypertension-induced abdominal aortic aneurysms. Therefore, we hypothesize that CTRP13 may also play an important role in the regulation of vascular biology, particularly vascular remodeling. However, the role of CTRP13 in renal tissue fibrosis has not been studied. Summary of the invention:
[0007] (1) Technical problems solved
[0008] Against this background, the present invention experimentally discovered that complement C1q tumor necrosis factor-related protein 13, or CTRP13, has an inhibitory effect on the Smad3 downstream signaling pathway, can inhibit the transdifferentiation of renal tubular epithelial cells, thereby preventing and / or treating renal tissue fibrosis. CTRP13 does not have the common liver and kidney toxicity or cytotoxic effects of other renal fibrosis inhibitors, and its safety can be expected. This indicates that CTRP13 can be used to prepare drugs for treating renal tissue fibrosis caused by diseases such as infection, drugs, aging, and hypertension.
[0009] (2) Technical solution
[0010] In order to solve the above technical problems, the present invention adopts the following technical solutions:
[0011] Application of CTRP13 in the preparation of drugs for treating renal tissue fibrosis.
[0012] Furthermore, the application is the use of CTRP13 as an inhibitor of Smad3 phosphorylation and Smad3 downstream signaling pathway in the preparation of drugs for treating renal tissue fibrosis.
[0013] The present invention also provides an inhibitor of Smad3 phosphorylation and Smad3 downstream signaling pathway, wherein the effective component of the inhibitor includes CTRP13.
[0014] Furthermore, the inhibitor is used for phenotypic transdifferentiation of renal tubular epithelial cells.
[0015] Furthermore, the inhibitor is used to prevent and / or treat renal tissue fibrosis.
[0016] Furthermore, the renal tissue fibrosis includes renal tissue fibrosis caused by infection, drugs, aging, and hypertension.
[0017] The present invention also provides a method for inhibiting the phosphorylation of Smad3 and the downstream signaling pathway of Smad3, comprising administering CTRP13 to an individual in need thereof.
[0018] According to an embodiment of the present invention, the phosphorylation of Smad3 and the downstream signaling pathway of Smad3 are inhibited to antagonize the phenotypic transdifferentiation of renal tubular epithelial cells, thereby preventing and / or treating renal tissue fibrosis.
[0019] (3) Beneficial effects
[0020] The beneficial effects produced by the present invention are:
[0021] (1) Complement C1q tumor necrosis factor-related protein 13 (CTRP13) has an inhibitory effect on the Smad3 downstream signaling pathway and can inhibit the transdifferentiation of renal tubular epithelial cells, thereby preventing and / or treating renal tissue fibrosis. This indicates that CTRP13 can be used to prepare drugs for the treatment of renal tissue fibrosis.
[0022] (2) CTRP13 does not have the common liver and kidney toxicity or cytotoxic effects of other renal fibrosis inhibitors, and its safety can be expected; this indicates that CTRP13 can be widely used as a drug for treating renal tissue fibrosis caused by diseases such as infection, drugs, aging, and hypertension. Description of the drawings:
[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments.
[0024] Figure 1a is the sham operation + vehicle treatment group, b is the sham operation + CTRP13 treatment group, c is the ligation + vehicle treatment group, and c is the ligation + CTRP13 treatment group. After treatment, the oil red-hematoxylin (HE) staining experiment was used to detect the renal morphology of mice after unilateral ureteral obstruction injury.
[0025] Figure 2 a is the sham operation + vehicle treatment group, b is the sham operation + CTRP13 treatment group, c is the ligation + vehicle treatment group, and c is the ligation + CTRP13 treatment group. After treatment, the Masson trichrome staining experiment was used to detect the fibrosis morphology after unilateral ureteral obstruction injury in mice.
[0026] Figure 3 a is the sham operation + vehicle treatment group, b is the sham operation + CTRP13 treatment group, c is the ligation + vehicle treatment group, and c is the ligation + CTRP13 treatment group. After treatment, the expressions of collagen fibrosis markers Collagen I, Vimentin, and αSMA were detected by immunofluorescence assay.
[0027] Figure 4 a is the sham operation + vehicle treatment group, b is the sham operation + CTRP13 treatment group, c is the ligation + vehicle treatment group, and c is the ligation + CTRP13 treatment group. After treatment, the expression of Collagen I, Vimentin, and αSMA in the kidney tissue of the model mice was detected by Western blotting experiments.
[0028] Figure 5 a is the sham operation + vehicle treatment group, b is the sham operation + CTRP13 treatment group, c is the ligation + vehicle treatment group, and c is the ligation + CTRP13 treatment group. After treatment, the serum urea nitrogen and creatinine levels of the model mice were detected, and the urine protein levels of mice 24 were also detected.
[0029] Figure 6 Mesotubular epithelial cells were treated with TGF-β and then stimulated with CTRP13 (100 ng / ml) and vehicle (DMSO). The figure shows the expression of E-cadherin, Vimentin, and αSMA by CTRP13 using real-time quantitative PCR.
[0030] Figure 7 After TGF-β treatment, renal tubular epithelial cells were stimulated with CTRP13 (100 ng / ml) and vehicle (DMSO), and the phosphorylation level of Smad3 was detected by Western blotting. Specific implementation method:
[0031] In order to enable those skilled in the art to more clearly 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.
[0032] The following examples use cell experiments to study the inhibitory effect of CTRP13 on renal tissue fibrosis.
[0033] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.
[0034] The CTRP13 used in the test of this example was purchased from Aviscera Bioscience, model number 00333-01-100. The following steps were used to detect renal tissue fibrosis and renal tubular epithelial cell phenotypic transdifferentiation in the following experiments.
[0035] The specific process is as follows:
[0036] Human renal proximal tubular epithelial cells (HK2) were cultured in DMEM / F12 medium supplemented with 10% fetal bovine serum (FBS), 1.2 g / L sodium bicarbonate, and 1% penicillin / streptomycin at 37°C in 5% CO₂. HK2 cells were digested with 0.2% trypsin (Sigma) and 0.02% EDTA and seeded into 6-well plates. When HK2 cells reached 80% confluence, they were starved overnight with serum-free DMEM / F12. After 24 hours of starvation, HK2 cells were treated with TGFβ-1 (Peprotw'h) and CTRP13.
[0037] Reverse transcription-polymerase chain reaction (RT-PCR) Total RNA from kidney tissue and HK2 cells was isolated using an RNA isolation kit (TaKaRa). 2 μg of total RNA was used for reverse transcription. The StepOnePlus real-time PCR system was used. 18sRNA was used as an internal control to adjust for intersample variability. Relative concentrations were calculated using the PCR method. Primers were designed using the PubMed online tool and synthesized by Shanghai Sangon Biotechnology Co., Ltd.
[0038] Protein expression was detected by Western blotting. Eggs were extracted using RIPA lysis buffer (Beyotime), and proteins were separated on 10% acrylamide gels. Protein bands were transferred to PVDF membranes (Millipore) and blocked with 10% skim milk for 1 hour. The membranes were incubated with primary and secondary antibodies. Bands were detected using enhanced chemiluminescence detection reagents and visualized using X-ray film (Bio-Rad). Protein expression levels were quantified by the ratio with GAPDH.
[0039] The animal study protocol was approved by the Animal Experimental Ethics Committee. Male C57 mice (18-21 g) were purchased from Huazhong University of Science and Technology. A unilateral ureteral obstruction (UUO) model was established: mice were anesthetized with 3% pentobarbital (1 mg / kg body weight) via intraperitoneal injection. After disinfection, the abdominal cavity was exposed, the left ureter was isolated, and two silk ligatures were used to tie the left ureter. Mice in the sham-operated group did not undergo ureteral ligation.
[0040] Example 1
[0041] C57 mice were subjected to unilateral ureteral obstruction (UUO) or sham surgery and then intraperitoneally injected with CTRP13 (10 mg / kg / day) or vehicle (DMSO). Fourteen days later, the mice were euthanized, and the injured kidneys underwent nephrectomy. The kidneys were fixed with 4% formaldehyde, embedded in paraffin, and then sectioned. Figure 1 A is the sham operation + vehicle treatment group, b is the sham operation + CTRP13 treatment group, c is the ligation + vehicle treatment group, and c is the ligation + CTRP13 treatment group. After treatment, the renal morphology of mice after unilateral ureteral obstruction injury was detected using oil red-hematoxylin (HE) staining. Figure 1 It can be seen that compared with the sham operation + vehicle treatment group, UUO injury in mice caused epithelial cell necrosis, inflammatory cell infiltration and renal tubular dilation; and CTRP13 can inhibit epithelial cell necrosis, inflammatory cell infiltration and renal tubular dilation caused by UUO injury.
[0042] Example 2
[0043] C57 mice were subjected to unilateral ureteral obstruction (UUO) or sham surgery and then intraperitoneally injected with CTRP13 (10 mg / kg / day) or vehicle (DMSO). Fourteen days later, the mice were euthanized, and the injured kidneys underwent nephrectomy. The kidneys were fixed with 4% formaldehyde, embedded in paraffin, and then sectioned. Figure 1 A is the sham operation + vehicle treatment group, b is the sham operation + CTRP13 treatment group, c is the ligation + vehicle treatment group, and c is the ligation + CTRP13 treatment group. After treatment, the Masson trichrome staining experiment was used to detect the fibrosis morphology of mice after unilateral ureteral obstruction injury. Figure 2 It can be seen that compared with the sham operation + vehicle treatment group, a large amount of collagen deposition was caused after UUO injury in mice; and CTRP13 can inhibit the large amount of collagen deposition caused by UUO injury.
[0044] Example 3
[0045] C57 mice were subjected to unilateral ureteral obstruction (UUO) or sham surgery and then intraperitoneally injected with CTRP13 (10 mg / kg / day) or vehicle (DMSO). Fourteen days later, the mice were euthanized, and the injured kidneys underwent nephrectomy. The kidneys were fixed with 4% formaldehyde, embedded in paraffin, and then sectioned. Figure 1 a is the sham operation + vehicle treatment group, b is the sham operation + CTRP13 treatment group, c is the ligation + vehicle treatment group, and c is the ligation + CTRP13 treatment group. After treatment, the expression of collagen fibrosis markers Collagen I, Vimentin, and αSMA were detected by immunofluorescence assay. Figure 3 It can be seen that compared with the sham operation + vehicle treatment group, the expression of Collagen I, Vimentin, and αSMA was significantly increased after UUO injury in mice; and CTRP13 can inhibit the expression level of corresponding collagen fibrosis phenotype proteins caused by UUO injury.
[0046] Example 4
[0047] C57 mice were subjected to unilateral ureteral obstruction (UUO) or sham surgery and then intraperitoneally injected with CTRP13 (10 mg / kg / day) or vehicle (DMSO). Fourteen days later, the mice were euthanized, and the injured kidneys underwent nephrectomy. The kidneys were fixed with 4% formaldehyde, embedded in paraffin, and then sectioned. Figure 1 A is the sham operation + vehicle treatment group, b is the sham operation + CTRP13 treatment group, c is the ligation + vehicle treatment group, and c is the ligation + CTRP13 treatment group. After treatment, the expression of Collagen I, Vimentin, and αSMA in the kidney tissue of the model mice was detected by Western blotting. Figure 4 It can be seen that compared with the sham operation + vehicle treatment group, the expression of Collagen I, Vimentin, and αSMA was significantly increased after UUO injury in mice; and CTRP13 can inhibit the expression level of corresponding collagen fibrosis phenotype proteins caused by UUO injury.
[0048] Example 5
[0049] C57 mice were subjected to unilateral ureteral obstruction (UUO) or sham surgery and then intraperitoneally injected with CTRP13 (10 mg / kg / day) or vehicle (DMSO). Fourteen days later, the mice were euthanized, and the injured kidneys underwent nephrectomy. The kidneys were fixed with 4% formaldehyde, embedded in paraffin, and then sectioned. Figure 1A is the sham operation + vehicle treatment group, b is the sham operation + CTRP13 treatment group, c is the ligation + vehicle treatment group, and c is the ligation + CTRP13 treatment group. After treatment, the serum urea nitrogen and creatinine levels of the model mice were detected, and the urine protein levels of the mice were also detected. Figure 5 It can be seen that compared with the sham operation + vehicle treatment group, UUO injury in mice caused a significant increase in the expression of urea nitrogen, creatinine, and 24 mouse proteinuria; and CTRP13 can inhibit the renal function damage caused by UUO injury.
[0050] Example 6
[0051] After the renal tubular epithelial cells were treated with TGFβ (5 ng / ml), they were stimulated with CTRP13 (100 ng / ml) and vehicle DMSO. The cells were collected, RNA was extracted, and the expression of E-cadherin, Vimentin, and αSMA was detected by real-time quantitative PCR. The results are shown in Figure 2. Figure 6 As shown. Figure 6 It can be seen that after treatment with TGFβ1, the expression of E-cadherin, Vimentin, and αSMA mRNA in renal tubular epithelial cells increased; after stimulation with CTRP13, the expression of E-cadherin, Vimentin, and αSMA mRNA induced by TGFβ1 decreased.
[0052] Example 7
[0053] After the renal tubular epithelial cells were treated with TGFβ1, they were stimulated with CTRP13 (100 ng / ml) and vehicle DMSO. The phosphorylation level of Smad3 was detected by Western blotting. The results were as follows: Figure 7 As shown. Figure 7 It can be seen that after TGFβ1 treatment of renal tubular epithelial cells, the phosphorylation level of Smad3 increased; after further treatment with CTRP13, the phosphorylation level of Smad3 decreased. Therefore, it is shown that TGFβ1 promotes the phosphorylation of Smad3, while CTRP13 inhibits the phosphorylation of Smad3.
[0054] From the above experimental results, we can see that CTRP13 can directly inhibit renal tissue fibrosis and tubular epithelial cell transdifferentiation.
[0055] In summary, the present invention experimentally discovered that complement C1q tumor necrosis factor-related protein 13, or CTRP13, has an inhibitory effect on the Smad3 downstream signaling pathway, can inhibit the transdifferentiation of renal tubular epithelial cells, and thus prevent and / or treat renal tissue fibrosis. CTRP13 does not have the common liver and kidney toxicity or cytotoxic effects of other renal fibrosis inhibitors, and its safety can be expected. This indicates that CTRP13 can be used to prepare drugs for treating renal tissue fibrosis caused by diseases such as infection, drugs, aging, and hypertension.
[0056] Finally, it should be noted that the above embodiments are intended only to illustrate the present invention and are not intended to limit the scope of protection of the present invention. In addition, after reading the technical contents of the present invention, those skilled in the art may make various changes, modifications, or variations to the present invention, and all such equivalent forms also fall within the scope of protection defined by the present application.
Claims
1. Application of CTRP13 in the preparation of drugs for the treatment of renal tissue fibrosis.
2. The use according to claim 1, characterized in that: The application is the use of CTRP13 as an inhibitor of Smad3 phosphorylation and Smad3 downstream signaling pathway in the preparation of drugs for treating renal tissue fibrosis.