Use of pgk1 protein in the preparation of a medicament for treating diabetic nephropathy

Abstract

The application discloses application of PGK1 protein in preparation of a medicine for treating diabetic nephropathy. It is found by the application that PGK1 protein knockdown helps to improve the kidney function of a sugar kidney model mouse, and PGK1 protein overexpression leads to poorer kidney function of the sugar kidney model mouse. The finding shows that the PGK1 protein can be used as a target for screening and preparing a medicine for treating diabetic nephropathy, and a PGK1 protein inhibitor has a prospect of being developed into a medicine for treating diabetic nephropathy.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to the application of PGK1 protein in the preparation of drugs for treating diabetic nephropathy. Background Technology

[0002] Diabetic nephropathy (DN) seriously endangers human health. It is not only a common complication of diabetes but also one of the most important kidney diseases leading to end-stage renal disease (ESRD), and has risen to become the leading cause of ESRD worldwide. The International Diabetes Federation estimates that the number of people with diabetes will increase to 700 million by 2045, and the prevalence of diabetic nephropathy, as a common complication of diabetes, is also increasing year by year. [Reference: Research progress on the main pathogenesis of diabetic nephropathy, Life Sciences, 2023]

[0003] Phosphoglycerate kinase 1 (PGK1) is the first enzyme in glycolysis to produce adenosine triphosphate (ATP). It catalyzes the transfer of the phosphate group of 1,3-bisphosphoglycerate to adenosine diphosphate (ADP), generating 3-phosphoglycerate and ATP. Besides participating in glycolysis, PGK1 also acts as a protein kinase regulator involved in various biological activities, including cell growth, division, differentiation, death, and DNA repair. In recent years, PGK1 overexpression has been associated with various cancers, including breast cancer, prostate cancer, glioma, liver cancer, lung cancer, and gastric cancer, and has become an important target in cancer research. Studies have found that PGK1 can affect the function of cancer-related transcription factors, thereby influencing tumor growth, proliferation, metastasis, angiogenesis, and drug resistance. Furthermore, the glycolysis step catalyzed by PGK1 generates ATP, providing energy and substances for tumor cell growth and proliferation, especially under hypoxic conditions, which is also considered an important reason for PGK1's involvement in tumor growth. However, the mechanism by which PGK1 participates in cancer development is very complex and remains unclear. [Reference: Zhou Jing, Therapeutic effect and molecular mechanism of alfuzosin in type 2 diabetic mice, Master's thesis, Lanzhou University, 2023]

[0004] In recent years, some studies have shown that activating PGK1 can activate glycolysis, producing some beneficial effects on the body. Studies have demonstrated that activating PGK1 can stimulate glycolysis, increase ATP levels, thereby slowing nerve damage and enhancing dopamine function, thus slowing the neurodegenerative changes in Parkinson's disease. Furthermore, studies have shown that terazosin can treat sepsis, colitis, and gastric ulcers by activating PGK1. Zhou Jing also found that alfuzosin can activate PGK1, enhance aerobic glycolysis, consume more glucose, and lower blood sugar. [Reference: Zhou Jing, Therapeutic effects and molecular mechanisms of alfuzosin in type 2 diabetic mice, Master's thesis, Lanzhou University, 2023]

[0005] There are currently no literature reports on the treatment of diabetic nephropathy using PGK1 inhibitors. Summary of the Invention

[0006] The purpose of this invention is to provide the application of PGK1 protein in the preparation of drugs for treating diabetic nephropathy.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution:

[0008] Application of PGK1 protein as a target in the preparation of drugs for the treatment of diabetic nephropathy.

[0009] The application of PGK1 protein as a target in the screening and discovery of drugs for the treatment of diabetic nephropathy.

[0010] Application of PGK1 protein inhibitors in the preparation of drugs for treating diabetic nephropathy.

[0011] Preferably, the drug uses a PGK1 protein inhibitor as the active ingredient and is formulated into a pharmaceutically acceptable dosage form using pharmaceutically acceptable excipients.

[0012] More preferably, the excipient is a solid, liquid, or semi-solid excipient.

[0013] More preferably, the dosage form includes tablets, capsules, and injections.

[0014] Beneficial effects:

[0015] This invention reveals that PGK1 protein knockdown helps improve kidney function in diabetic nephropathy model mice, while PGK1 protein overexpression leads to further deterioration of kidney function in these mice. This finding suggests that PGK1 protein can serve as a target for screening and developing drugs to treat diabetic nephropathy, and that PGK1 protein inhibitors hold promise for development into therapeutic drugs for diabetic nephropathy. Attached Figure Description

[0016] Figure 1The levels of PGK1 protein expression in the kidneys of mice in each group during the knockdown experiment are shown in: (A) Western blot plot; (B) Western blot statistical plot (n=4).

[0017] Figure 2 The expression levels of PGK1 protein in the kidneys of mice in each group during the overexpression experiment are shown in: (A) Western blot plot; (B) Western blot statistical plot (n=4).

[0018] Figure 3 Serum creatinine (A), blood urea nitrogen (B), and cystatin C (C) levels in each group of mice during the knockdown experiment (n=6).

[0019] Figure 4 The serum creatinine (A), blood urea nitrogen (B), and cystatin C (C) levels in each group of mice during the overexpression experiment (n=6) were measured.

[0020] Figure 5 The degree of glomerular mesangial expansion in each group of mice during the knockdown experiment is shown in: (A) representative H&E staining pathological images; (B) glomerular mesangial expansion score (n=6).

[0021] Figure 6 The degree of glomerular mesangial expansion in each group of mice during the overexpression experiment is shown in the following figures: (A) representative H&E staining pathological images; (B) glomerular mesangial expansion score (n=6).

[0022] Figure 7 The degree of renal fibrosis in each group of mice during the knockdown experiment is shown in: (A) representative Sirius map; (B) fibrosis score (n=6).

[0023] Figure 8 The degree of renal fibrosis in mice in each group during the overexpression experiment is shown in: (A) representative Sirius diagram; (B) fibrosis score (n=6).

[0024] Figure 9 The following figures represent the renal ROS levels of mice in each group during the knockdown experiment: (A) representative DHE staining image; (B) DHE staining score (n=6).

[0025] Figure 10 The ROS levels in the kidneys of mice in each group during the overexpression experiment are shown in: (A) representative DHE staining image; (B) DHE staining score (n=6).

[0026] Figure 11 The degree of macrophage infiltration in the kidneys of mice in each group during the knockdown experiment is shown in: (A) representative F4 / 80 staining map; (B) F4 / 80 staining score (n=6).

[0027] Figure 12 The degree of macrophage infiltration in the kidneys of mice in each group during the overexpression experiment is shown in: (A) representative F4 / 80 staining map; (B) F4 / 80 staining score (n=6). Detailed Implementation

[0028] The following describes the substantive content of the present invention in detail with reference to embodiments, but this does not limit the scope of protection of the present invention.

[0029] I. Experimental Materials

[0030] C57BLKS / J db / db and C57BLKS / J db / m mice were purchased from Changzhou Cavens Laboratory Animal Co., Ltd.

[0031] AAV9-Ksp-shPGK1 and its control AAV9-Ksp-shCon, AAV9-Ksp-PGK1 overexpression plasmid and its control AAV9-Ksp-control plasmid were purchased from Paizhen Biotechnology.

[0032] II. Experimental Methods

[0033] 1. Selection of model animals

[0034] The db / db mouse is derived from the C57BLKS / J inbred strain through autosomal recessive inheritance. Significant obesity and elevated fasting blood glucose, along with increased water intake and urine output, are observed as early as 6 weeks after birth, peaking at 10-12 weeks, and complications such as diabetic nephropathy develop. The 10-12 week C57BLKS / J db / db mouse is commonly used in this field as a diabetic nephropathy model, while the C57BLKS / Jdb / m mouse serves as a negative control model for diabetic nephropathy.

[0035] 2. Animal grouping and PGK1 protein knockdown test

[0036] Eleven-week-old C57BLKS / J db / db and db / m mice were pre-acclimatized for one week. They were then anesthetized with 0.3% sodium pentobarbital, and the renal pelvis was located after opening the kidneys. 50 μl each of AAV9-Ksp-shPGK1 (5'-GTCCAAACTAGGAGATGTCTA-3') or AAV9-Ksp-shCon (5'-TTCTCCGAACGTGTCACGT-3') were injected into the renal pelvis to achieve specific PGK1 knockdown in renal tubular epithelial cells. After injection, the sutures were closed, and the mice were fed for another 6 weeks. Serum and kidney tissue were then collected for serological and renal tissue biochemical analysis. Grouping, naming, and treatment are shown in the table below.

[0037]

[0038] 3. Animal grouping and PGK1 protein overexpression experiment

[0039] Eleven-week-old C57BLKS / J db / db and db / m mice were pre-acclimatized for one week. They were then anesthetized with 0.3% sodium pentobarbital, and the renal pelvis was located after opening the kidneys. 50 μl each of the AAV9-Ksp-PGK1 overexpression plasmid or the AAV9-Ksp-control plasmid were injected into the renal pelvis to achieve specific overexpression of PGK1 in renal tubular epithelial cells. After injection, the sutures were closed, and the mice were fed for another 6 weeks. Serum and kidney tissue were then collected for serological and renal tissue biochemical analysis. Grouping, naming, and processing are shown in the table below.

[0040]

[0041] 4. Measurement of PGK1 protein expression level

[0042] Western blot was used to detect the protein expression level of PGK1 (ab199438, Abcam) in mouse kidney tissue. A suitable amount of mouse kidney tissue was cut, lysed with RIPA lysis buffer, and then centrifuged. The supernatant was collected, and the protein content was determined to quantify the total protein to the same level. 5× protein loading buffer was added to the protein sample, and the mixture was boiled for denaturation for 10 min. After cooling to room temperature, the sample was loaded for SDS-PAGE gel electrophoresis. After wet transfer, the membrane was blocked, and incubated with primary and secondary antibodies. Finally, β-actin (ab6276, Abcam) was used as an internal control to determine the relative protein content. ImageJ software was used to analyze the gray values ​​of the bands.

[0043] 5. Measurement of serum urea nitrogen (BUN) and creatinine (CRE) levels

[0044] Blood was collected from the heart, and whole blood was allowed to stand at room temperature for 30 min, then centrifuged at 3000 r / min for 15 min to obtain mouse serum samples. The levels of BUN and Cre in mouse serum were determined using a blood urea nitrogen kit (C013-2-1, Nanjing Jiancheng) and a creatinine kit (C011-2-1, Nanjing Jiancheng), respectively.

[0045] 6. Determination of serum cystatin C levels

[0046] The level of serum Cystatin C in mice was determined using an ELISA kit (EK0679, Boster Biologics).

[0047] 7. Kidney tissue pathological examination

[0048] 7.1 HE staining, Sirius red staining

[0049] Mice were euthanized by cervical dislocation, and half of each kidney was fixed in formalin solution at 4°C for 24 hours. Kidney tissue from each group was then embedded in paraffin and sectioned to a thickness of 4 μm. Hematoxylin-eosin (HE) staining and Sirius red staining were performed. Pathological changes in kidney structure and interstitial fibrosis were observed and histologically analyzed using a light microscope. Glomerular matrix proliferation, mesangial expansion, saccule wall adhesion, inflammatory cell infiltration, and glomerular basement membrane thickening were observed. Collagen fiber deposition in the renal interstitial region was observed under a light microscope, and ImageJ software was used for quantitative analysis of the collagen fibers (stained red).

[0050] 7.2 DHE probe staining

[0051] Kidney paraffin sections were dewaxed and hydrated, then stained with the reactive oxygen species probe DHE (810253P, Sigma, 5 μM) at 37°C in the dark for 1 hour. The sections were then mounted with an anti-fluorescence quencher and photographed under a fluorescence microscope. A macro program for positive expression was set up using Image Pro Plus software, and the expression levels of DHE in different groups were analyzed under the same conditions.

[0052] 7.3, F4 / 80 immunofluorescence staining

[0053] Kidney paraffin sections were dewaxed, hydrated, and then subjected to high-pressure antigen heat retrieval. After blocking, the sections were incubated overnight at 4°C with F4 / 80 antibody (ab300421, Abcam) at a concentration of 1:200. After washing, the sections were incubated with fluorescent secondary antibody (Goat Anti-Rabbit IgG H&L (Alexa)). 594)(ab150080) was incubated at a concentration of 1:1000 at 37℃ for 1 h, and mounted with DAPI (10236276001, Sigma, 5 μM). Five fields of view were randomly selected from each slide under a microscope and photographed and saved. The macro program for positive expression was set using ImagePro Plus software. Under the same conditions, the expression level of F4 / 80 in different groups was analyzed.

[0054] 8. Data Processing

[0055] Experimental results are expressed as (Mean ± SD). Student's t was used to compare two groups, and Tne-way ANOVA was used to compare multiple groups. P < 0.05 was considered statistically significant.

[0056] III. Experimental Results

[0057] 1. Results of PGK1 protein knockdown

[0058] The results are as follows Figure 1As shown in the figure, compared with the control mouse-Con shRNA group (A1), the expression level of PGK1 protein in mice in the glycosuria-reperfusion-induced kidney disease (A3) group was significantly upregulated (P<0.05), indicating that PGK1 protein expression was upregulated in the glycosuria-reperfusion-induced kidney disease model. Compared with the glycosuria-reperfusion-induced kidney disease (A3) mouse-Con shRNA group (A4) mouse-PGK1 shRNA group was significantly downregulated (P<0.05), indicating that PGK1 shRNA achieved knockdown of PGK1 protein, achieving the expected goal.

[0059] 2. Results of PGK1 protein overexpression

[0060] The results are as follows Figure 2 As shown in the figure, compared with the control mouse-PGK1 control group (B1), the PGK1 protein expression level of the glycosuria-reperfusion mouse-PGK1 control group (B3) was significantly upregulated (P<0.05), indicating that PGK1 protein expression was upregulated in the glycosuria-reperfusion model. Compared with the glycosuria-reperfusion mouse-PGK1 overexpression group (B3), the PGK1 protein expression level of the glycosuria-reperfusion mouse-PGK1 overexpression group (B4) was significantly upregulated (P<0.05), indicating that the AAV9-Ksp-PGK1 overexpression plasmid achieved overexpression of PGK1 protein, achieving the expected purpose.

[0061] 3. Results of serum BUN, Cre, and Cystatin C level measurements

[0062] The results of serum marker level measurements in the knockdown test are as follows: Figure 3 As shown in the figure. Compared with the control mouse-Con shRNA group (A1), the serum levels of BUN, Cre, and Cystatin C in mice in the glycosuria-reperfusion mouse-Con shRNA group (A3) were significantly upregulated (P<0.05), indicating that the serum levels of BUN, Cre, and Cystatin C are upregulated in the glycosuria-reperfusion model. Compared with the glycosuria-reperfusion mouse-Con shRNA group (A3), the serum levels of BUN, Cre, and Cystatin C in mice in the glycosuria-reperfusion mouse-PGK1 shRNA group (A4) were significantly downregulated (P<0.05), indicating that PGK1 protein knockdown can effectively reduce the serum levels of BUN, Cre, and Cystatin C in glycosuria-reperfusion mice.

[0063] The results of serum marker level determination in the overexpression assay are as follows: Figure 4As shown in the figure, compared with the control mouse-PGK1 control group (B1), the serum levels of BUN, Cre, and Cystatin C in the glycosuria-reperfusion mouse-PGK1 control group (B3) were significantly upregulated (P<0.05), indicating that the serum levels of BUN, Cre, and Cystatin C were upregulated in the glycosuria-reperfusion model. Compared with the glycosuria-reperfusion mouse-PGK1 control group (B3), the serum levels of BUN, Cre, and Cystatin C in the glycosuria-reperfusion mouse-PGK1 overexpression group (B4) were significantly further upregulated (P<0.05), indicating that PGK1 protein overexpression leads to a further increase in the serum levels of BUN, Cre, and Cystatin C in glycosuria-reperfusion mice.

[0064] Under normal circumstances, the concentrations of BUN, Cre, and Cystatin C in the blood are very stable. By detecting the levels of BUN, Cre, and Cystatin C in the blood, renal tubular filtration function can be accurately assessed. Figure 3 , Figure 4 The results showed that PGK1 protein knockdown helped improve kidney function in diabetic kidney model mice, while PGK1 protein overexpression led to worse kidney function in diabetic kidney model mice.

[0065] 4. Kidney histopathological examination results

[0066] The results of the knockdown test on the degree of expansion of the glomerular mesangial area in mice are as follows: Figure 5 As shown in the figure, compared with the control mouse-ConshRNA group (A1), the degree of glomerular mesangial expansion in mice with glycosuria-reperfusion kidney disease (A3) was significantly enhanced (P<0.05), indicating that the degree of glomerular mesangial expansion in mice with glycosuria-reperfusion kidney disease was significantly enhanced. Compared with the glycosuria-reperfusion kidney disease (A3) mouse-ConshRNA group (A4), the degree of glomerular mesangial expansion in mice with glycosuria-reperfusion kidney disease (A4) was significantly reduced (P<0.05), indicating that PGK1 protein knockdown can effectively alleviate the degree of glomerular mesangial expansion in mice with glycosuria-reperfusion kidney disease.

[0067] The results of the overexpression assay for the degree of expansion of the glomerular mesangial area in mice are as follows: Figure 6 As shown in the figure, compared with the control group (B1), the degree of glomerular mesangial expansion was significantly enhanced in the glycosuria-prone kidney mouse-PGK1 control group (B3) (P<0.05), indicating that the degree of glomerular mesangial expansion was significantly enhanced in the glycosuria-prone kidney model. Compared with the glycosuria-prone kidney mouse-PGK1 overexpression group (B3), the degree of glomerular mesangial expansion was further enhanced in the glycosuria-prone kidney mouse-PGK1 overexpression group (B4) (P<0.05), indicating that PGK1 protein overexpression leads to a further enhancement of glomerular mesangial expansion in glycosuria-prone kidney mice.

[0068] Results of the knockdown test on the degree of renal fibrosis in mice are as follows: Figure 7 As shown in the figure, compared with the control mouse-Con shRNA group (A1), the degree of renal fibrosis in the glycosuria-reperfusion mouse-Con shRNA group (A3) was significantly enhanced (P<0.05), indicating that the degree of renal fibrosis in mice with glycosuria-reperfusion model was significantly enhanced. Compared with the glycosuria-reperfusion mouse-Con shRNA group (A3), the degree of renal fibrosis in the glycosuria-reperfusion mouse-PGK1 shRNA group (A4) was significantly reduced (P<0.05), indicating that PGK1 protein knockdown can effectively alleviate the degree of renal fibrosis in glycosuria-reperfusion mice.

[0069] The results of the overexpression assay for the degree of renal fibrosis in mice are as follows: Figure 8 As shown in the figure, compared with the control mouse-PGK1 control group (B1), the degree of renal fibrosis in the glycosuria-reperfusion mouse-PGK1 control group (B3) was significantly enhanced (P<0.05), indicating that the degree of renal fibrosis in mice in the glycosuria-reperfusion model was significantly enhanced. Compared with the glycosuria-reperfusion mouse-PGK1 overexpression group (B3), the degree of renal fibrosis in the glycosuria-reperfusion mouse-PGK1 overexpression group (B4) was further enhanced (P<0.05), indicating that PGK1 protein overexpression leads to a further enhancement of renal fibrosis in glycosuria-reperfusion mice.

[0070] Results of ROS level measurement in mouse kidneys during knockdown assay: Figure 9 As shown in the figure, compared with the control mouse-Con shRNA group (A1), the renal ROS level of mice in the glycosuria-reperfusion mouse-Con shRNA group (A3) was significantly increased (P<0.05), indicating that the renal ROS level of mice in the glycosuria-reperfusion model was significantly increased. Compared with the glycosuria-reperfusion mouse-Con shRNA group (A3), the renal ROS level of mice in the glycosuria-reperfusion mouse-PGK1 shRNA group (A4) was significantly decreased (P<0.05), indicating that PGK1 protein knockdown can effectively reduce the renal ROS level of mice with glycosuria-reperfusion.

[0071] The results of ROS level measurement in mouse kidneys during the overexpression assay are as follows: Figure 10 As shown in the figure, compared with the control mouse-PGK1 control group (B1), the renal ROS level of glycosuria mice-PGK1 control group (B3) was significantly increased (P<0.05), indicating that the renal ROS level of mice in the glycosuria model was significantly increased. Compared with the glycosuria mice-PGK1 control group (B3), the renal ROS level of mice in the glycosuria mice-PGK1 overexpression group (B4) was further increased (P<0.05), indicating that PGK1 protein overexpression leads to a further increase in the renal ROS level of glycosuria mice.

[0072] Results of mouse kidney macrophage infiltration in knockdown assay: Figure 11As shown in the figure, compared with the control mouse-ConshRNA group (A1), the degree of macrophage infiltration in the kidneys of mice in the glycosuria-reperfusion mouse-ConshRNA group (A3) was significantly enhanced (P<0.05), indicating that the degree of macrophage infiltration in the kidneys of mice in the glycosuria-reperfusion model was significantly enhanced. Compared with the glycosuria-reperfusion mouse-ConshRNA group (A3), the degree of macrophage infiltration in the kidneys of mice in the glycosuria-reperfusion mouse-PGK1 shRNA group (A4) was significantly reduced (P<0.05), indicating that PGK1 protein knockdown can effectively alleviate the degree of macrophage infiltration in the kidneys of mice with glycosuria.

[0073] The results of the overexpression assay for the degree of macrophage infiltration in mouse kidneys are as follows: Figure 12 As shown in the figure, compared with the control mouse-PGK1 control group (B1), the degree of macrophage infiltration in the kidneys of mice with glycosuria-reperfusion kidney (B3) was significantly enhanced (P<0.05), indicating that the degree of macrophage infiltration in the kidneys of mice in the glycosuria-reperfusion kidney model was significantly enhanced. Compared with the glycosuria-reperfusion kidney-PGK1 control group (B3), the degree of macrophage infiltration in the kidneys of mice with glycosuria-reperfusion kidney-PGK1 overexpression group (B4) was further enhanced (P<0.05), indicating that PGK1 protein overexpression leads to a further enhancement of macrophage infiltration in the kidneys of mice with glycosuria-reperfusion kidney.

[0074] The degree of glomerular mesangial expansion, the degree of renal fibrosis, renal ROS levels, and the degree of renal macrophage infiltration are important indicators for evaluating renal function. Figures 5-12 The results showed that PGK1 protein knockdown helped improve kidney function in diabetic kidney model mice, while PGK1 protein overexpression led to worse kidney function in diabetic kidney model mice.

[0075] In summary, PGK1 protein knockdown helps improve kidney function in diabetic nephropathy model mice, while PGK1 protein overexpression leads to further deterioration of kidney function in these mice. This finding suggests that PGK1 protein can serve as a target for screening and developing drugs to treat diabetic nephropathy, and that PGK1 protein inhibitors hold promise for development into therapeutic drugs for diabetic nephropathy.

[0076] The purpose of the above embodiments is to specifically illustrate the substantive content of the present invention, but those skilled in the art should know that the scope of protection of the present invention should not be limited to the specific embodiments.

Claims

1. Application of PGK1 protein inhibitors in the preparation of drugs for treating diabetic nephropathy; among which, The PGK1 protein inhibitor is AAV9-Ksp-shPGK1, and the sequence of shPGK1 is: 5'-GTCCAAACTAGGAGATGTCTA-3'.

2. The application according to claim 1, characterized in that: The drug uses a PGK1 protein inhibitor as its active ingredient and is formulated into a pharmaceutically acceptable dosage form using pharmaceutically acceptable excipients.

3. The application according to claim 2, characterized in that: The excipients are solid, liquid, or semi-solid excipients.

4. The application according to claim 3, characterized in that: The dosage form is tablet, capsule, or injection.

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