Preparation of aldobk147 mutant, kidney stone prevention and treatment agent and application

Abstract

This invention has discovered that the pathogenesis of kidney stones is accompanied by a significant increase in the lactation modification level of histidine at position 147 of ALDOB. Furthermore, when histidine at position 147 is mutated, the lactation modification at position 147 of the mutant amino acid disappears. In vivo experiments show that this mutation can significantly inhibit the accumulation of calcium oxalate crystals in mouse kidneys, preventing the occurrence of kidney stones. Based on this, this invention provides a novel agent and pathway for the prevention and treatment of kidney stones.

Description

Technical Field

[0001] This invention relates to the field of kidney stone diagnosis and prevention, and more particularly to the preparation of kidney stone prevention and treatment agents using ALDOB K147 mutants and their applications. Background Technology

[0002] Kidney stones are a common and frequently occurring disease in urology, with an increasing incidence rate worldwide. Kidney stones primarily affect young adults aged 30-50, with a recurrence rate as high as 50%. Among all types of kidney stones, calcium oxalate (CaOx) stones are the most common, accounting for approximately 80%. Kidney stones seriously impact patients' health and quality of life, and have become a significant public health issue that cannot be ignored.

[0003] Lactation, a widespread and important type of post-translational modification of proteins, plays a crucial role in disease development and immune regulation. Under normal physiological conditions, the concentration of lactate in blood and tissues is 1.5–3.0 mmol / L. Due to increased glycolysis caused by hypoxia, lactate concentrations can reach as high as 10 mmol / L. In some diseased tissues, lactate concentrations can even reach 20–30 mmol / L. The degree of protein lactation is inextricably linked to anaerobic glycolysis in cells, playing a vital role in various life activities and disease processes, and pointing to a new direction for research in metabolism and immunity. In kidney stones, under conditions of crystal adhesion and oxidative stress caused by inflammation, pyruvate, an intermediate metabolite of glycolysis in renal epithelial cells, cannot enter the mitochondria for oxidation. Instead, it is converted to lactate by lactate dehydrogenase (LDH). However, whether (and how) lactation participates in mediating the formation of an inflammatory environment and thus promoting kidney stone formation remains poorly understood. Summary of the Invention

[0004] Our research found that the pathogenesis of kidney stones is accompanied by a significant increase in the lactation modification level of histidine at position 147 of ALDOB. Furthermore, when histidine at position 147 is mutated, the lactation modification of amino acid at position 147 of the mutant disappears. In vivo experiments showed that this mutation can significantly inhibit the accumulation of calcium oxalate crystals in the kidneys of mice and prevent the occurrence of kidney stones.

[0005] Based on the above findings, this invention provides the application of an ALDOB histidine 147 lactation modification level detection agent in the preparation of a kidney stone diagnostic agent. The protein reference sequence of ALDOB is shown in SEQ ID NO: 1.

[0006] The present invention also provides the application of a site-directed mutagen of the ALDOB gene in the preparation of a drug for the prevention and treatment of kidney stones. The site-directed mutagen of the ALDOB gene is a mutagen that induces a point mutation at histidine 147 of the ALDOB gene. The protein reference sequence of ALDOB is shown in SEQ ID NO: 1.

[0007] The present invention also provides a drug for preventing and treating kidney stones, comprising a site-directed mutagen of the ALDOB gene, wherein the site-directed mutagen of the ALDOB gene is a mutagen that induces a point mutation at histidine 147 of the ALDOB gene, and the protein reference sequence of the ALDOB is shown in SEQ ID NO: 1.

[0008] In one specific embodiment, the site-directed mutagen induces the mutation of histidine at position 147 of ALDOB into arginine.

[0009] In one specific implementation, the site-directed mutagen is a Cre / loxP-based site-directed mutagen.

[0010] The present invention also provides an engineered renal epithelial cell in which the original ALDOB in the genome is knocked out and ALDOB with a point mutation at histidine 147 is expressed, the protein reference sequence of which is shown in SEQ ID NO: 1.

[0011] In one specific implementation, the 147th histidine of the expressed ALDOB is mutated to arginine or threonine. Attached Figure Description

[0012] Figure 1 Single-cell nuclear sequencing analysis of kidney tissue from a rat model of calcium oxalate kidney stones was performed. (A) shows HE staining and Von Kossa staining analysis of kidney tissue from rats with calcium oxalate kidney stones; (B) shows a single-cell sequencing t-SNE plot illustrating the major cell groups in the kidney tissues of the control and experimental groups; (C) shows a heatmap of expression of marker genes specifically expressed in each cell subpopulation; (D) shows a statistical graph of the proportion of each cell subpopulation in the kidney tissues of the control and experimental groups, with the red arrow indicating macrophages; (E and F) show inflammatory factors. Tnfa and Il1b Expression levels in macrophages of rat kidney tissue in the control and experimental groups; (G) indicates differentially expressed gene function enrichment analysis showed that the activity of glycolysis and inflammation-related signaling pathways in renal tubular epithelial cells was significantly upregulated. ***, P < 0.001.

[0013] Figure 2Lactomic analysis of proteins revealed a significantly elevated level of ALDOB K147 lactation modification in kidney stone tissue. The findings are as follows: (A) Metabolomics analysis of kidney tissue from the control and experimental groups; (B) Urine lactate levels in the control and treated groups; (C) Urine lactate levels in healthy individuals and kidney stone patients; (D) Western blot analysis of pan-lactic acidification modification levels in kidney tissue from the control and experimental groups; (E) Differential analysis of protein lactomic modification in kidney tissue from the control and experimental groups; the right figure shows the top five sites of differential lactation modification; (F) Mass spectrometry peptide mapping, indicating lactation modification at the ALDOB K147 site in the kidney tissue of the experimental group; (G) Quantitative mass spectrometry analysis of ALDOB K147 lactation modification levels in kidney tissue from the control and experimental groups; (H) Aldob expression levels in various cell subpopulations from single-cell sequencing results. **, P<0.01; ***, P<0.001.

[0014] Figure 3 A specific antibody against ALDOB K147 was used to confirm a significant upregulation of lactation modification at this site in the kidney stone model. (A) shows the preparation of an anti-ALDOB K147la antibody, which can specifically detect the lactated ALDOB K147la peptide; (B) shows Western blot results indicating that the antibody can specifically recognize the ALDOB K147la site in HK-2 (human renal epithelium) and TCMK-1 (mouse renal epithelium) cells; (C) shows a point mutation experiment confirming that K147 is the major lactation site of ALDOB; (D) shows the ALDOB K147 lactation modification level in the kidney tissues of control and experimental rats detected by Western blot; (E) shows the levels of ALDOB and ALDOB K147la in the kidney tissues of control and experimental rats detected by immunofluorescence; and (F) shows the ALDOB K147la level in HK-2 cells treated with CaOx to simulate an in vitro kidney stone model via Western blot.

[0015] Figure 4Lactation modification of ALDOB K147 in renal tubular epithelial cells promotes the formation of kidney stones and inflammatory activation of macrophages. Among them, (A) is a schematic diagram of the construction of the Cre-AldobK147R conditional point mutation genetically engineered mouse model; (B) shows the calcium oxalate kidney stone models prepared using Cre-AldobK147R mice and WT mice, respectively, and the comparison of kidney stone crystal formation between the two groups by Von kossastaining; the right figure is a statistical graph of staining positivity; (C) shows the difference in macrophage infiltration in the kidney tissue of the two groups of kidney stone models analyzed by CD68 immunofluorescence staining; (D) shows the expression of TNF-α and IL-β genes in macrophages sorted by flow cytometry in the kidney tissue of the two groups of kidney stone models by Real-time PCR; (E) shows the reintroduction of wild-type and ALDOB K147R point mutations in ALDOB knockout HK-2 cells, respectively, and the detection of ALDOB expression by Western blot, confirming successful reintroduction; (F) shows the treatment of wild-type and ALDOB cells with CaOx. Transcriptome sequencing analysis of K147R cells revealed that the top five enriched pathways in HK-2 cells were significantly enhanced after inoculation with wild-type ALDOB compared to the K147R group. (G) indicates that macrophages treated with wild-type cell culture supernatant showed significantly upregulated expression of inflammatory factors compared to K147R cell culture supernatant. ***, P < 0.001. Detailed Implementation

[0016] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0017] 1. Single-cell nuclear sequencing reveals changes in cellular components and functional states in kidney stones. In our previous work, the project team established a rat model of kidney stones using 3-month-old male SD rats by administering 1% ethylene glycol in drinking water and 1% ammonium chloride via gavage for 14 days. After anesthesia, both kidneys were removed, and HE staining results showed that, compared to the normal control group, a large number of crystals were present in the kidney tissue sections of the treated rats, appearing dark brown under calcium salt staining conditions. Figure 1 A), indicating that the rat kidney stone animal model was successfully established.

[0018] To gain a deeper understanding of the functional characteristics of each cellular component in the animal model of kidney stones, we performed single-cell nuclear sequencing on the model and grouped these cells according to the expression of specific marker genes. Figure 1 BC). We found that macrophages were significantly enriched in the kidney stone model ( Figure 1 D), and functional analysis revealed that this group of cells highly expressed [D]. Il-1β , Tnfα Inflammatory molecules ( Figure 1E, F), suggesting that this cell group plays an important role in the formation of kidney stones. We also focused on the functional changes of renal epithelial cells during stone injury. Functional analysis revealed that their glycolysis and inflammation-related signaling pathways were significantly enhanced in the kidney tissue of the experimental group rats. Figure 1 G). The above results indicate that the kidney stone model showed increased infiltration of inflammatory macrophages, enhanced glycolysis and inflammatory response in renal tubular epithelial cells.

[0019] 2. Protein lactation modification proteomics revealed a significantly increased level of ALDOB K147 lactation modification in kidney stone tissue. Given the enhanced glycolysis in the kidney tissue of rats with calcium oxalate kidney stones, our team further investigated the differences in metabolites using metabolomics. We found that the levels of β-D-glucuronic acid, creatinine, and lactate in the kidney tissue of the stone-bearing rats were significantly higher than those in the normal control group. Figure 2 A). Consistent with this result, we found that lactic acid levels in the urine of both the rat model of kidney stones and patients with kidney stones were significantly higher than those in the control group (A). Figure 2 B, C).

[0020] Lactic acid, as a product of glycolysis, can provide lactyl groups to lactate proteins, thereby regulating their biological functions and playing an important physiological role. By extracting kidney tissue proteins from control and stone-forming rats and performing Western blotting with anti-pan-lactation antibodies, we found that the lactation modification level of proteins in the treated group was significantly increased. Figure 2 D). The project team further analyzed the changes in the proteomic lactation modification profile of kidney tissue in a rat kidney stone model using proteomic analysis. We found that the level of proteomic lactation modification was significantly increased in the stone group, with the most significant upregulation of lactation modification expression at the ALDOB K147 site. Figure 2 EG). Single-cell sequencing results indicate that ALDOB is mainly expressed in proximal tubular epithelial cells of the kidney tissue ( Figure 2 H). The above results indicate that with the increase of glycolysis level, the level of protein lactation modification in kidney stone tissue increases, with the most significant upregulation of ALDOB K147 lactation modification level.

[0021] 3. Preparation and validation of specific antibodies modified by lactation at the ALDOB K147 site. To further investigate the role of ALDOB K147 lactation modification in renal epithelial cells, we prepared an ALDOB K147 lactation modification-specific antibody (…). Figure 3 A). K147 lactation modification disappeared after ALDOB knockout, verifying the specificity of this antibody. Figure 3 B).

[0022] We further overexpressed wild-type (WT) or mutant (K147R or K147T) ALDOB protein in HK-2 cells with ALDOB knockout, and found that ALDOB lactation modification disappeared after K147 was mutated to R or T, confirming that K147 is the main lactation modification site of ALDOB. Figure 3 C).

[0023] Using this antibody, protein blotting and immunofluorescence experiments confirmed that the lactation modification level at the ALDOB K147 site was significantly increased in an animal model of kidney stones, but the overall protein expression level did not change significantly. Figure 3 D, E). Consistent with in vivo experiments, in vitro experiments using CaOx to treat HK-2 cells revealed that although ALDOB protein levels did not change significantly, its lactation modification level increased significantly. Figure 3 F). The above experiments confirmed that the lactation level at the ALDOBK147 site was significantly increased in both in vivo and in vitro kidney stone models.

[0024] 4. Lactation modification of ALDOB K147 in renal tubular epithelial cells promotes kidney stone formation and inflammatory activation of macrophages. Next, we will verify the role of ALDOB K147 lactation modification in kidney stone formation. To answer this question, we constructed a [system / mechanism] using the Cre / loxP system. Aldob K147R Point mutation knock-in mice, through and Cdh16 -Cre mouse hybridization established specific renal epithelial cells Aldob Cre- Aldob K147R This point mutation can mimic delactation modification ( Figure 4 A). We are in Cre- Aldob K147R A kidney stone model was established in male mice and their littermate controls (wild-type male mice). We found that inhibiting ALDOB K147 lactation modification significantly inhibited the accumulation of calcium oxalate crystals in the mouse kidneys. Figure 4 B). Contrary to the aforementioned single-cell transcriptome sequencing data, immunofluorescence experiments showed Cre- Aldob K147R Reduced infiltrating macrophages in mice ( Figure 4 C); Flow cytometry sorting of macrophages revealed a significant reduction in the expression of inflammatory factors such as IL-1β and TNF-α. Figure 4 D).

[0025] We further confirmed using in vitro experiments that ALDOB K147 lactation modification in renal epithelial cells has a pro-inflammatory phenotype. We reintroduced wild-type and ALDOB K147R point mutations into ALDOB knockout HK-2 cells, respectively. Figure 4 E), followed by CaOx treatment to construct a kidney stone cell model. Transcriptome sequencing results showed that compared with the K147R group, HK-2 cells showed significantly increased inflammatory signaling pathway activity after inoculation with wild-type ALDOB (E). Figure 4 F), and the expression of inflammatory factors was significantly upregulated after macrophages were treated with wild-type cell culture supernatant. Figure 4 G). The above in vitro and in vivo results indicate that ALDOB K147 lactation modification plays an important role in the occurrence of kidney stones and the recruitment of macrophages for inflammatory activation.

[0026] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. 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 ALDOB histidine 147 lactation modification level detection agent in the preparation of kidney stone diagnostic agent. The protein reference sequence of ALDOB is shown in SEQ ID NO:

1.

2. The application of a site-directed mutagen of the ALDOB gene in the preparation of a drug for the prevention and treatment of kidney stones, wherein the site-directed mutagen of the ALDOB gene is a mutagen that induces a point mutation in histidine at position 147 of the ALDOB gene, and the protein reference sequence of the ALDOB is shown in SEQ ID NO:

1.

3. A medicine for preventing and treating kidney stones, characterized in that, The invention includes a site-directed mutagen for the ALDOB gene, wherein the site-directed mutagen for the ALDOB gene is a mutagen that induces a point mutation at histidine residue 147 of the ALDOB gene, and the protein reference sequence of the ALDOB is shown in SEQ ID NO:

1.

4. The application according to claim 2 or the medicament for preventing and treating kidney stones according to claim 3, characterized in that, The site-directed mutagen induces the mutation of histidine at position 147 of ALDOB into arginine or threonine.

5. The application according to claim 2 or the agent for preventing and treating kidney stones according to claim 3, characterized in that, The site-directed mutagen is a site-directed mutagen based on the Cre / loxP system.

6. An engineered renal epithelial cell, characterized in that, The original ALDOB in the renal epithelial cell genome was knocked out, and ALDOB with a point mutation at histidine 147 was expressed. The protein reference sequence of the ALDOB is shown in SEQ ID NO:

1.

7. The engineered renal epithelial cells according to claim 6, characterized in that, The 147th histidine of the expressed ALDOB is mutated to arginine or threonine.

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