Kidney stone prevention and treatment medicament prepared from LDHB K156 mutagenic agent and application

Abstract

According to the invention, a human renal cortex proximal tubule epithelial cell HK2-CaOx model and a rat kidney stone animal model are adopted, differential modified proteins are analyzed and verified through a protein modification omics technology, and key factors and key sites of lactic acid modification participating in regulation and control of kidney stone generation are identified; single cell sequencing is combined with space transcriptome analysis, identification and subdivision of kidney cell subgroups, and differential expression ligand and receptor pairs and key signal channels of macrophages and renal tubular epithelial cells are analyzed through cell communication. On the basis, the key function of the LDHB protein lactic acid modification site K156 in the kidney stone progress process is identified, and a new molecular marker and a treatment target are provided for early diagnosis of kidney stone and intervention of kidney stone.

Description

Technical Field

[0001] This invention relates to the field of kidney stone diagnosis and prevention, and more particularly to kidney stone prevention and treatment agents prepared by LDHB K156 mutagen and their applications. Background Technology

[0002] Kidney stones have a high incidence rate, seriously affecting patients' physical health and quality of life, and have become a significant public health issue that cannot be ignored. Kidney stones mainly affect young adults aged 30-50, with a recurrence rate as high as 50%. However, the molecular mechanisms of kidney stone formation and progression remain unclear, and research on these mechanisms is of great importance for the treatment and prevention of kidney stone disease.

[0003] Lactate dehydrogenase (LDH) is an NAD-dependent kinase with three subunits: LDHA, LDHB, and LDHC. It is a zinc-containing metalloprotein with a molecular weight of 135-140 kDa. LDH is a crucial oxidoreductase in glycolysis, reversibly catalyzing the oxidation of lactate to pyruvate, the final product of anaerobic glycolysis. LDH is widely distributed in human tissues, with the highest concentration in the kidneys, followed by cardiac and skeletal muscle. Clinically, LDH testing is commonly used to diagnose myocardial infarction, liver disease, and certain malignant tumors.

[0004] Studies have shown that knocking down LDH can alleviate primary type 1 hyperoxaluria and inhibit the progression of kidney stones. In a renal ischemia-reperfusion injury model, LDHB protein expression was significantly decreased. Single-cell RNA sequencing analysis showed that the number of cells expressing LDHB decreased after injury, but the average expression level of positive cells increased transiently after injury and then gradually decreased during the recovery period.

[0005] Currently, no relationship has been found between LDHB lactation and kidney stones. Summary of the Invention

[0006] This invention provides the application of a detection agent for the lactation modification level of histidine 156 of LDHB in the preparation of a diagnostic agent for kidney stones. The protein reference sequence of LDHB is shown in SEQ ID NO: 1.

[0007] The present invention also provides the application of a site-directed mutagen of the LDHB gene in the preparation of a drug for the prevention and treatment of kidney stones. The site-directed mutagen of the LDHB gene is a mutagen that induces a point mutation at histidine 156 of the LDHB gene. The protein reference sequence of the LDHB gene is shown in SEQ ID NO: 1.

[0008] The present invention also provides a drug for preventing and treating kidney stones, comprising a site-directed mutagen of the LDHB gene, wherein the site-directed mutagen of the LDHB gene is a mutagen that induces a point mutation at histidine 156 of the LDHB gene, and the protein reference sequence of the LDHB is shown in SEQ ID NO: 1.

[0009] In one specific embodiment, the site-directed mutagen induces the histidine at position 156 of the LDHB to be a site-directed mutagen for arginine.

[0010] The present invention also provides an engineered renal epithelial cell in which the original LDHB in the genome is knocked out and expresses LDHB with a point mutation at histidine 156, the protein reference sequence of which is shown in SEQ ID NO: 1.

[0011] In one specific implementation, the 156th histidine of the expressed LDHB is mutated to arginine or threonine.

[0012] This invention utilizes the HK2-CaOx model of human renal cortical proximal tubular epithelial cells and a rat model of kidney stones. Through proteomics analysis and validation of differentially modified proteins, it identifies key factors and sites involved in the regulation of kidney stone development through lactation modification. Single-cell sequencing combined with spatial transcriptomics analysis is employed to identify and subdivide renal cell subsets. Cell communication analysis is used to identify differentially expressed receptor-ligand pairs and key signaling pathways in macrophages and renal tubular epithelial cells. Based on this, we identified the crucial role of the K156 lactation modification site of the LDHB protein in the progression of kidney stones, providing a novel molecular marker and therapeutic target for the early diagnosis and intervention of kidney stones. Attached Figure Description

[0013] Figure 1 The results show the staining of kidney tissue in a rat model of calcium oxalate kidney stones. In this diagram, A represents HE staining; B represents Von-Kossa silver staining.

[0014] Figure 2 Lactation modification proteomics and candidate protein identification were performed. A represents the pan-lactic acidification modification level in kidney tissue of the control and stone groups; B represents the Pearson correlation coefficient of the samples; C represents the volcano plot of differentially expressed protein sites; and D represents differentially expressed lactation modification proteins.

[0015] Figure 3 This study validates key protein modification sites. A shows the lactation levels of candidate proteins in the kidney tissues of control and treatment rats (stone model group). B shows the characteristic peak of the Kla156 lactation site of LDHB.

[0016] Figure 4The data represent the overexpression and knockdown of LDHB protein. A represents the expression levels of endogenous and exogenous LDHB protein in various cell lines; B represents the knockdown of endogenous LDHB expression by LDHB shRNA; and C represents the effect of CaOx stimulation on LDHB protein expression in cells.

[0017] Figure 5 Lactation modification of LDHB K156 inhibits CD206 expression. A shows the decrease in CD206 expression after LDHB protein knockdown; B shows the CD206 expression level after transfection with LDHB, LDHB K156R, and LDHB K156T vectors; C shows the expression of CD206 in LDHB WT and K156R transfected cells as detected by flow cytometry.

[0018] Figure 6 Lactation modification of LDHB K156 in renal tubular epithelial cells promotes kidney stone formation and macrophage inflammatory activation. Where A represents... cdh16-ldhb K156R Schematic diagram of constructing a conditional point mutation genetically engineered mouse model; B represents the different methods used. cdh16-ldhb K156R mice and ldhb K156R -flox A mouse model of calcium oxalate kidney stones was established; the formation of kidney stone crystals in the two groups was compared by Von kossastaining; and the difference in macrophage infiltration in the renal tissue of the two kidney stone models was analyzed by CD206 immunofluorescence staining.

[0019] Figure 7 The IL-17 signaling pathway in macrophages is significantly activated upon lactate stimulation. In the diagram, A shows cell morphology; B shows the expression levels of CD206 and CD86 in cells; C shows the changes in gene expression levels in M0 macrophages induced by lactate stimulation; D shows the activation of the IL-17 signaling pathway induced by lactate stimulation; E represents the Top 20 GSEA pathway; and F represents the Top 20 KEGG pathway. Detailed Implementation

[0020] 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.

[0021] 1. Establishing a rat model of kidney stones In previous work, our team established a rat model of kidney stones using 3-month-old male SD rats via oral administration of 1% ethylene glycol and 1% ammonium chloride for 14 days. After CO2 anesthesia, the rats were euthanized by cervical dislocation, and both kidneys were quickly removed and fixed with 10% formalin buffer. The tissues were dehydrated, embedded in paraffin, and routinely sectioned and stained. HE staining results showed a large number of crystals present in the kidney tissue sections of the treated group rats. Figure 1 (A is indicated by the green arrow), the black part stained with calcium salts is the crystal. These results demonstrate that we have stably established a rat model of kidney stones.

[0022] Given that combined proteomics and transcriptomics analysis showed that differentially expressed genes are mostly involved in the regulation of metabolic, immune, and apoptosis-related pathways, we aimed to detect the proteomic level of kidney tissue from rats with kidney stones. We extracted proteins from kidney tissues of control and stone-forming rats and performed Western blotting with anti-pan-lactation antibodies, finding differences in the lactation modification levels of proteins between the treatment and control groups. Figure 2 A). Pearson's correlation coefficients were calculated for each pair of all samples, and the results showed that the differences between the control group and the treatment group were relatively significant. Figure 2 B). The above results indicate that protein lactation modification may be involved in regulating the occurrence and development of kidney stones.

[0023] Subsequently, we digested the extracted protease to obtain peptides, performed high-performance liquid chromatography (HPLC) fractionation and enrichment of modified peptides, and commissioned Hangzhou Jingjie Biotechnology Co., Ltd. to perform mass spectrometry analysis. Lactic acid modification proteomics data identified a total of 1221 lactation modification sites, corresponding to 463 proteins, of which 278 proteins were quantifiable. Figure 2 C). Differential site analysis, such as Figure 2 In group D, the expression levels of 7 lactation-modified proteins were downregulated and 4 were upregulated.

[0024] 2. Screening of key protein modification sites We used protein immunoprecipitation combined with Western blotting and tissue immunofluorescence to validate the results of mass spectrometry detection and bioinformatics analysis. The results showed that, consistent with proteomics findings, LDHB lactation modification levels were significantly increased in the kidneys of rats with kidney stones. Figure 3 A). Simultaneously, mass spectrometry also identified the characteristic peak of the Kla156 lactation site of LDHB (A). Figure 3 B).

[0025] In the study identifying all lactation-modified proteins, LDHB protein size (35-40 kDa) and Figure 2The protein bands in group A that showed significant differences were of uniform size. LDHB is a crucial oxidoreductase in glycolysis, reversibly catalyzing the oxidation of lactate to pyruvate; this enzyme is widely present in human tissues. In this study, we selected LDHB as a candidate protein to investigate how LDHB lactation mediates macrophage polarization in the regulation of kidney stone development.

[0026] We constructed the vectors LDHB wildtype, LDHB(K156R), LDHB(K156T), and an empty vector as negative controls. We transfected the expression vectors into HEK293 (human embryonic kidney cells), MDCK (NBL-2 canine kidney cells), and HK2 (human renal cortical proximal tubule epithelial cells) cells, and detected endogenous and overexpressed LDHB protein. Figure 4 A). We synthesized LDHB shRNA to knock down endogenous LDHB protein in cells ( Figure 4 B), and puromycin was used to screen for stable knockdown cell lines for subsequent experiments. Meanwhile, we co-incubated HK2 cells with 200 μg / ml CaOx for 24 h, and Western blotting showed no significant difference in LDHB protein levels (B). Figure 4 C).

[0027] 3. Lactation modification of LDHB K156 in renal tubular epithelial cells promotes kidney stone formation and inflammatory activation of macrophages. We knocked down LDHB protein in THP1 cells, then treated the cells with CaOx and examined the expression of inflammatory genes. The results showed that CD206 expression levels were reduced. Figure 5 A); Cells were transfected with LDHB, LDHB K156R, and LDHB K156T vectors, and then treated with CaOx. The expression of inflammatory genes was detected. Immunoblotting results showed that CD206 expression was increased in cells transfected with LDHB and decreased in cells transfected with LDHB K156T. Figure 5 B); similarly, flow cytometry results showed that CD206 expression levels in LDHB WT cells were higher than those in K156R cells (B). Figure 5 C).

[0028] We will further investigate the role of LDHB K156 lactation modification in kidney stone formation. To answer this question, we constructed a [presumably a specific assay / method] using the Cre / loxP system. ldhb K156R Point mutation knock-in mice, through and cdh16 -Cre mouse hybridization established specific renal epithelial cells ldhbA point mutation mouse model of lysine (K) to arginine (R) at position 156 of the gene ( cdh16-ldhb K156R This point mutation can mimic delactation modification ( Figure 6 A). We are cdh16- ldhb K156R Among male mice and their littermate wild males ( ldhb K156R -flox Constructing a kidney stone model ( Figure 6 B). We found that inhibiting LDHB K156 lactation significantly suppressed the accumulation of calcium oxalate crystals in mouse kidneys. Immunofluorescence assays showed... cdh16- ldhb K156R Increased M2 macrophage infiltration in mice ( Figure 6 C).

[0029] 4. Lactic acid stimulation induces activation of the IL-17 signaling pathway in macrophages. We induced THP1 cells to become M0 macrophages, transfected them with vectors (LDHB, LDHB K156R, LDHB K156T, and a control empty vector), and continued to add cytokines to induce their transformation into M1 / M2. After characterizing M1 and M2 macrophages, we performed transcriptome sequencing and differential proteome analysis on cells from each transfection group, finding that the IL-17 signaling pathway was highly activated (…). Figure 7 The results suggest that LDHB K156 lactation may regulate macrophage transformation by activating the IL-17 signaling pathway, thus affecting the progression of kidney stones.

[0030] 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 the detection agent for lactation modification level of histidine 156 of LDHB in the preparation of diagnostic agents for kidney stones. The protein reference sequence of LDHB is shown in SEQ ID NO:

1.

2. Application of a site-directed mutagen of the LDHB gene in the preparation of a drug for the prevention and treatment of kidney stones, wherein the site-directed mutagen of the LDHB gene is a mutagen that induces a point mutation in histidine at position 156 of the LDHB gene, and the protein reference sequence of the LDHB gene 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 LDHB gene, wherein the site-directed mutagen for the LDHB gene is a mutagen that induces a point mutation at histidine residue 156 of the LDHB gene, and the protein reference sequence of the LDHB 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 histidine at position 156 of the LDHB to be transformed 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 LDHB in the renal epithelial cell genome was knocked out, and LDHB with a point mutation at histidine 156 was expressed. The protein reference sequence of the LDHB is shown in SEQ ID NO:

1.

7. The engineered renal epithelial cells according to claim 6, characterized in that, The histidine at position 156 of the expressed LDHB is mutated to arginine or threonine.

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