CN114134222B - Lupus nephritis diagnosis marker and application thereof - Google Patents

Lupus nephritis diagnosis marker and application thereof Download PDF

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CN114134222B
CN114134222B CN202111310527.0A CN202111310527A CN114134222B CN 114134222 B CN114134222 B CN 114134222B CN 202111310527 A CN202111310527 A CN 202111310527A CN 114134222 B CN114134222 B CN 114134222B
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protein
lupus nephritis
proteins
ndufa4
col6a2
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CN114134222A (en
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戴勇
郑凤屏
汤冬娥
张欣洲
蔡晚霞
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Shenzhen Linyan Medical Co ltd
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Abstract

The invention discloses a lupus nephritis diagnosis marker and application thereof. In a first aspect of the invention, the invention provides the application of a reagent for quantitatively detecting at least one marker in C8b, NDUFA4 and COL6A2 in the preparation of products for diagnosis, disease monitoring and prognosis judgment of lupus nephritis. According to the application of the embodiment of the application, at least the following beneficial effects are achieved: the trace kidney tissue samples of different parts are obtained by the laser capturing micro-cutting technology, so that the markers which are differentially expressed in various kidney tissues are screened out and used as the diagnosis markers of lupus nephritis, the defect of the existing lupus nephritis diagnosis indexes is overcome, and the method has good clinical diagnosis value.

Description

Lupus nephritis diagnosis marker and application thereof
Technical Field
The application relates to the technical field of lupus nephritis diagnosis, in particular to a lupus nephritis diagnosis marker and application thereof.
Background
Systemic Lupus Erythematosus (SLE) is a systemic, systemic disease that can damage various organs of the body, and when lupus immune complexes reach the kidneys, lupus Nephritis (LN) can result. LN can be classified as a disease of glomerulonephritis, which is also a major risk factor for overall morbidity and mortality of SLE. Although potent anti-inflammatory and immunosuppressive therapies may be employed for LN, the prognosis for most patients will still end up with chronic long-term renal disease (Chronic Kidney Disease, CKD) or end-stage renal disease (End Stage Renal Disease, ESRD).
The main means of diagnosis of LN clinically today are kidney biopsies, which are the gold standard for LN diagnosis, and serological examinations. LNs are currently classified as type 6 (I-VI). According to literature reports, prognosis of LN is related to diagnosis time. In other words, early diagnosis of early treatment, LN prognosis is better. The patient should immediately take a kidney biopsy when they have significant clinical symptoms of nephritis to expedite treatment decisions and minimize the risk of irreversible kidney damage due to inflammation, which also suggests an important role for LN for early diagnosis.
Finding new diagnostic markers for LN that can replace kidney biopsies is one of the current research hotspots for LN. With the progress of new research techniques, the development of research for searching LN-specific diagnostic biomarkers in blood or urine has been driven by the research of LN in the group of transcriptomics, proteomics, etc. Therefore, there is a need to find more effective LN diagnostic markers by means of the above-mentioned means.
Disclosure of Invention
The present application aims to solve at least one of the technical problems existing in the prior art. Therefore, the application provides lupus nephritis markers with good diagnostic value, and the reagent for quantitatively detecting the markers can be used for diagnosis or prognosis judgment of lupus nephritis.
In a first aspect of the application, the application of a reagent for quantitatively detecting at least one marker in C8b, NDUFA4 and COL6A2 in preparation of a product for diagnosis, monitoring or prognosis of lupus nephritis is provided.
According to the application of the embodiment of the application, at least the following beneficial effects are achieved:
the trace kidney tissue samples of different parts are obtained by the laser capturing micro-cutting technology, so that the markers which are differentially expressed in various kidney tissues are screened out and used as the diagnosis markers of lupus nephritis, the defect of the existing lupus nephritis diagnosis indexes is overcome, and the method has good clinical diagnosis value.
Wherein C8b (complete C8 Beta Chain) is the Beta Chain of Complement component 8. C8 consists of α, β and γ subunits, which are one component of the membrane attack complex, mediate cell lysis, and initiate membrane permeation of the complex.
NDUFA4 (NDUFA 4 Mitochondrial Complex Associated) is a protein belonging to the complex I9 KDA subunit family. This complex I of the mammalian mitochondrial respiratory chain consists of 45 different subunits. The protein has NADH dehydrogenase activity and oxidoreductase activity.
COL6A2 (Collagen Type VI Alpha Chain) is a collagen type VI α2Chain, a beaded silk collagen that can be found in most connective tissues. The collagen has important interactions in the tissue matrix components.
In some embodiments of the present application, the reagents quantitatively detect at least two of them, including C8b and NDUFA4, C8b and COL6A2, NDUFA4 and COL6A2, and all three markers.
In some embodiments of the present application, the agent quantitatively detects the marker at the gene level or protein level. The quantitative detection of nucleic acid at the gene level is carried out by methods including, but not limited to, polymerase Chain Reaction (PCR), isothermal amplification reaction (e.g., loop-mediated isothermal amplification LAMP, recombinase polymerase amplification RPA, etc.), probe hybridization technique, RNA blotting, etc. Reagents for quantitative detection of markers at the protein level are performed by methods including, but not limited to, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (IRA), immunohistochemical staining, western blot, electrophoresis, liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), and the like.
In some embodiments of the present application, the reagent for quantitatively detecting at least one marker of C8b, NDUFA4, COL6A2 at the gene level is selected from the group consisting of primers, probes, and gene chips. Wherein, the primer refers to a primer capable of specifically amplifying C8b, NDUFA4 and COL6A2 genes, the probe refers to a probe capable of specifically recognizing C8b, NDUFA4 and COL6A2 genes or transcripts of the genes, and the gene chip refers to a composite structure formed by immobilizing an array of the probes on a substrate material (specifically including but not limited to a polymer such as nylon membrane, nitrocellulose membrane, glass and the like).
In some embodiments of the present application, the reagent that quantitatively detects the marker at the protein level is an antibody. The antibody is an antibody capable of specifically recognizing at least one marker protein of C8b, NDUFA4 and COL6A2, and specifically comprises at least one of a monoclonal antibody and a polyclonal antibody.
In some embodiments of the present application, the reagent quantitatively detects at least one of C8b in the glomerular sample, NDUFA4 in the tubular sample, COL6A2 in the renal interstitial sample at the protein level.
In some embodiments of the present application, the lupus nephritis is diagnosed when at least one of the expression level of C8b protein in the glomerular tissue sample is up-regulated relative to normal, the expression level of NDUFA4 protein in the glomerular tissue sample is down-regulated relative to normal, and the expression level of COL6A2 protein in the interstitial tissue sample is up-regulated relative to normal. In some embodiments, when two or three of the above conditions occur, a diagnosis is made as having lupus nephritis. The level of up-and-down regulation relative to a normal person refers to a level of up-regulation determined by a level of expression of a marker in a sample (e.g., a normal person sample) satisfying a clinical diagnosis-related criterion not suffering from lupus nephritis, and a level of expression of a marker in a sample of a subject above a certain threshold is determined by up-regulation determined by a level below a certain threshold.
In some embodiments of the present application, the disease progression of lupus nephritis in a patient is monitored or prognosis is determined by monitoring changes in the expression levels of C8b protein, NDUFA4 protein, COL6A2 protein.
In some embodiments of the present application, diagnosing includes diagnosing at least one of pathological grading, activity (AI), chronicity (CI) of lupus nephritis.
Lupus nephritis is mainly classified into six types, and is classified by kidney pathology type, specifically, as follows, type I: slight membranous lupus nephritis, normal glomerulus under light microscope, but immunofluorescence and/or electron microscopy visible membranous region immune complex deposition. Type II: the glistening nephritis is characterized in that the proliferation of simple glistening cells or the increase of glistening matrix can be seen under the light microscope, immune complex deposition in the companion membrane region; small amounts of subepithelial or subendothelial immune complex deposition were visible under immunofluorescence and electron microscopy. Type III: focal lupus nephritis, a sub-active (a) or inactive (C) pathology, is a focal (affected glomeruli < 50%) segmental or glomerular intraglomerular hyperplasia, membranous hyperplasia and moderately severe mesangial hyperplasia or with crescent formation, typical focal subendothelial immune complex deposition with or without mesangial changes. Type IV: diffuse lupus nephritis, active or inactive lesions. The diffuse glomerular capillary hyperplasia, membranous hyperplasia and moderately severe mesangial hyperplasia (with the affected glomerulus not less than 50%) or the crescent glomerular nephritis, with or without membranous lesion, are typical diffuse subendothelial immune complex deposition. V type: membranous lupus nephritis. Diffuse thickening of glomerular basement membrane is seen with diffuse or segmental deposition of subcutaneous immune complexes with or without membranous lesions. Type VI: severe sclerosis lupus nephritis. More than 90% of the glomeruli exhibit glomerulosclerosis and no active lesions remain. Clinical Activity (AI) and Chronic Index (CI) of lupus nephritis reflect to some extent the extent of kidney damage, AI and CI scores given by the National Institutes of Health (NIH) are currently widely used.
In a second aspect of the present application, there is provided a method of screening for a marker of lupus nephritis, the method comprising the steps of:
obtaining kidney tissue samples of different parts from the tissue slices by adopting a laser capturing micro-cutting technology; extracting peptide fragments from a kidney tissue sample; performing DIA quantitative detection on the peptide fragment; and carrying out bioinformatics analysis according to the detection result to obtain the differential expression protein and screening out the marker.
The screening method according to the embodiment of the application has at least the following beneficial effects:
the laser capture microdissection technique (LCM) is a technique that can cut any region of interest with the aid of a microscope in a short period of time (typically 1-1.5 hours) and can be accurate on a single cell, thus allowing for investigation of tissue space. Understanding the molecular and pathological changes in different regions of the kidney in kidney disease is a profound understanding. Thus, LCM was used to isolate glomeruli, tubules and renal interstitium in kidney tissues of LN patients and normal control groups, and proteomic studies were performed to screen appropriate markers by bioinformatic analysis.
In some embodiments of the present application, methods from differential expression of proteins to screening for markers include, but are not limited to, using specific expression of differential expression proteins in collected kidney tissue or other samples by a suitable algorithm with modeling of whether the person from whom the sample was derived is ill, and the specific method may be replaced with a linear regression method or other supervised machine learning nonlinear algorithm.
In some embodiments of the present application, the different kidney tissue samples include at least one of a kidney glomerular tissue sample, a kidney tubule tissue sample, a kidney interstitium tissue sample.
In a third aspect of the present application, there is provided a method of screening a drug for treating lupus nephritis, the method comprising the steps of: the medicine to be screened acts on the lupus nephritis model, the expression quantity of at least one marker in C8b, NDUFA4 and COL6A2 is detected, and the medicine with obvious treatment effect on the lupus nephritis is screened according to the change condition of the expression quantity.
Additional aspects and advantages of the application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the application.
Drawings
FIG. 1 is a Metascape result of glomeruli participating in proteins of the complement system in example 1 of the present application, wherein A is a histogram of biological processes, B is a network interaction map of biological processes, and C is a protein network interaction map.
FIG. 2 is a Metascape result of a tubular metabolic pathway protein in example 1 of the present application, wherein A is a histogram of biological processes, B is a network interaction map of biological processes, and C is a protein network interaction map.
FIG. 3 is Metascape results for ECM receptor related proteins of the renal interstitial tissue in example 1 of the present application, wherein A is a histogram of biological processes, B is a network interaction graph of biological processes, and C is a protein network interaction graph.
FIG. 4 shows the results of the immunohistochemical analysis of C8B in example 2 of the present application, A is the staining pattern at different magnification, and B is the IRS-score comparison result.
FIG. 5 shows the results of an immunohistochemical experiment of NDUFA4 in example 2 of the present application, A is a staining chart at different magnification, and B is the result of IRS-score comparison.
FIG. 6 shows the results of immunohistochemical experiments on COL6A2 in example 2 of the present application, A is a staining chart at different magnification, and B is the result of IRS-score comparison.
FIG. 7 is a ROC curve of C8b, NDUFA4 and COL6A2 as LN biomarker for biological diagnosis in example 2 of the present application, A-C represent ROC curves of C8b, NDUFA4 and COL6A2 distinguishing between light and moderate severity activity indices, and D-F represent ROC curves of C8b, NDUFA4 and COL6A2 distinguishing between light and moderate severity chronicity indices.
Fig. 8 is a ROC curve of the combination of C8b, NDUFA4 and COL6A2 as LN biomarker in example 2 of the present application, a-C being any two combinations to distinguish between light and moderate and severe activity indices, D being three combinations to distinguish between light and moderate and severe activity indices.
Fig. 9 is a ROC curve of the combination of C8b, NDUFA4 and COL6A2 as LN biomarker in example 2 of the present application, a-C being ROC curves of any two combinations to distinguish between mild and moderate chronic indices, D being ROC curves of three combinations to distinguish between mild and moderate chronic indices.
Detailed Description
The conception and technical effects produced by the present application will be clearly and completely described below in connection with the embodiments to fully understand the objects, features and effects of the present application. It is apparent that the described embodiments are only some embodiments of the present application, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort based on the embodiments of the present application are within the scope of the present application.
The following detailed description of embodiments of the present application is exemplary and is provided merely for purposes of explanation and not to be construed as limiting the application.
In the description of the present application, the meaning of a number is one or more, the meaning of a number is two or more, and greater than, less than, exceeding, etc. are understood to exclude the present number, and the meaning of a number above, below, within, etc. are understood to include the present number. The description of the first and second is for the purpose of distinguishing between technical features only and should not be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated or implicitly indicating the precedence of the technical features indicated.
In the description of the present application, unless explicitly defined otherwise, terms such as arrangement, installation, connection, etc. should be construed broadly and the specific meaning of the terms in the present application can be reasonably determined by a person skilled in the art in combination with the specific contents of the technical solution.
In the description of the present application, a description with reference to the terms "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present application. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
Example 1: proteomics research
Experimental method
1. Determining a sample
Tissue sections of 21 LN kidney tissue samples and 11 normal human kidney tissue samples were taken together for laser microdissection and DIA protein quantification. Wherein the pathological diagnosis of the kidney puncture examination in the LN group accords with LN, and the pathological result is according to the revised standard of LN typing by the International society of Kidney/Kidney pathological society (ISN/RPS) in 2003. Inclusion criteria included: (1) age 16 to 55 years; (2) no immunosuppressant treatment or hormonal treatment; (3) no acute or chronic infectious diseases such as tuberculosis, etc.; (4) Has no other immune related diseases, such as dermatomyositis, ankylosing spondylitis, etc. Eliminating IgA nephropathy, purpura nephropathy, hepatitis B virus related nephropathy, diabetes mellitus nephropathy, acute glomerulonephritis, chronic glomerulonephritis, drug related kidney injury, etc.
LCM sampling
Using the Leica LMD7000, germany, a laser capture microdissection system was used to find tissue with a 4-fold objective, after adjustment to a 10-fold objective, the glomeruli, tubules and tubules were each found by a computer screen, and then the required area of interest was circled on the computer display screen by a mouse, with the cutting energy set at 38, aperture set at 14, and speed set at 4. Then, the switch of the laser generator is pressed, the laser cuts along the drawn graph, the target cells and the surrounding tissue cells are separated, and the cut target cells fall into 200 mu l of PCR tube caps pre-arranged on the collector object stage under the action of gravity. This is repeated until the desired sample is cut. The cut samples were collected in PCR tubes and kept in dry ice for protein extraction and subsequent experiments.
3. Proteomics research
3.1 protein extraction
Centrifuging the sample tube for 10 minutes at 20000g at normal temperature; adding 20 microliters of 50mM ammonium bicarbonate aqueous solution containing Dithiothreitol (DTT) at a concentration of 10 mM; carrying out metal bath reaction for 30min at 95 ℃; immediately adding Iodoacetamide (IAM) after the temperature of the mixture is reduced to room temperature, so that the final concentration of the mixture is 50mM, and standing the mixture at room temperature in a dark place for 30min; the sample tube was placed in a water bath sonicator for 20 minutes.
3.2 proteolysis
Adding 0.2ug of Trypsin enzyme into each sample tube for enzymolysis; shaking and mixing for 30s, and performing enzymolysis in a water bath kettle at 37 ℃ for 14-16 h after short centrifugation.
3.3 desalting of peptide fragments
Activating: taking a new C18 column, passing 1mL of methanol through the column, and controlling the flow rate to be 3 drops/s;
balance: column chromatography with 1mL of 0.1% FA at a flow rate of 3 drops/s;
loading: diluting the protein liquid sample to 1mL by SDS-free L3, and allowing the flow rate to pass through a column for 1 drop/s;
washing: column chromatography with 1mL of 0.1% FA at a flow rate of 3 drops/s and repeated 3 times in total;
eluting: slowly eluting with 800L of 75% ACN at a flow rate of 0.5 drops/s;
and (5) pumping: the eluate was lyophilized.
3.4 high Performance liquid chromatography separation
The liquid phase of the sample is separated by using an Shimadzu LC-20AD liquid phase system, wherein a separation column is a 5 mu m 4.6x250mm Gemini C18 column. The experimental method is as follows, after mixing equal amounts of peptide fragments from all samples, diluting with 5% Acetonitrile (ACN) and injecting sample, and gradient eluting with a flow rate of 1 mL/min, wherein mobile phase B is 95% ACN, and the elution flow is as follows: 5% mobile phase B10 min,5% to 35% mobile phase B40 min,35% to 95% mobile phase B1min, mobile phase B lasting 3min,5% mobile phase B equilibrated for 10min. The elution peak was monitored at 214nm and one component was collected every minute, and 10 components were obtained by combining the samples with the chromatogram elution peak pattern, followed by freeze-drying.
3.5DIA quantitative detection
The pumped peptide sample is redissolved by mobile phase A (2% ACN,0.1% Formic Acid (FA)), centrifuged for 10min with 20000g, and the supernatant is sampled and separated by high performance liquid chromatography. The sample was first run into a trap column for enrichment and desalting, and then serially connected to a C18 column (150 μm inside diameter, 1.8 μm column size, about 35cm column length) for separation at a 500nL/min flow rate by the following effective gradient: 0-5 min,5% mobile phase B (98% ACN,0.1% FA); 5-130 min, and linearly increasing the mobile phase B from 5% to 25%; 130-150 min, the mobile phase B rises from 25% to 35%; 150-160 min, the mobile phase B rises from 35% to 80%; 160-175 min,80% mobile phase B; 175-175.5 min, the mobile phase B is reduced from 80% to 5%; 175.5-180 min,5% mobile phase B. The tail end of the high performance liquid phase separation is directly connected with a mass spectrometer and is detected.
3.5.1 library building detection
The liquid phase separated peptide was ionized by the nano ESI source and then entered into a tandem mass spectrometer for data dependent acquisition (Data Dependent Acquisition, DDA) mode detection. Main parameter setting: the ion source voltage is set to 2kV; the scanning range of the primary mass spectrum is 350-1,500 m/z; resolution was set to 60000 and the maximum ion implantation time (MIT) was 50ms; the secondary mass spectrum fragmentation mode is HCD, and the fragmentation energy is set to 30; the resolution was set to 15,000, the maximum ion implantation time (MIT) was 50ms, and the dynamic exclusion time was set to 30s. The initial m/z of the secondary mass spectrum is fixed to be 100; the screening conditions of the secondary fragmentation parent ions are as follows: charge 2+ to 6+, parent ion with peak intensity exceeding the intensity of 2E4 row 30. The AGC is set to: primary 1E5, secondary 2E4.
3.5.2DIA Mass Spectrometry detection
The peptide fragments after liquid phase separation are ionized by a nano ESI source and then enter a tandem mass spectrometer for DIA mode detection. Main parameter setting: the ion source voltage is set to 2kV; the scanning range of the primary mass spectrum is 400-1500 m/z; resolution is set to 60000; the maximum ion implantation time (MIT) is 50ms; dividing 400-1500m/z into 44 windows for continuous window fragmentation and signal acquisition. The ion fragmentation mode is HCD, the maximum ion implantation time is 54ms, fragment ions are detected in Orbitrap, the resolution is set to 30000, and the fragmentation energy is 30; AGC is set to 5E4.
4. Bioinformatics analysis
The DDA data of the off-line machine was identified using a Max Quant-integrated Andromeda engine, and then a profile library was built using the results. For large-scale DIA data, the m Prophet algorithm is used for completing analysis quality control of the data, so that a large number of reliable quantitative results are obtained.
4.1 database selection
The database is selected from the UniProt protein database (https:// www.uniprot.org /), and UniProt is the most informative and widely available protein database. It is formed by integrating the data of three databases of Swiss-Prot, trEMBL and PIR-PSD.
4.2DDA data analysis
Identification of DDA data was accomplished using Max Quant as a spectral library for subsequent DIA analysis. During operation, the original machine-down data is used as an input file, corresponding parameters and a database are configured, and then identification and quantitative analysis are carried out. Wherein identification information satisfying FDR < 1% will be used to build the final spectral library.
4.3DIA data analysis
The DIA data of the off-line machine is corrected for retention time (retention time) by using peptide fragments. And then, based on a Target-decoy model applicable to SWATH-MS, performing false positive control with FDR less than or equal to 1%, thereby obtaining a remarkable quantitative result.
4.4MSstats variance analysis
According to the set comparison group, MSstats are adopted to preprocess the data, and then the significance test is carried out based on the model. Thereafter, differential protein screening was performed according to fold difference >1.5 and P value <0.05 as judgment criteria for significant differences. And simultaneously, enrichment analysis is completed on the differential protein.
4.5 annotation analysis
Proteins were analyzed by GO, KOG, KEGG Pathway, PPI annotation analysis, subcellular localization analysis, metacape analysis, etc.
Experimental results
5.1 identification of peptide fragments and proteins
After collection of experimental samples of LCM, proteomic studies were performed on kidney regions. The DIA mode was used to obtain MS data, one sample was removed to identify 0, and a total of 49658 peptides, 4056 proteins, were identified in the remaining 95 samples. In addition, the results show that most samples identify more than 500 proteins, suggesting that the DIA protein identification technology has a certain depth. While some samples contain only tens of proteins, it is speculated that the possible reasons for the small number of proteins identified in the partial region are that the sample itself is expressed in a small amount or that degradation occurs during the protein extraction process.
5.2 differential protein screening
After obtaining the proteins in each region of each sample, we screened for differential proteins at Fold Change (FC) >1.5, P <0.05, and experimental results showed that 478 differentially expressed proteins were identified in total in the glomeruli in LN group, 167 proteins were highly expressed, 311 proteins were lowly expressed, and a total of 2383 proteins were not statistically different. 532 differentially expressed proteins were identified in the LN group kidney tubule, with 125 proteins having increased expression levels and 407 proteins having decreased expression levels, and 3153 proteins in total, were not statistically different. In LN group kidney interstitium 653 differentially expressed proteins were identified, 573 proteins were up-regulated, 80 proteins were down-regulated, 1330 proteins were not statistically different.
5.2.1 glomerular differential protein analysis
GO enrichment analysis was performed on these 478 glomerular differential proteins. The results showed that the most differential proteins involved in the biological process were cellular processes (418). The most diverse protein-involved cellular components are cells (cell) and cell regions (cell part), both of which are 428. The most diverse proteins are involved in the molecular function of binding, 361. The cellular regions that showed the most protein localization at the cell sub-localization results were cytoplasmic, followed by nuclei, respectively, and the results indicated that glomerular differential proteins function mainly in the cytoplasm and nuclei of glomerular cells.
KOG annotation results indicated that the differential proteins in the glomeruli are mainly involved in cellular processes and signal transduction (cellular processes and signaling), with 389 proteins, 102 of which are involved in the signal transduction mechanism (signal transduction mechanisms). The KEGG pathway analysis showed that glomerular differential proteins are involved in LN pathogenesis via complement system (complement and coagulation cascades), SLE, PI3K-Akt, and the Hippo signaling pathway. Abnormal activation of the complement system is closely related to the immune response of the body. Thus, differential proteins in the glomeruli can be involved in the disease process of LN through the complement system.
5.2.2 analysis of tubular differential protein.
GO enrichment analysis was performed on these 532 tubular differential proteins. The results showed that 449 proteins were involved in cellular processes, 387 proteins were involved in metabolic processes, 488 proteins were involved in the formation of cellular components, and 362 proteins were involved in the binding function of molecular functions. Subcellular localization results, showing that the tubular differential protein is mainly concentrated in mitochondria and cytoplasm, of which 198 are present in mitochondria, suggest that the tubular differential protein performs biological functions in the mitochondria of cells. Taken together with the above results, it is speculated that the tubular differential protein is primarily associated with metabolism.
KOG annotation showed that 252 proteins were enriched on metabolic-related pathways, 97 of which were involved in energy metabolism (Energy production and conversion). There were 185 involved in cell processes and signal transduction (pink), with minimal differential protein enrichment on information storage and processing functions. The KEGG pathway analysis results show that the tubular differential protein is mainly involved in the kidney metabolic processes, such as amino acid metabolism, fatty acid metabolism, carbon metabolism, and the like. The above results further suggest that the differential proteins of the tubules are involved in the pathogenesis of LN mainly by regulating renal metabolism.
5.2.3 analysis of renal interstitial differentiation protein
GO enrichment analysis was performed on these 653 tubular-differentiated proteins. The results showed that 592 proteins were involved in cellular processes, 622 proteins were involved in cell formation, and 544 proteins performed a binding function. Cell sublocalization results showed 247 proteins localized to the cytoplasm, well above 131 on the nucleus.
KOG annotation results indicated that 386 proteins were involved in cellular processes and signal transduction, of which 98 were involved in protein post-translational modifications, protein turnover and chaperones. The KEGG channel analysis result shows that the renal interstitial differential protein participates in a plurality of signal transduction channels, including SLE, PI3K-AKT and other signal channels. Wherein unlike glomerular and tubular protein differential proteins, interactions of extracellular matrix receptors are specific in the renal interstitial KEGG enrichment pathway (ECM receptor interaction). In conclusion, the renal interstitial differentiation protein can be involved in the progress of LN through the interaction of extracellular matrix receptors. Differential proteins in the renal interstitium may be associated with renal fibrosis, while demonstrating that different regions of the kidney may be involved in the pathogenesis of LN through different pathways and actions.
5.3Metascape analysis results
The interaction between proteins in the glomerular complement system pathway, the tubular metabolic pathway, and the renal interstitial extracellular matrix receptor interaction pathway was analyzed using metacape.
The metacape results of glomeruli involved in proteins of the complement system are shown in figure 1, the main biological process of these proteins is the regulation of the complement cascade (regulation of complement cascades), and interactions between these proteins exist. Based on the MCODE algorithm, there is a close interaction of complement molecules C5, C6, C8A, C8B, C G and C9. These complement molecules are suggested to influence the pathogenesis of LN that the complement system participates by modulating the complement cascade. Thus, it is possible to use it as a diagnostic marker for lupus nephritis.
The metacape results for the tubular metabolic pathway proteins are shown in fig. 2, which are involved in metabolic processes in kidney tissue, primarily by participation in the oxidative phosphorylation system in mitochondria (OXPHOS system in mitochondria). PPI results show that there is a close correlation between ubiquitin oxidoreductase core subunit (NDUF) family molecules, which are key molecules for the formation of mitochondrial complexes. The above results further suggest that the family of ubiquitin oxidoreductase subunits (including NDUFA2, NDUFA4, NDUFB1, NDUFB5, NDUFS3, NDUFS4, NDUFS 7) in tubular differential proteins may influence metabolism by oxidative phosphorylation of the cellular mitochondrial system, thereby participating in disease progression of LN. Thus, it is possible to use it as a diagnostic marker for lupus nephritis.
Metascape results for ECM receptor associated proteins of the renal interstitium as shown in FIG. 3, these proteins can affect kidney fibrosis by affecting extracellular matrix proteoglycans (ECM proteoglycans) to affect extracellular matrix receptor interactions. The PPI results show that among the proteins interacting with extracellular matrix, collagen COL1A1, COL2A1, COL1A2, COL4A2, COL6A2, and COL6A3 have close effects. These results suggest that we may have an important role for these collagens in kidney fibrosis. Thus, it is possible to use it as a diagnostic marker for lupus nephritis.
Example 2: immune group test
To verify the reliability of protein identification and to explore the potential of some proteins as biological diagnostic markers, IHC was used to verify the expression of the proteins. In glomerular differential proteins, protein C8b, log thereof, is selected from complement molecular proteins involved in the complement system based on GO enrichment analysis, KEGG pathway analysis, protein-protein interactions, and the like 2 FC is 2.71, p=0.005, and the biological process in which C8b participates is to regulate activation of the complement system and immune response.
Among the proteins differentially expressed in the tubular, NDUFA4 was screened from the ubiquitin oxidoreductase subunit family involved in cellular oxidative phosphorylation in the metabolic pathway based on GO enrichment analysis, KEGG pathway analysis, and protein-protein interactions, etc., and proteomic results showed that NDUFA4 was a protein with low expression in LN patients. Log of it 2 FC is-2.11, p=0.003.
In proteins differentially expressed in renal interstitium, col6A2 was selected from the collagen family based on GO enrichment analysis, KEGG pathway analysis, and protein-protein interactions, log thereof 2 FC is 4.06, P<0.001。
The experimental method comprises the following steps:
paraffin-embedded tissues were cut to a thickness of 4 μm and placed on a slide, dried and stored for later use.
The method steps for detecting the three proteins in the tissue by the immunohistochemical staining method are briefly described as follows:
1) Dewaxing, namely dewaxing in dimethylbenzene for three times, wherein each time lasts for 5 minutes;
2) Dehydration, alcohol gradient dehydration (100% alcohol-95% alcohol 2 times-70% alcohol);
3) Washing, namely washing for 5 minutes by using Milli-Q pure water;
4) Antigen retrieval, namely boiling and repairing for 20 minutes in a microwave oven by using sodium citrate buffer solution;
5) Washing with Milli-Q pure water for 5 minutes followed by PBS for 5 minutes;
6) Incubation with 2.5% horse serum reduced non-specific background;
7) Primary antibody was diluted with horse serum, incubated overnight at 4 degrees, three antibody dilutions were all 1:100;
8) The next day, wash 3 times with PBS for 5 minutes each;
9) Incubation with 3% hydrogen peroxide for 5min reduced non-specific background staining by endogenous peroxidase;
10 3 washes with PBS buffer for 5 minutes each;
11 Secondary antibody incubation for 30min;
12 3 washes with PBS buffer for 5 minutes each;
13 DAB color development;
14 Rinsing, namely rinsing continuously after 2 minutes of Mayor's hematrixylene counterstaining;
15 Dehydrated, 95% alcohol 2 times, 100% alcohol 2 times;
16 Dry and seal with DPX.
2.5 immune response scoring
And photographing and preserving the tissue after sealing the tissue under a microscope. Immunohistochemical scoring methods (The Immunoreactive Score, IRS) refer to the existing literature and invite two pathologists completely blinded to this experimental design to do, ensuring the accuracy of the scoring. The scoring criteria table is shown in table 1 below.
TABLE 1 IRS scoring criteria Table
Clinical data and IRS are expressed as mean±sd. The IRS mean comparison for C8b, NDUFA4 and COL6A2 was tested using the Mann-Whitney U test, since the numbers of LN groups and control groups were not identical. Correlation analysis of IHC scores and clinical data for C8b, NDUFA4 and COL6A2 uses a spline correlation analysis and calculates correlation coefficients. The diagnostic and differential pathotyping potential of C8b, NDUFA4 and COL6A2 were calculated using ROC curves. All statistics were analyzed using SPSS 22.0, and were considered statistically significant when P-values were less than 0.05.
In this example, 44 LN patients and 6 normal control samples were included. All LN patients were diagnosed by renal biopsy and clinical index.
The results of the C8B test are shown in fig. 4, a is a staining chart at different magnification, and B is an IRS score comparison. As can be seen from the figure, C8b was mainly expressed on glomeruli in LN group kidney tissue, and the expression level of C8b in LN group was significantly higher than that in normal control group (P < 0.05). This is similar to the proteomics results in example 1, confirming the reliability of the data.
The results of the NDUFA4 test are shown in FIG. 5, A is a staining chart at different magnification, and B is an IRS score comparison. As can be seen from the figure, NDUFA4 is mainly expressed on the tubular of kidney tissue, and the expression level of NDUFA4 in LN is significantly lower than that in the normal control group (P < 0.05). These results are consistent with the proteomics study in example 1, validating the results.
The detection results of COL6A2 are shown in FIG. 6, A is a staining chart under different magnification, and B is IRS scoring comparison. As can be seen from the figure, COL6A2 was mainly expressed on the renal tubules and renal interstitium of the kidney tissue, and COL6A2 was significantly higher in LN than in the control group (P < 0.05). These results are consistent with the proteomics study in example 1, validating the results.
To initially investigate the potential of C8b, NDUFA4 and COL6A2 as LN biomarkers, ROC curve analysis was applied to investigate the potential of these three molecules as LN hierarchical diagnostic markers, LN activity index scores 1-2 were classified as mild, 3-9 as moderate, > 10 as severe, LN chronicity index 0-1 as mild, 2 as moderate, and > 3 as severe. The results are shown in fig. 7, where a-C represent ROC curves for C8b, NDUFA4 and COL6A2 to distinguish between light and moderate and heavy activity indices, D-F represent ROC curves for C8b, NDUFA4 and COL6A2 to distinguish between light and moderate severe chronicity indices, and the ROC curves are outside y=x. As can be seen from the graph, the area under ROC curve is less than 0.8, wherein the area under C8b predicted activity index curve is 0.606, the area under ndufa4 predicted activity index curve is 0.741, and the area under col6a2 predicted activity index curve is 0.603. The area under the C8b predicted chronicity index curve is 0.502, the area under the NDUFA4 predicted chronicity index curve is 0.504, and the area under the COL6A2 predicted chronicity index curve is 0.505. Therefore, these three proteins are of lower value as independent hierarchical diagnostic markers.
For this reason, regression analysis is performed on these protein combinations to make ROC curves for predicting activity, and the results are shown in fig. 8, where a to C are two combined ROC curves, and D is three combined ROC curves, where ROC curves are other than y=x. As can be seen from the graph, the area under the ROC curve of the two-by-two combinations of these proteins is 0.8-0.9, wherein the area under the C8b+NDUFA4 curve is 0.86, the area under the C8b+COL6A2 curve is 0.81, the area under the NDUFA4+COL6A2 curve is 0.858, and the area under the ROC curve of the three proteins is as high as 0.904. The above results indicate that these proteins, when combined, have potential as diagnostic biomarkers for LN pathology activity stratification.
Similarly, regression analysis of these protein combinations was performed to make ROC curves for prediction of chronicity, and the results are shown in fig. 9, where a to C are two-combination ROC curves, and D is three-combination ROC curves, where ROC curves are other than y=x. As can be seen from the figure, the area under the C8b+NDUFA4 curve is 0.732, the area under the C8b+COL6A2 curve is 0.832, the area under the NDUFA4+COL6A2 curve is 0.888, and the area under the three protein-bound ROC curve is 0.92.
Therefore, according to the expression level of the protein, two pathological parameters of chronic lupus nephritis and activity can be accurately reflected, so that the LN pathological change degree of a subject and the pathological change of the kidney are judged, and the purposes of monitoring the disease progress and predicting the disease prognosis are achieved.
Example 3
The embodiment provides a screening method of a drug for lupus nephritis, which comprises the following steps:
the medicine to be screened acts on lupus nephritis model;
detecting the expression level of at least one marker in C8b, NDUFA4 and COL6A 2;
and screening out the medicine according to the change condition of the expression quantity.
The combination of C8b, NDUFA4 and COL6A2 has potential as a biological diagnosis marker for LN pathological activity and chronicity grading, so that whether the drug to be screened plays a corresponding therapeutic role can be judged according to the expression condition of the related markers before and after the drug to be screened is used.
From the above results, it can be seen that C8b, NDUFA4 and COL6A2 can be used as diagnostic and prognostic markers for lupus nephritis patients, and have good diagnostic value.
The present application has been described in detail with reference to the embodiments, but the present application is not limited to the embodiments described above, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present application. Furthermore, embodiments of the present application and features of the embodiments may be combined with each other without conflict.

Claims (7)

1. Use of a reagent for quantitatively detecting a marker combination, C8b, NDUFA4 and COL6A2, in the preparation of a diagnostic or monitoring product for lupus nephritis.
2. The use according to claim 1, wherein the reagent quantitatively detects the marker at the gene level or protein level.
3. The use according to claim 2, wherein said reagent for quantitatively detecting said marker at the gene level is selected from the group consisting of primers, probes and gene chips.
4. The use according to claim 2, wherein the reagent for quantitatively detecting the marker at the protein level is an antibody.
5. The use of claim 2, wherein the reagent quantitatively detects C8b in glomerular samples, NDUFA4 in tubular samples, COL6A2 in renal interstitial samples at the protein level.
6. The use of claim 5, wherein the lupus nephritis is diagnosed when at least one of the expression level of C8b protein in the glomerular sample is up-regulated relative to normal, the expression level of NDUFA4 protein in the tubular sample is down-regulated relative to normal, and the expression level of COL6A2 protein in the interstitial sample is up-regulated relative to normal.
7. The use according to any one of claims 1 to 6, wherein said diagnosis comprises diagnosing at least one of pathological grading, mobility, chronicity of lupus nephritis.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103492590A (en) * 2011-02-22 2014-01-01 卡里斯生命科学卢森堡控股有限责任公司 Circulating biomarkers
CN106170700A (en) * 2014-01-20 2016-11-30 玛丽亚·阿黛尔·西尔维亚·丹尼杰奥派德4有限公司 Prediction in the therapeutic of lupus nephritis is followed up a case by regular visits to, the in vitro method diagnosing and monitoring
CN106324131A (en) * 2016-08-12 2017-01-11 中国人民解放军第四军医大学 Method for screening labeled protein of esophageal squamous cell carcinoma front LGIEN and HGIEN
KR20180033821A (en) * 2016-09-26 2018-04-04 울산대학교 산학협력단 A biomarker composition for diagnosis of lupus nephritis comprising Immunoglobulin binding protein 1

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012103455A1 (en) * 2011-01-28 2012-08-02 Pangea Biosciences, Inc. Biomarkers and their uses in cancer detection and therapy
ES2528672B1 (en) * 2013-07-10 2015-11-24 Consejo Superior De Investigaciones Científicas (Csic) Urinary neurophilin-1 (NRP-1) as a prognostic marker of nephritis and lupus nephritis
US20210104321A1 (en) * 2018-11-15 2021-04-08 Ampel Biosolutions, Llc Machine learning disease prediction and treatment prioritization

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103492590A (en) * 2011-02-22 2014-01-01 卡里斯生命科学卢森堡控股有限责任公司 Circulating biomarkers
CN106170700A (en) * 2014-01-20 2016-11-30 玛丽亚·阿黛尔·西尔维亚·丹尼杰奥派德4有限公司 Prediction in the therapeutic of lupus nephritis is followed up a case by regular visits to, the in vitro method diagnosing and monitoring
CN106324131A (en) * 2016-08-12 2017-01-11 中国人民解放军第四军医大学 Method for screening labeled protein of esophageal squamous cell carcinoma front LGIEN and HGIEN
KR20180033821A (en) * 2016-09-26 2018-04-04 울산대학교 산학협력단 A biomarker composition for diagnosis of lupus nephritis comprising Immunoglobulin binding protein 1

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Selective pharmacological inhibition of DDR1 prevents experimentally‑induced glomerulonephritis in prevention and therapeutic regime;Solange Moll等;Moll et al. J Transl Med;1-20 *
狼疮性肾炎生物标志物;张凯聪;张丽;梁馨苓;;肾脏病与透析肾移植杂志(05);69-73 *

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