CN114134222A - Diagnostic marker for lupus nephritis and application thereof - Google Patents

Diagnostic marker for lupus nephritis and application thereof Download PDF

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CN114134222A
CN114134222A CN202111310527.0A CN202111310527A CN114134222A CN 114134222 A CN114134222 A CN 114134222A CN 202111310527 A CN202111310527 A CN 202111310527A CN 114134222 A CN114134222 A CN 114134222A
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protein
lupus nephritis
marker
col6a2
sample
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CN114134222B (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 application of a reagent for quantitatively detecting at least one marker in C8b, NDUFA4 and COL6A2 in the preparation of products for diagnosing, monitoring and 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 application obtains the trace kidney tissue samples of different parts by the laser capture microdissection technology, thereby screening out the markers differentially expressed in various kidney tissues to be used as the diagnostic markers of the lupus nephritis, making up the defects of the existing lupus nephritis diagnostic indexes, and having good clinical diagnostic value.

Description

Diagnostic marker for lupus nephritis 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 cause Lupus Nephritis (LN) when lupus immune complexes reach the kidneys. LN can be classified as a disease of glomerulonephritis, which is also a major risk factor for overall morbidity and mortality in SLE. Despite the availability of powerful anti-inflammatory and immunosuppressive therapies for LN, the prognosis in most patients will End up with Chronic Kidney Disease (CKD) or End-Stage Kidney Disease (ESRD).
The current clinical major diagnostic tools for LN are kidney biopsy, which is the gold standard for LN confirmation, and serological examination. LNs are currently classified as type 6 (I-VI) in total. According to literature reports, the prognosis of LN is related to the time of diagnosis. In other words, early diagnosis of early treatment, the prognosis of LN is better. Renal biopsy should be performed immediately when patients have overt clinical symptoms of nephritis to speed up treatment decisions and minimize the risk of inflammation-induced irreversible renal damage, which also suggests an important role for early diagnosis of LN.
The search for new diagnostic markers for LN that can replace kidney biopsy is one of the current research hotspots for LN. With the development of new research technologies, LN omics studies, such as transcriptomics, proteomics, etc., have promoted the development of research for finding LN-specific diagnostic biomarkers in blood or urine. Therefore, there is a need to find more effective LN diagnostic markers by the above means.
Disclosure of Invention
The present application is directed to solving at least one of the problems in the prior art. Therefore, the application provides lupus nephritis markers with good diagnostic value, and a reagent for quantitatively detecting the markers can be used for diagnosing or prognosing lupus nephritis.
In a first aspect of the application, an application of a reagent for quantitatively detecting at least one marker in C8b, NDUFA4 and COL6A2 in preparing a product for diagnosing, monitoring or prognosticating lupus nephritis is provided.
According to the application of the embodiment of the application, at least the following beneficial effects are achieved:
the application obtains the trace kidney tissue samples of different parts by the laser capture microdissection technology, thereby screening out the markers differentially expressed in various kidney tissues to be used as the diagnostic markers of the lupus nephritis, making up the defects of the existing lupus nephritis diagnostic indexes, and having good clinical diagnostic value.
Wherein C8b (supplement C8 Beta Chain) is the Beta Chain of Complement component 8. C8 consists of α, β and γ subunits, which are a component of the membrane attack complex, mediate cell lysis, and initiate membrane permeation of the complex.
NDUFA4(NDUFA4 Mitochondrial complete 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 2Chain) is a Collagen Type VI Alpha 2Chain, a string of string-of-filaments 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 ndifa 4, C8b and COL6a2, ndifa 4 and COL6a2, and all three markers.
In some embodiments of the present application, the agent quantitatively detects the marker at the gene level or at the protein level. The reagent for quantitatively detecting nucleic acid at the gene level is carried out by methods including, but not limited to, Polymerase Chain Reaction (PCR), isothermal amplification reaction (such as loop-mediated isothermal amplification (LAMP), Recombinase Polymerase Amplification (RPA) and the like), probe hybridization technique, northern blotting and the like. Reagents for quantitatively detecting markers at the protein level are performed by methods including, but not limited to, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (IRA), immunohistochemical staining, western blotting, 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, ndifa 4, COL6a2 at the gene level is selected from the group consisting of primers, probes, and gene chips. The primer is a primer capable of specifically amplifying C8b, NDUFA4 and COL6A2 genes, the probe is a probe capable of specifically recognizing C8b, NDUFA4 and COL6A2 genes or transcripts of the genes, and the gene chip is a composite structure formed by an array in which the probes are fixed on a substrate material (specifically including but not limited to a polymer such as a nylon membrane, a 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 a glomerular sample, ndifa 4 in a tubular sample, COL6a2 in an interstitial sample at the protein level.
In some embodiments of the present application, lupus nephritis is diagnosed when at least one of an up-regulation of the expression level of C8b protein in a glomerular tissue sample relative to a normal human, a down-regulation of the expression level of NDUFA4 protein in a tubular tissue sample relative to a normal human, and an up-regulation of the expression level of COL6a2 protein in a renal interstitial tissue sample relative to a normal human occurs. In some embodiments, when two or three of the above occur, lupus nephritis is diagnosed. The term "up-down level" in a relatively normal human means that, with respect to a threshold value, a critical value (e.g., median value, minimum value, maximum value, or a multiple thereof) of the expression level of a marker in a sample (e.g., normal human sample) satisfying a standard relevant to clinical diagnosis of lupus nephritis, the expression level of the marker in the sample of a subject is determined to be up-regulated when the expression level is higher than a certain threshold value, and is determined to be down-regulated when the expression level is lower than the certain threshold value.
In some embodiments of the present application, the disease progression or prognosis of lupus nephritis in a patient is monitored by monitoring changes in the expression levels of C8b protein, NDUFA4 protein, COL6a2 protein.
In some embodiments of the present application, diagnosing comprises diagnosing at least one of a pathological grade, Activity (AI), Chronicity (CI) of lupus nephritis.
Lupus nephritis is mainly classified into six types, and is classified through the pathological type of kidney, specifically, the type i is as follows: in mild mesangial lupus nephritis, glomeruli are normal under a light microscope, but immunofluorescence and/or electron microscopy can show that the immune complex of the mesangial region is deposited. Type II: mesangial proliferative lupus nephritis, which can be seen under light microscope with different degrees of simple mesangial cell proliferation or increased mesangial matrix and mesangial area immune complex deposition; immunofluorescence and electron microscopy showed a small amount of subendothelial or subendothelial immune complex deposition. Type III: focal lupus nephritis, with active (a) or inactive (C) lesions, intraglomerular hyperplasia, membranous hyperplasia and moderately severe mesangial hyperplasia in focal segments or globuli (< 50% of affected glomeruli) or with crescentic formation, typically focal subendothelial immune complex deposition with or without mesangial changes. And IV, type: diffuse lupus nephritis, active or inactive lesions. Diffuse (affected glomerulus is more than or equal to 50 percent) segmental or spherical glomerular capillary intravascular hyperplasia, membranous hyperplasia and moderate and severe mesangial hyperplasia, or crescentic glomerulonephritis, typical diffuse subendothelial immune complex deposition with or without mesangial lesion. Type V: membranous lupus nephritis. The glomerular basement membrane is diffusely thickened and diffuse or segmental subepithelial immune complex deposition is seen with or without mesangial lesions. Type VI: severe sclerosis type lupus nephritis. Over 90% of the glomeruli exhibit a globular sclerosis, no more active lesions. The clinical Activity (AI) and the Chronic Index (CI) of lupus nephritis reflect to some extent the degree of kidney damage, and currently, AI and CI scores given by the National Institute of Health (NIH) are widely used.
In a second aspect of the present application, there is provided a method for screening a marker for lupus nephritis, comprising the steps of:
obtaining kidney tissue samples of different parts from the tissue slices by adopting a laser capture microdissection technology; extracting peptide fragments from a kidney tissue sample; quantitatively detecting DIA of the peptide fragment; and (4) performing bioinformatics analysis according to the detection result to obtain the differential expression protein and screening out the marker.
The screening method provided by the embodiment of the application has at least the following beneficial effects:
laser Capture Microdissection (LCM) is a technique that allows any region of interest to be dissected with the aid of a microscope in a relatively short time (typically 1-1.5h), can be precise on a single cell, and can therefore be used to study the spatiality of tissue. There is a greater understanding of the molecular and pathological changes in different regions of the kidney in renal disease. Thus, the LN patients and normal controls were isolated by LCM for glomeruli, tubules, and renal interstitium in renal tissue, and proteomic studies were performed to screen out appropriate markers by biological assay.
In some embodiments of the present application, methods from differential expression of proteins to screening for markers include, but are not limited to, modeling whether a person from whom a sample is derived is diseased or not by using the specific expression profile of the differentially expressed proteins in collected kidney tissue or other samples via a suitable algorithm, which may be replaced by a linear regression method or other supervised machine learning non-linear algorithm.
In some embodiments of the present application, the different renal tissue samples comprise at least one of a glomerular tissue sample, a tubular tissue sample, a renal interstitial tissue sample.
In a third aspect of the present application, there is provided a method for screening a drug for treating lupus nephritis, the method comprising the steps of: the drug to be screened acts on a lupus nephritis model, the expression level of at least one marker in C8b, NDUFA4 and COL6A2 is detected, and the drug with obvious therapeutic action on lupus nephritis is screened according to the change condition of the expression level.
Additional aspects and advantages of the present 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 present application.
Drawings
FIG. 1 is the results of Metascape of the glomeruli involved in the complement system protein in example 1 of the present application, wherein A is a histogram of the biological process, B is a network interaction diagram of the biological process, and C is a network interaction diagram of the protein.
FIG. 2 is the Metascape results for tubular metabolic pathway proteins in example 1 of the present application, wherein A is a histogram of biological processes, B is a network interaction diagram of biological processes, and C is a protein network interaction diagram.
Fig. 3 is a Metascape result of an ECM receptor-related protein of renal mesenchyme in example 1 of the present application, wherein a is a histogram of biological processes, B is a network interaction diagram of biological processes, and C is a protein network interaction diagram.
FIG. 4 shows the results of immunohistochemistry for C8B in example 2 of the present application, where A is a staining pattern at different magnifications and B is the result of comparison of IRS scores.
FIG. 5 shows the results of immunohistochemistry experiments with NDUFA4 in example 2 of the present application, where A is the staining pattern at different magnifications and B is the comparison of IRS scores.
FIG. 6 is the results of immunohistochemistry experiments with COL6A2 in example 2 of the present application, A is a staining pattern at different magnifications, and B is the result of comparison of IRS scores.
FIG. 7 is a ROC curve with C8b, NDUFA4 and COL6A2 as LN bio-diagnostic markers in example 2 of the present application, A-C representing the ROC curve with C8b, NDUFA4 and COL6A2 distinguishing mild-to-moderate and severe activity indices, and D-F representing the ROC curve with C8b, NDUFA4 and COL6A2 distinguishing mild-to-moderate and severe chronic progression indices.
FIG. 8 is a ROC curve for the combination of C8b, NDUFA4, and COL6A2 as LN biomarker in example 2 of the present application, with A-C being any two combinations to distinguish between mild-to-moderate and severe activity indices and D being three combinations to distinguish between mild-to-moderate and severe activity indices.
FIG. 9 is a ROC curve for the combination of C8b, NDUFA4 and COL6A2 as a marker for LN biomarkers in example 2 of the present application, with A-C being the ROC curve for any two combinations to distinguish mild and moderate-severe chronization indices and D being the ROC curve for three combinations to distinguish mild and moderate-severe chronization indices.
Detailed Description
The conception and the resulting technical effects of the present application will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, and not all embodiments, and other embodiments obtained by those skilled in the art without inventive efforts based on the embodiments of the present application belong to the protection scope of the present application.
The following detailed description of embodiments of the present application is provided for the purpose of illustration only and is not intended to be construed as a limitation of the application.
In the description of the present application, the meaning of a plurality is one or more, the meaning of a plurality is two or more, and the above, below, exceeding, etc. are understood as excluding the present number, and the above, below, within, etc. are understood as including the present number. If the first and second are described for the purpose of distinguishing technical features, they are not to be understood 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 otherwise expressly limited, terms such as set, mounted, connected and the like should be construed broadly, and those skilled in the art can reasonably determine the specific meaning of the terms in the present application by combining the detailed contents of the technical solutions.
In the description of the present application, reference to the description of the terms "one embodiment," "some embodiments," "an illustrative embodiment," "an example," "a specific example," 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, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. 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 methods
1. Determining a sample
A total of 21 LN kidney tissue samples, 11 normal human kidney tissue samples were included in the histological sections for laser microdissection and DIA protein quantification. The pathological diagnosis of the renal puncture examination in the LN group conforms to LN, and the pathological result is based on the revision standard of LN typing by the International society for renal sciences/Kidney pathology (ISN/RPS) in 2003. Inclusion criteria included: (1) age 16 to 55 years; (2) no immunosuppressant and hormone treatments were performed; (3) no acute or chronic infectious diseases, such as pulmonary tuberculosis; (4) there are no other immune related diseases, such as dermatomyositis, ankylosing spondylitis, etc. The medicine can be used for treating IgA nephropathy, purpura nephropathy, hepatitis B virus related nephropathy, diabetic nephropathy, acute glomerulonephritis, chronic glomerulonephritis and drug related renal damage.
LCM sampling
A Leica LMD7000, Germany laser capture microdissection system is applied, a 4-time objective is used for finding tissues, glomeruli, renal tubules and renal tubules are respectively found through a computer screen after the objective is adjusted to be 10 times, then a required target area is circled on a computer display screen through a mouse, wherein the cutting energy is set to be 38, the aperture is set to be 14, and the speed is set to be 4. Then, the switch of the laser generator is pressed, the laser cuts along the drawn figure, the target cells and the surrounding tissue cells are separated, and the cut target cells fall into a 200 mu l PCR tube cap which is pre-arranged on the collector stage due to the gravity. The process is repeated until the required sample is cut. And collecting the cut sample in a PCR tube, placing the PCR tube in dry ice for storage, and performing protein extraction and subsequent experiments.
3. Proteomics research
3.1 protein extraction
Centrifuging the sample tube at 20000g for 10 minutes at normal temperature; adding 20 microliter of 50mM ammonium bicarbonate water solution containing Dithiothreitol (DTT) with the concentration of 10 mM; reacting in metal bath at 95 ℃ for 30 min; immediately adding Iodoacetamide (IAM) after the temperature is reduced to room temperature to make the final concentration of the iodoacetamide be 50mM, and standing for 30min in a dark place at room temperature; the sample tube was placed in a water bath sonicator for 20 minutes.
3.2 proteolytic cleavage
Adding 0.2ug of Trypsin enzyme into each sample tube for enzymolysis; and oscillating and uniformly mixing for 30s, centrifuging for a short time, and performing enzymolysis in a water bath kettle at 37 ℃ overnight for 14-16 h.
3.3 desalting of peptide fragments
And (3) activation: taking a new C18 column, and passing the column through 1mL of methanol at the flow rate of 3 drops/s;
balancing: passing through the column with 1mL of 0.1% FA at a flow rate of 3 drops/s;
loading: diluting the protein liquid sample by using SDS-free L3 to 1mL, and passing through the column at the flow rate of 1 drop/s;
washing: passing through a column with 1mL of 0.1% FA at a flow rate of 3 drops/s, and repeating for 3 times;
and (3) elution: slowly eluting with 800L 75% ACN at flow rate of 0.5 drop/s;
and (3) draining: the eluent is frozen and dried by suction.
3.4 high Performance liquid chromatography separation
The samples were subjected to liquid phase separation using Shimadzu LC-20AD liquid phase system with a 5 μm 4.6X250mm Gemini C18 column. The experimental method is as follows, after mixing equal amount of peptide fragments from all samples, diluting with 5% Acetonitrile (ACN) and injecting, and eluting with a flow rate gradient of 1 mL/min, wherein the mobile phase B is 95% ACN, and the elution process is as follows: 5% of mobile phase B for 10min, 5% to 35% of mobile phase B for 40min, 35% to 95% of mobile phase B for 1min, the duration of the mobile phase B is 3min, and 5% of the mobile phase B is balanced for 10 min. The elution peak was monitored at 214nm and one fraction was collected per minute, combined with the chromatogram elution peak pattern and the sample was pooled to give 10 fractions, which were then freeze dried.
3.5 quantitative detection of DIA
The dried peptide fragment sample was reconstituted with mobile phase A (2% ACN, 0.1% Formic Acid (FA)), centrifuged at 20000g for 10min, and the supernatant was injected and separated by HPLC. The sample was first enriched and desalted in a trap column and then serially connected to a C18 column (150 μm internal diameter, 1.8 μm column size, about 35cm column length) and separated at a flow rate of 500nL/min by the following effective gradient: 0-5 min, 5% mobile phase B (98% ACN, 0.1% FA); the mobile phase B linearly rises from 5% to 25% in 5-130 min; 130-150 min, and the mobile phase B rises from 25% to 35%; 150-160 min, and increasing the mobile phase B from 35% to 80%; 160-175 min, 80% of mobile phase B; 175-175.5 min, reducing the content of the mobile phase B from 80% to 5%; 175.5-180 min, 5% mobile phase B. And the tail end of the high performance liquid phase separation is directly connected with a mass spectrometer and is detected.
3.5.1 detection of library construction
The peptide fragments subjected to liquid phase separation are ionized by a nano ESI source and then enter a tandem mass spectrometer for Data Dependent Acquisition (DDA) mode detection. Setting main parameters: the ion source voltage was set to 2 kV; the scanning range of the primary mass spectrum is 350-1,500 m/z; the resolution was set to 60000 and the maximum ion implantation time (MIT) was 50 ms; the secondary mass spectrum fragmentation mode is HCD, and the fragmentation energy is set to be 30; the resolution was set to 15,000, the maximum ion implantation time (MIT) was 50ms, and the dynamic exclusion time was set to 30 s. The initial m/z of the secondary mass spectrum is fixed to be 100; the screening conditions of the parent ions for secondary fragmentation are as follows: parent ions with a charge of 2+ to 6+ and a peak intensity exceeding that of 2E4 line up in the first 30. The AGC is set as: primary 1E5, secondary 2E 4.
3.5.2DIA Mass Spectrometry detection
The peptide fragments separated by the liquid phase are ionized by nano ESI source and then enter a tandem mass spectrometer for DIA mode detection. Setting main parameters: the ion source voltage was set to 2 kV; the scanning range of the primary mass spectrum is 400-1500 m/z; resolution was set to 60000; a maximum ion implantation time (MIT) of 50 ms; divide 400-. The ion fragmentation mode is HCD, the maximum ion implantation time is 54ms, fragment ions are detected in Orbitrap, the resolution ratio is set to be 30000, and the fragmentation energy is 30; AGC is set to 5E 4.
4. Bioinformatics analysis
The off-line DDA data is identified using the Max Quant integrated Andromeda engine, and then the results are used to build a spectrogram library. For large-scale DIA data, analysis and quality control of the data are completed by using an m Prophet algorithm, so that a large number of reliable quantitative results are obtained.
4.1 database selection
Database selection UniProt protein database (https:// www.uniprot.org /), UniProt is the most informative and widely available protein database. The system is formed by integrating data of three databases of Swiss-Prot, TrEMBL and PIR-PSD.
4.2DDA data analysis
Identification of DDA data was done using Max Quant as a spectrogram library for subsequent DIA analysis. During operation, original off-line 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 next DIA data is corrected for retention time (retention time) using the peptide fragment. And then, based on a Target-decoy model applicable to SWATH-MS, false positive control is completed with FDR less than or equal to 1%, so that a significant quantitative result is obtained.
4.4MSstats differential analysis
The data were preprocessed using mstats according to a set comparison set and then significance checked 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 difference. Meanwhile, the enrichment analysis is completed on the differential protein.
4.5 Annotation analysis
And analyzing and processing the protein by means of GO, KOG, KEGG Pathway, PPI annotation analysis, subcellular localization analysis, Metascape analysis and the like.
Results of the experiment
5.1 identification of peptide fragments and proteins
After collection of LCM experimental samples, proteomics studies were performed on the kidney region. The DIA model was used to obtain MS data, except that one sample was identified as 0, and a total of 49658 peptides, 4056 proteins, were identified in the remaining 95 samples. In addition, the results show that most samples identified more than 500 proteins, suggesting that the DIA protein identification technique has a certain depth. Some samples contain only dozens of proteins, so we speculate that the possible reasons for the low number of proteins identified in a part of the region are that the sample expresses little by itself or that the protein extraction process is degraded.
5.2 differential protein screening
After obtaining the proteins in each region of each sample, we screened for differential proteins with Fold difference (FC) >1.5 and P <0.05, and the experimental results showed that 478 differentially expressed proteins were identified in the glomeruli of LN group, 167 proteins were highly expressed and 311 proteins were lowly expressed, and 2383 proteins were not statistically different. 532 differentially expressed proteins were identified in the group of LN renal tubules, of which 125 proteins were expressed in higher amounts and 407 proteins were expressed in lower amounts, and a total of 3153 proteins were not statistically different. 653 differentially expressed proteins were identified in the renal mesenchyme of the LN group, 573 proteins were up-regulated, 80 proteins were down-regulated, and 1330 proteins were not statistically different.
5.2.1 glomerular differential protein assay
GO enrichment analysis was performed on these 478 glomerular differential proteins. The results showed that the most different proteins involved in the biological process were cellular processes (418). The cellular components in which the most different proteins are involved are the cells (cells) and the cellular regions (cell part), which are 428 in common. The molecular function involved by the most different proteins is binding, 361. The cellular regions where the cellular sub-localization results showed the most protein localization were cytoplasm followed by nucleus, respectively, indicating that the glomerular differentiated proteins function primarily in the cytoplasm and nucleus of the glomerular cells.
KOG annotation results indicated that the differential proteins in glomeruli are mainly involved in cellular processes and signaling (cellular processes and signaling), and there are 389 proteins, 102 of which are involved in signal transduction mechanisms. The KEGG pathway analysis result shows that glomerular differential protein is involved in the pathogenesis of LN through complement and aggregation cassettes (complement and aggregation cassettes), SLE, PI3K-Akt and Hippo signaling pathways. Abnormal activation of the complement system is closely related to the immune response of the body. Thus, differential proteins in the glomeruli may be involved in the disease process of LN through the complement system.
5.2.2 renal tubule differential protein assay.
GO enrichment analysis was performed on these 532 tubular differential proteins. The results showed that 449 proteins are involved in cellular processes, 387 proteins are involved in metabolic processes, 488 proteins are involved in the formation of cellular components, and 362 proteins are involved in the binding function of molecular functions. Subcellular localization results showed that tubular differential proteins were mainly concentrated in the mitochondria and cytoplasm, 198 of them, suggesting that tubular differential proteins perform biological functions in the mitochondria of cells. In combination with the above results, it is presumed that tubular differential proteins are mainly involved in metabolism.
KOG annotation results indicated that 252 proteins were enriched in metabolic-related pathways, of which 97 were involved in Energy metabolism (Energy production and conversion). 185 (pink) are involved in cellular processes and signal transduction, with minimal differential protein enrichment for information storage and processing functions. The KEGG pathway analysis result shows that the tubular differential protein is mainly involved in the processes of kidney metabolism, such as amino acid metabolism, fatty acid metabolism, carbon metabolism and the like. The above results further suggest that tubular differential proteins are involved in the pathogenesis of LN, primarily by regulating kidney metabolism.
5.2.3 analysis of renal interstitial differential proteins
GO enrichment analysis was performed on this 653 tubular differential proteins. The results showed that 592 proteins were involved in cellular processes, 622 proteins were involved in cellular formation, and 544 proteins performed binding functions. The cellular sub-localization results showed that 247 proteins localized to the cytoplasm, much higher than 131 on the nucleus.
KOG annotation results indicated that 386 proteins are involved in cellular processes and signal transduction, and 98 of them are 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 distinct from the glomerular and tubular protein differential proteins is an extracellular matrix receptor interaction (ECM receptor interaction) in the renal interstitial KEGG enrichment pathway. Taken together, the renal interstitial differential proteins can be involved in LN progression through extracellular matrix receptor interactions. Differential proteins in the renal interstitium may be associated with renal fibrosis, suggesting that different regions of the kidney may be involved in the pathogenesis of LN through different pathways and actions.
5.3Metascape analysis results
Metascape was used to analyze the interaction between proteins in the glomerular complement system pathway, the tubular metabolic pathway, and the renal interstitial extracellular matrix receptor interaction pathway.
The results of Metascape of glomeruli involved in complement system proteins are shown in FIG. 1, and the major biological process of these proteins is the regulation of the complement cascade (regulation of complement cascades), and the interactions between these proteins exist. Based on the MCODE algorithm, there is a close interaction of complement molecules C5, C6, C8A, C8B, C8G and C9. These complement molecules are suggested to influence the complement system's involvement in the pathogenesis of LN by modulating the complement cascade. Therefore, it is likely to be used as a diagnostic marker for lupus nephritis.
Metascape results for tubular metabolic pathway proteins are shown in FIG. 2, and these proteins are involved in the metabolic processes of kidney tissues mainly through the involvement of the oxidative phosphorylation system (OXPHOS system in mitochondria) 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 ubiquitin oxidoreductase subunit family in tubular differential proteins (including ndifa 2, ndifa 4, ndifb 1, ndifb 5, ndifs 3, ndifs 4, ndifs 7) may influence metabolism through oxidative phosphorylation of the cell mitochondrial system and thus participate in disease progression of LN. Therefore, it is likely to be used as a diagnostic marker for lupus nephritis.
Metascape results for renal interstitial ECM receptor-associated proteins, which affect extracellular matrix receptor interactions by affecting extracellular matrix proteoglycans (ECM proteolycans), are shown in FIG. 3, and thus affect kidney fibrosis. The PPI results showed that among the proteins with which the extracellular matrix interacts, there are close effects of the collagens COL1a1, COL2a1, COL1a2, COL4a2, COL6a2, and COL6 A3. These results suggest that we may have an important role for these collagens in kidney fibrosis. Therefore, it is likely to be used as a diagnostic marker for lupus nephritis.
Example 2: immunohistochemical validation
To verify the reliability of protein identification and to explore the potential of some proteins as a biomarker for biological diagnosis, IHC was used to verify protein expression. Among the glomerular differential proteins, based on GO enrichment analysis, KEGG pathway analysis, protein-protein interaction and the like, protein C8b is screened from complement molecular proteins participating in the complement system, and the log of the protein C8b is obtained2FC is 2.71, P is 0.005, and C8b is involved in regulating activation of complement system and immune response.
Among proteins differentially expressed in renal tubules, based on GO enrichment analysis, KEGG pathway analysis, protein-protein interaction analysis and other analysis, NDUFA4 is screened from a ubiquitin oxidoreductase subunit family participating in cell oxidative phosphorylation reaction in a metabolic pathway, and the proteomic result shows that NDUFA4 is a low-expression protein in an LN patient. Log of it2FC was-2.11 and P ═ 0.003.
In proteins differentially expressed in renal interstitium, based on analysis such as GO enrichment analysis, KEGG pathway analysis and protein-protein interaction, Col6A2 is screened from collagen family, and log of Col6A2 is obtained2FC is 4.06, P<0.001。
The experimental method comprises the following steps:
the paraffin embedded tissue is cut into 4 μm thickness and placed on a glass slide, dried and stored for later use.
The procedure for detection of the above three proteins in tissues by immunohistochemical staining is briefly described as follows:
1) dewaxing, namely dewaxing in xylene for three times, and 5 minutes each time;
2) dehydrating, and dehydrating with alcohol gradient (100% alcohol-95% alcohol for 2 times-70% alcohol);
3) washing with Milli-Q pure water for 5 minutes;
4) performing antigen retrieval, namely boiling a sodium citrate buffer solution in a microwave oven for 20 minutes;
5) washing with Milli-Q pure water for 5 minutes and then PBS for 5 minutes;
6) incubation with 2.5% horse serum reduced non-specific background;
7) primary antibodies were diluted with horse serum and incubated overnight at 4 degrees, with three antibody dilutions all at 1: 100, respectively;
8) the next day, washing with PBS 3 times for 5 minutes each;
9) 3% hydrogen peroxide is incubated for 5 minutes, so that nonspecific background staining caused by endogenous peroxidase is reduced;
10) washing with PBS buffer solution for 3 times, 5min each time;
11) incubating the secondary antibody for 30 minutes;
12) washing with PBS buffer solution for 3 times, 5min each time;
13) DAB color development;
14) washing, and continuously washing after 2 minutes of Mayor's hematoxylene counterstaining;
15) dehydrating with 95% ethanol for 2 times and 100% ethanol for 2 times;
16) dried and mounted with DPX.
2.5 immune response score
The mounted tissue was photographed and stored under a microscope. Immunohistochemical scoring (The immunohistochemical Score, IRS) refers to The existing literature and invites two pathologists completely blinded to this experimental design to ensure The accuracy of scoring. The scoring criteria are shown in table 1 below.
TABLE 1 IRS Scoring Standard Table
Figure BDA0003340995370000111
Figure BDA0003340995370000121
Clinical data and IRS are expressed as mean ± SD. The IRS mean comparisons of C8b, NDUFA4 and COL6A2 were tested using Mann-Whitney U, due to the inconsistent number of LN and control groups. Correlation analysis of IHC scores and clinical data for C8b, ndifa 4, and COL6a2 spearman correlation analysis was used and correlation coefficients were calculated. The diagnostic potential and differential pathotyping potential of C8b, ndifa 4 and COL6a2 were calculated using ROC curves. All statistics were analyzed using SPSS 22.0 and statistical significance was considered when P values were less than 0.05.
In this example, a total of 44 LN patients and 6 normal control group samples were included. All LN patients were confirmed by renal biopsy and clinical indices.
The results of the C8B test are shown in FIG. 4, where A is the staining at different magnifications and B is the comparison of IRS scores. As can be seen from the figure, C8b is mainly expressed on glomeruli in kidney tissue of LN group, and the expression level of C8b in LN group is significantly higher than that in normal control group (P < 0.05). This is similar to the proteomic results in example 1, confirming the reliability of the data.
The results of the NDUFA4 test are shown in FIG. 5, where A is the staining pattern at different magnifications and B is the comparison of IRS scores. It can be seen from the figure that NDUFA4 is mainly expressed on the renal tubules of the kidney tissue, and NDUFA4 is expressed in LN in a significantly lower amount than in the normal control group (P < 0.05). These results are consistent with the results of the proteomics study in example 1, and the results were verified.
The results of the COL6A2 tests are shown in FIG. 6, where A is the staining pattern at different magnifications and B is the comparison of IRS scores. As can be seen from the figure, COL6a2 was mainly expressed on the tubules and renal interstitium of the kidney tissue, and the expression of COL6a2 was significantly higher in LN than in the control group (P < 0.05). These results are consistent with the results of the proteomics study in example 1, and the results were verified.
In order to preliminarily discuss the potential of C8b, NDUFA4 and COL6A2 as LN biological diagnostic markers, ROC curve analysis is applied to discuss the potential of the three molecules as LN grading diagnostic markers, the LN activity index score is 1-2 and classified as mild, the score is 3-9 and classified as moderate, the score is 10 or more and classified as severe, the LN chronicity index score is 0-1 and classified as mild, the score is 2 and classified as moderate, and the score is 3 or more and classified as severe. The results are shown in fig. 7, wherein a to C represent ROC curves for distinguishing mild-moderate and severe activity indexes of C8b, ndifa 4 and COL6a2, D to F represent ROC curves for distinguishing mild-moderate and severe chronic activity indexes of C8b, ndifa 4 and COL6a2, and the ROC curves are shown except for y ═ x in the figure. As can be seen, the areas under the ROC curves are all less than 0.8, with the C8b predicted activity index area under the curve being 0.606, the ndifa 4 predicted activity index area under the curve being 0.741, and the COL6a2 predicted activity index area under the curve being 0.603. C8b predicted the area under the curve for the chronic index to be 0.502, ndefa 4 predicted the area under the curve for the chronic index to be 0.504, and COL6a2 predicted the area under the curve for the chronic index to be 0.505. Therefore, these three proteins are of low value as independent hierarchical diagnostic markers.
For this purpose, these combinations of proteins were subjected to regression analysis to generate ROC curves for predicting activity, and the results are shown in fig. 8, where a to C are two combinations of ROC curves, D is three combinations of ROC curves, and the ROC curves are shown except for y ═ x in the figure. As can be seen from the figure, the area under the ROC curve of the combination of the two 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 have the potential to be combined as a biomarker for staging the pathological activity of LN.
Similarly, the results of regression analysis of these combinations of proteins were shown in fig. 9, in which a to C were ROC curves for two combinations, D was a ROC curve for three combinations, and the ROC curves were obtained except for y ═ x. As can be seen from the figure, the area under the C8b + ndifa 4 curve is 0.732, the area under the C8b + COL6a2 curve is 0.832, the area under the ndifa 4+ COL6a2 curve is 0.888, and the area under the ROC curve for binding of three proteins is 0.92.
Therefore, according to the expression level of the protein, the chronic and active pathological parameters of lupus nephritis can be accurately reflected, so that the LN pathological degree of a subject and the pathological change of the kidney can be judged, and the purposes of monitoring the disease progression and predicting the disease prognosis can be achieved.
Example 3
The embodiment provides a method for screening a drug for lupus nephritis, which comprises the following steps:
reacting the medicine to be screened with a 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 the potential of being used as a biological diagnostic marker for LN pathological activity and chronic differentiation grading, so whether the medicine to be screened plays a corresponding therapeutic role can be judged according to the expression conditions of related markers before and after the medicine to be screened is used.
The results are combined to show that C8b, NDUFA4 and COL6A2 can be used as diagnostic and prognostic markers of 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 those skilled in the art without departing from the gist of the present application. Furthermore, the embodiments and features of the embodiments of the present application may be combined with each other without conflict.

Claims (10)

1. The application of a reagent for quantitatively detecting at least one marker in C8b, NDUFA4 and COL6A2 in the preparation of a product for diagnosing, monitoring or prognosticating lupus nephritis.
2. The use of claim 1, wherein the agent quantitatively detects the marker at the gene level or at the protein level.
3. The use according to claim 2, wherein said reagents for quantitative detection of said markers at the gene level are selected from the group consisting of primers, probes and gene chips.
4. The use according to claim 2, wherein said reagent for quantitatively detecting said marker at the protein level is an antibody.
5. The use of claim 2, wherein the reagent quantitatively detects at least one of C8b in a glomerular sample, ndifa 4 in a tubular sample, COL6a2 in an interstitial renal sample at the protein level.
6. The use of claim 5, wherein lupus nephritis is diagnosed when at least one of an up-regulation of the expression level of C8b protein in a glomerular sample relative to a normal person, a down-regulation of the expression level of NDUFA4 protein in a tubular sample relative to a normal person, and an up-regulation of the expression level of COL6A2 protein in a renal interstitial sample relative to a normal person occurs.
7. The use of any one of claims 1 to 6, wherein the diagnosis comprises diagnosing at least one of the pathological grade, activity, or chronicity of lupus nephritis.
8. The method for screening the marker of lupus nephritis is characterized by comprising the following steps:
obtaining kidney tissue samples of different parts from the tissue slices by adopting a laser capture microdissection technology;
extracting a peptide fragment from the kidney tissue sample;
quantitatively detecting the DIA of the peptide fragment;
and (4) performing bioinformatics analysis according to the detection result to obtain the differential expression protein and screening out the marker.
9. The screening method of claim 8, wherein the different renal tissue samples comprise at least one of a glomerular tissue sample, a tubular tissue sample, and a renal interstitial tissue sample.
10. The screening method of the medicine for treating lupus nephritis is characterized by comprising the following steps: the drug to be screened acts with a lupus nephritis model, the expression level of at least one marker in C8b, NDUFA4 and COL6A2 is detected, and the drug is screened according to the change condition of the expression level.
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