CN110244053B

CN110244053B - Molecular marker for diagnosing lupus nephritis and pulmonary hypertension disease and application thereof

Info

Publication number: CN110244053B
Application number: CN201910386642.2A
Authority: CN
Inventors: 李秋钰
Original assignee: Peking University Third Hospital Peking University Third Clinical Medical College
Current assignee: Peking University Third Hospital Peking University Third Clinical Medical College
Priority date: 2019-05-09
Filing date: 2019-05-09
Publication date: 2022-03-11
Anticipated expiration: 2039-05-09
Also published as: CN110244053A

Abstract

The object of the present invention is to provide molecular markers for diagnosing lupus nephritis and pulmonary hypertension and uses thereof. The use of complement factor H and complement Bb fragments as molecular markers for diagnosing or predicting lupus nephritis and pulmonary hypertension can be simple and accurate. Diagnosis or prediction of pulmonary hypertension or its risk in patients with lupus nephritis. Compared with invasive examination methods such as right heart catheterization, it is more suitable for clinical promotion.

Description

Molecular marker for diagnosing lupus nephritis and pulmonary hypertension disease and application thereof

Technical Field

The invention relates to a disease molecular marker, in particular to a molecular marker for diagnosing lupus nephritis and pulmonary hypertension diseases and application thereof.

Background

Systemic Lupus Erythematosus (SLE) is one of the most common autoimmune diseases in China, kidney is one of the most easily affected organs of systemic lupus erythematosus, lupus nephritis (1upus nephritis, LN) is immune complex nephritis caused by the systemic lupus erythematosus affecting the kidney, and is the most common secondary glomerular disease. Pulmonary Arterial Hypertension (PAH) refers to the change of pulmonary vascular function and/or structure caused by various reasons, is characterized by progressive increase of pulmonary vascular resistance, is one of the main factors of the poor prognosis of SLE patients as a common and serious complication of SLE.

The pathogenic mechanism of SLE-PAH is mainly dysfunction of vascular endothelium, which not only has the function of physical barrier, but also can regulate the vasodilatation and contraction of blood vessels, and also plays an important role in promoting blood coagulation and intercellular interaction. The initial stage of pulmonary hypertension vasculopathy can be accompanied by endothelial cell dysfunction, and the following factors are involved: 1) pulmonary vasculitis caused by inflammatory reactions with hypocomplement-emia. 2) Pulmonary vascular diseases mainly caused by the proliferation of pulmonary vascular endothelial cells and smooth muscle cells have generally low primary pathogenic activity, are related to vascular diseases, and have low reversible degree of primary pathogenic treatment. 3) It is mainly caused by pulmonary small vessel thrombosis, such as antiphospholipid syndrome, etc., and also relates to various reasons such as complement system dysfunction, etc.

In the prior art, in order to diagnose whether a patient has pulmonary hypertension, invasive examination is required, right ductal examination is the gold standard for determining the pulmonary hypertension, and the right ductal examination can accurately obtain the hemodynamic characteristics of the pulmonary circulation and the right heart system, but has inherent defects due to the invasiveness.

The complement system belongs to the first "line of defense" of the natural immune system, coexisting in three activation pathways: the classical pathway, the alternative pathway and the lectin pathway are involved in the participation of more than 30 known plasma proteins. In order to prevent damage to the body itself from over-activation of the complement system, there are numerous inhibitors of complement components that fine-tune their activation. Thus, the complement system plays an important role in the regulation of the body's immune system.

In the pathogenic process of systemic lupus erythematosus, the classical theory holds that immune complex activation of complement is the most important factor, but there are many clinical features that cannot be explained with confidence. Further, the scholars propose: after the immune complex locally activates the classical pathway of complement in the target organ, the further activation of the alternative pathway enables inflammatory components such as C3a and C5a to be further released, so that the formation of a membrane attack complex is caused, and the target organ is finally damaged, therefore, the alternative pathway of the complement system has important clinical significance in the pathogenic process of the systemic lupus erythematosus.

Factor H, as a major regulatory protein of the alternative complement pathway, plays a central role in preventing the alternative complement pathway from being over-activated. The gene encoding factor H is located at 1q32, a 150kDa glycoprotein consisting of 20 Short Consensus Repeats (SCRs) domains, 1213 amino acid residues, of which the liver is the major synthetic organ. Furthermore, kidney membrane cells, retinal pigment epithelial cells, platelets, peripheral mononuclear cells, glial cells, fibroblasts, endothelial cells, and the like may also be expressed. Two major functional domains of factor H are located at both ends of the protein: SCR1-4 at the N-terminal is mainly used for inhibiting the formation of C3 convertase (C3bBb) of the alternative pathway and assisting factor I in degrading C3bBb, thereby inhibiting the over-activation of the alternative complement pathway; and SCR19-20 at C terminal can combine with mucopolysaccharide on host cell surface to complete target recognition, and through combination with these sites, factor H is assisted to inhibit excessive activation of host cell surface alternative complement pathway. In addition, the H factor can be combined with fragments, DNA, histone and other components of a plurality of apoptotic cells to regulate and eliminate the apoptotic cells. Factor H therefore plays an important "brake" in regulating the complement system to participate in the natural immune defense, inhibiting over-activation of complement, and clearance of apoptotic substances.

In summary, there is still a need for a simple and accurate diagnosis of whether a patient with systemic lupus erythematosus is complicated with pulmonary hypertension, which can be used for non-invasive tests such as diagnosis using molecular markers and/or as targets for treating systemic lupus erythematosus and pulmonary hypertension/lupus nephritis and pulmonary hypertension.

Disclosure of Invention

In order to solve the defects of the prior art, the invention provides a molecular marker for lupus nephritis and pulmonary hypertension diseases, wherein the marker is complement factor H, and compared with a patient with normal pulmonary arterial pressure of lupus nephritis, the expression level of the complement factor H is obviously reduced.

Furthermore, the invention also provides a molecular marker of lupus nephritis and pulmonary hypertension disease, wherein the marker is complement Bb fragment, and compared with a patient with normal pulmonary arterial pressure of lupus nephritis, the expression level of the complement Bb fragment is obviously increased.

Further, the molecular markers are complement factor H and complement Bb fragments.

The invention also provides an application of the molecular marker in diagnosing or predicting lupus nephritis and pulmonary hypertension diseases, and the application is to prepare a diagnostic reagent.

Further, the molecular marker is complement factor H, and the expression level of the complement factor H is obviously reduced compared with that of a patient with normal pulmonary arterial pressure of lupus nephritis.

Further, the marker is complement Bb fragment, and compared with a patient with normal pulmonary arterial pressure of lupus nephritis, the expression level of the complement Bb fragment is obviously increased.

Still further, the markers are complement factor H and complement Bb fragments.

Still further, the diagnostic reagent further comprises a reagent related to the detection of anti-RNP antibody, anti-cardiolipin antibody, D-dimer, and/or hemoglobin.

The invention provides a diagnostic kit, which comprises related reagents for determining molecular markers for diagnosing or predicting lupus nephritis and pulmonary hypertension diseases.

Further, the molecular marker is complement factor H.

Further, the molecular marker is complement Bb fragment.

Still further, the kit may further comprise reagents related to the detection of anti-RNP antibodies, anti-cardiolipin antibodies, D-dimers, and/or hemoglobin.

The invention also provides a method for diagnosing or predicting lupus nephritis and pulmonary hypertension diseases, which comprises the steps of detecting the expression level of the molecular marker of a patient to be diagnosed, comparing the expression level with the expression level of the molecular marker of a patient with normal pulmonary arterial pressure of lupus nephritis, and diagnosing or predicting lupus nephritis and pulmonary hypertension when the expression level of the molecular marker is remarkably different.

Still further, the markers are complement factor H and complement Bb fragments.

Still further, the method comprises detecting the expression level of anti-RNP antibodies, anti-cardiolipin antibodies, D-dimer, and or hemoglobin and comparing the expression level to that of a patient with lupus nephritis pulmonary arterial hypertension.

Still further, an expression level of the complement factor H of 241 μ g/ml or less indicates that the lupus nephritis patient has or has a high risk of having a complicated pulmonary hypertension.

Further, an expression level of the complement Bb fragment of greater than or equal to 1.26 μ g/ml indicates that the lupus nephritis patient has or is at high risk of having a complicated pulmonary hypertension.

Advantageous effects

The invention confirms that the alternative complement pathway plays an important role in the complicated pulmonary hypertension disease of lupus nephritis patients.

The factor H, Bb can be used as a molecular marker of lupus nephritis and pulmonary hypertension diseases, and compared with invasive examination methods such as right-heart catheter examination, the factor H, Bb can accurately and efficiently diagnose whether lupus nephritis patients have pulmonary hypertension or predict the risk of the pulmonary hypertension complicated.

Drawings

FIG. 1: patient enrollment into the enrollment flowchart.

FIG. 2: immunofluorescent staining results of complement pathway activation-related components including Bb, C3D, and C5B-9 in lung tissues of LN-PAH patients, co-staining of the extravascular major component laminin, fig. 2A, 2B, 2C relating to Bb, C3D, and C5B-9, respectively, and fig. 2D being results of co-localization range for Pearson correlation coefficient evaluation.

Detailed Description

The present invention is described in more detail below to facilitate an understanding of the present invention.

It should be understood that the terms or words used in the specification and claims should not be construed as having meanings defined in dictionaries, but should be interpreted as having meanings that are consistent with their meanings in the context of the present invention on the basis of the following principles: the concept of terms may be defined appropriately by the inventors for the best explanation of the invention.

Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the laboratory manual, or as recommended by the manufacturer.

Example 1 lupus nephritis and differential expression of pulmonary hypertension complement pathway-associated proteins

Data and method

Study subjects:

first, the case of 389 patients with lupus nephritis, diagnosed during hospitalization via chest echocardiography and assessed by renal pathology observation for all types of renal vascular disease including vascular immune complex deposition, arteriosclerosis, thrombotic microangiopathy, non-inflammatory necrotizing angiopathy, true vasculitis, was retrospectively evaluated, at the first hospital of the university of beijing, from month 1 to 5 in 2002 and confirmed by renal biopsy. Furthermore, 24 patients with lupus nephritis associated with pulmonary arterial hypertension (LN-PAH) were selected for study according to the criteria of FIG. 1. The diagnosis of lupus nephritis is based on the American society for rheumatology (ESC), PAH adopts the criteria specified in the European society for Heart disease (ESC)2015, and excludes congenital heart disease, portal hypertension, pulmonary vein occlusion, pulmonary embolism, chronic obstructive pulmonary disease, and history of left heart failure.

The method comprises the following steps:

the subjects were divided into LN-PAH group (24 cases) and LN-non-PAH group (328 cases) and normal population group (100 cases).

Detecting clinical indexes of the LN-PAH group and the LN-non-PAH group, wherein the clinical indexes comprise: age, sex, fever, malar erythema, photosensitivity, alopecia, oral ulcer, arthritis, pleurisy, Raynaud's phenomenon, nervous system damage, anemia, leukopenia, thrombocytopenia, lupus activity score. The above diagnosis and evaluation were performed according to standard methods.

Detecting laboratory indexes of the LN-PAH group and the LN-non-PAH group, wherein the laboratory indexes comprise: hemoglobin, blood creatinine, D dimer, and autoantibody spectrum related indexes. The index analysis is performed according to standard methods or according to kit instructions.

The expression of major components of the complement pathway in plasma of the LN-PAH group and LN-non-PAH group was examined by enzyme-linked immunoassay, and the concentration of major complement components in specific plasma was determined by enzyme-linked immunoassay, including complement fragment C5a (Quidel Corporation, San Diego, CA), C3a (Quidel Corporation, San Diego, CA), Bb (Quidel Corporation, San Diego, CA), soluble C5b-9(SC5b-9, Quidel Corporation, San Diego, CA), properdin (Uscnk life science Inc, Wuhan, China), and C3(Quidel Corporation, San Diego, CA). All complement components were determined according to the manufacturer's instructions. In addition, plasma C1q, MBL, C4BP were also determined according to standard methods.

The detection of plasma complement factor H levels was improved according to prior art experimental methods as follows: goat anti-human factor H polyclonal antibody (Calbiochem, Germany) was diluted to 10. mu.g/ml with 0.05M carbonate buffer pH9.6, pre-coated with an enzyme-labeled plate, and incubated overnight at 4 ℃. And adding the diluted H factor standard substance and the patient plasma sample into the reaction hole respectively, and incubating for 1H at 37 ℃. After washing, mouse anti-human factor H monoclonal antibody (Utilical Co., U.S.A.) was added at a dilution of 1: 500, and incubated at 37 ℃ for 1 hour. After washing, alkaline phosphatase-labeled goat anti-mouse IgG (Sigma) was added and incubated at 37 ℃ for 30 min. Finally, alkaline phosphatase substrate is added for color development, and an absorbance value is measured by a microplate reader at the wavelength of 405 nm. Plasma complement factor H levels were quantified against standards by plotting a standard curve.

Immunofluorescence assay: determining the expression of Bb, C3d and C5b-9 in the lung tissue of LN-PAH patients, in particular: lung tissues of LN-PAH patients were taken, fixed-dehydrated-embedded to make paraffin blocks, sectioned at 8um thickness, deparaffinized to water and antigen retrieval, immunofluorescence to detect the deposition of complement alternative pathway activation product Bb, terminal common pathway product C3d and complement activation terminal complex C5b-9 at tissue vessels, Confocal imaging and grey scale statistics.

And (3) data analysis:

continuous variables are described as mean ± Standard Deviation (SD) or median (IQR, quartet), and differences between groups are analyzed by two-factor analysis using variance test and nonparametric test. Categorical variables are expressed as percentages and analyzed by the chi-square test. Hardy-Winberg equilibrium was analyzed using x2 fitness. Univariates and multivariate used logistic regression analysis to assess survival. The results are expressed as a ratio of 95% confidence intervals. Statistical analysis of the data was calculated using SPSS 12.0 software. A two-tailed p-value of less than 0.05 is considered statistically significant.

The experimental results are as follows:

of the 24 patients in the LN-PAH group, 2 were male and 22 were female, with the mean age at the time of the experiment being 35.29. + -. 15.13 years; of 328 patients in the LN-non-PAN group, 53 were male and 275 were female, and the mean age at the time of the experiment was 32.83. + -. 11.30; there was no significant difference in age and gender between the LN-PAH patient group and the LN-non-PAH patient group (both p-values were 0.758).

The specific relevant information of the LN-PAH patient group is shown in Table 1.

Table 1: lupus combined pulmonary hypertension patient related information

The clinical evaluation data and laboratory examination results of the LN-PAH group patients and LN-non-PAH group patients are shown in tables 2 and 3, respectively.

Table 2: clinical data comparison between lupus patients with pulmonary hypertension complicated with lupus and patients with normal pulmonary arterial hypertension complicated with lupus

Table 3: laboratory data comparison of patients suffering from severe sore combined pulmonary hypertension and patients suffering from severe sore normal pulmonary arterial pressure

As can be seen from table 2, the LN-PAH patient group had more thrombocytopenia (p < 0.001), lower symptoms of zygomatic erythema (p ═ 0.01), alopecia (p ═ 0.017), and arthritis (p ═ 0.03) than the LN-non-PAH patient group, indicating that the above symptoms were not synchronized with the occurrence of pulmonary hypertension.

The results of comparing the complement levels in patients with lupus combined pulmonary hypertension to those with lupus pulmonary hypertension are shown in Table 4.

Table 4: comparison of complement levels in patients with lupus complicated with pulmonary hypertension and patients with lupus pulmonary hypertension

As can be seen from Table 4, the levels of serum mannose lectin, C3a, C5a and soluble C5b-9 in the normal population were 1532. + -. 1020ng/ml, 100.87. + -. 70.55ng/ml, 9.32. + -. 7.88ng/ml and 467.41. + -. 545.23ng/ml, respectively.

The serum mannose lectin, C3a, C5a and soluble C5b-9 levels in both LN-PAH patients and LN-non-PAH patients were significantly elevated (P values P < 0.01, respectively) compared to the normal population.

C1q, the first component of the classical complement activation pathway, was not significantly different in LN-PAH patients from those in LN-non-PAH patients (p ═ 0.206).

Mannose lectin is an inducer of the lectin complement activation pathway, and as such, the expression levels in LN-PAH patients are not significantly different from those in LN-non-PAH patients (p. 0.874).

C4BP is the major fluid phase inhibitor of complement activation and is capable of inhibiting by enhancing the failure of the C3 convertase C4b2a of the classical/lectin pathway and also by enhancing the failure of the alternative pathway C3 convertase C3 bBb. The expression level of C4BP in LN-PAH patients was not significantly different from that in LN-non-PAH patients (p ═ 0.308).

The results of the above experiments indicate that LN-PAH patients and LN-non-PAH patients cannot be distinguished by the classical pathway and the lectin pathway related components, and that the risk of merging pulmonary hypertension in lupus patients cannot be diagnosed or predicted by the classical pathway and the lectin pathway related components.

Properdin plays a key role in the stabilization of the alternative pathway C3 convertase, Bb is an active fragment of factor B in the alternative pathway, while complement factor H is an abundant plasma complement regulatory factor that inhibits the formation of C3 convertase and promotes the failure of C3 convertase, and inactivates C3B as a co-factor with complement factor I, by which complement factor H can inhibit the activation of the alternative pathway.

Whether the alternative pathway is activated can be known by measuring the expression levels of Bb, properdin and factor H in plasma. The results show (see table 4) that the expression level of Bb was significantly increased in LN-PAH patients compared to the expression level in LN-non-PAH patients (1.26 ± 0.758 μ g/ml vs.0.74 ± 0.186 μ g/ml, p ═ 0.049); the expression level of factor H in LN-PAH patients was significantly reduced compared to that in LN-non-PAH patients (241.00 ± 116.502 μ g/ml vs.417.13 ± 188.017 μ g/ml, p ═ 0.024), while the expression level of properdin in LN-PAH patients was not significantly different from that in LN-non-PAH patients (p ═ 0.102).

The above results indicate that in LN-PAH patients, the alternative complement pathway is over-activated, mainly by overexpression of Bb and a reduction in the level of factor H expression.

The activation of the classical complement pathway is mainly reflected in the conversion of C3 to C3a and C3b, thereby forming a C5 convertase multienzyme which is capable of cleaving C5 into C5a and C5b, and the final complement complex C5b-9 is obtained by the assembly of C5b and C9 due to the activation of the complement system, and therefore, C3, C3a, C5a and soluble C5b-9 have been studied, which can reflect the complement activation in the circulatory system.

The results showed that the expression levels of C3, C3a, C5a and soluble C5b-9 in LN-PAH patients were not significantly different from the expression levels in LN-non-PAH patients, indicating that the degree of activation of the classical complement pathway was not significantly different in the two groups of patients.

To further confirm the localization and expression of complement pathway-associated components in lung tissue of LN-PAH patients, C3d, C5b-9, and Bb deposition was examined by immunofluorescence and revealed (see FIG. 2) that C3d, C5b-9, which are markers of the classical complement pathway, and Bb fragments, which are specific markers of the alternative complement pathway, were all positive in lung tissue, in combination with the differential expression of C3d, C5b-9, Bb in the aforementioned LN-PAH patients and LN-non-PAH patients, further confirming the presence of overactivation of the alternative complement pathway in LN-PAH patients.

Vascular injury is a common feature of SLE, and lupus and pulmonary hypertension are considered lesions caused by their vascular endothelium. One accepted hypothesis of SLE vasculopathy is that immune complexes on the vascular endothelium trigger inflammatory responses, including activation of the complement cascade, forming C5b-9 membrane complement attack that ultimately destroys the vascular basement membrane and inflammatory cell infiltration.

The complement pathway mechanisms in LN-PAH are contradictory, on the one hand, genetic homozygous deficiency in the early classical or alternative pathway complement components predispose patients to lupus, and on the other hand, complement over-activation leads to inflammation and tissue damage.

Previous studies have only demonstrated that activation of the alternative complement pathway can reflect SLE activity, the severity of vascular lesions, especially those that predominate with thrombotic microangiopathy. Modulation of the alternative complement pathway over-activation can effectively inhibit SLE development and progression.

However, the present inventors found that increased expression levels of Bb, C3d and C5b-9, as well as decreased levels of factor H, indicate that alternative complement pathway activation plays a major role in LN-PAH, and that Bb, C3d, C5b-9 are deposited in lung tissue of LN-PAH patients.

Therefore, whether the lupus nephritis patient is combined with the pulmonary hypertension or not can be diagnosed or the risk of combined pulmonary hypertension can be predicted by detecting the expression level of Bb and factor H in plasma.

Example 2 diagnosis of lupus nephritis and pulmonary hypertension Using complement pathway differential proteins

7 patients with lupus nephritis newly acquired from the first hospital of Beijing university are selected, and whether pulmonary hypertension is caused by the lupus nephritis is diagnosed by using complement factor H and complement Bb fragments as molecular markers, and the right heart catheter examination result is used as a control.

The results are shown in table 5:

patient's health	1	2	3	4	5	6	7
								Factor H (ug/ml)	253.00	326.12	469.56	500.78	189.65	385.46	156.27
Bb(μg/ml)	1.20	0.72	0.69	0.61	1.29	0.69	2.11
								Right Heart catheter test (+/-)	+	-	-	-	+	-	+

The result shows that whether the lupus nephritis patient is combined with the pulmonary hypertension disease can be accurately diagnosed or predicted by using the complement factor H and the complement Bb fragment as the molecular marker.

The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.

Claims

1. Use of a reagent for detecting the expression level of a molecular marker or a molecular marker for the preparation of a reagent for diagnosing or predicting lupus nephritis and pulmonary hypertension disease, characterized in that: the molecular marker is complement factor H, and compared with a patient with normal pulmonary arterial pressure caused by lupus nephritis, the expression level of the complement factor H is obviously reduced.

2. Use according to claim 1, characterized in that: the molecular marker is complement Bb fragment, and compared with a patient with normal pulmonary arterial pressure caused by lupus nephritis, the expression level of the complement Bb fragment is obviously increased.

3. Use according to claim 1, characterized in that: the molecular markers are complement factor H and complement Bb fragments, and compared with a patient with normal pulmonary arterial pressure of lupus nephritis, the expression level of the complement factor H is obviously reduced, and the expression level of the complement Bb fragments is obviously increased.

4. Use according to any one of claims 1 to 3, characterized in that: the reagents also include components associated with the detection of anti-RNP antibodies, anti-cardiolipin antibodies, D-dimers, and hemoglobin.