CN115192563B - Use of C3a/C3aR pathway antagonists for the treatment of primary membranous nephropathy - Google Patents

Abstract

The use of a C3a/C3aR pathway antagonist for the treatment of primary membranous nephropathy is provided. The present invention relates to the use of a C3a/C3aR pathway antagonist in the manufacture of a medicament for treating membranous nephropathy in a subject. The C3aR antagonist may be selected from the group consisting of: n2- [ (2, 2-biphenylethoxy) acetyl ] -L-arginine, 5- (bis (4-chlorophenyl) methyl) -3-methylthiophene-2-carbonyl) -L-arginine, or a pharmaceutically acceptable salt thereof. The invention has the advantages of less side effect, protection effect on other viscera, no anaphylactic reaction and the like.

Description

Use of C3a/C3aR pathway antagonists in the treatment of primary membranous nephropathy

Technical Field

The present invention relates to the field of medicine, and in particular to the use of a C3a/C3aR pathway antagonist in treating primary membranous nephropathy.

Background Art

Primary membranous nephropathy (PMN) is an autoimmune glomerular disease and the most common cause of nephrotic syndrome in adults. Its incidence is about 2000/1 million per year, and it is increasing year by year. In my country, due to environmental pollution and other reasons, its incidence rate is increasing more rapidly, reaching an increase of 13% per year. PMN mainly affects middle-aged and elderly people, with an average age of onset of 50-60 years old, and males account for 2/3. Without treatment, about 30-40% of patients will eventually progress to end-stage renal disease (ESRD) and require renal replacement therapy or kidney transplantation. Currently, the KIDGO guidelines recommend rituximab (CD20 monoclonal antibody), calcineurin inhibitors (CNI) and cyclophosphamide as first-line medications. The existing first-line treatment drugs have a wide spectrum of immunosuppressive effects, which results in more side effects while exerting therapeutic effects. CD20 monoclonal antibodies are prone to allergic reactions, and immunodeficiency caused by inhibition of B cells significantly increases the risk of infection in patients. CNI and cyclophosphamide have adverse reactions such as increased risk of infection, bone marrow suppression, and hepato-nephrotoxicity. The pathological feature of PMN is the deposition of immune complexes in the subepithelial region of the glomerular capillary wall.

C3a is an important component of the complement system. As an anaphylatoxin, it can produce pro-inflammatory or anti-inflammatory effects in different cells under different pathological conditions after binding to its receptor. The complement pathway is a crucial part of immunotherapy, and research on drugs that intervene in the complement pathway is in the ascendant and has broad prospects. However, although there are a large number of clinical trials on anti-C5, C5a, and C5aR1 drugs, and C5 monoclonal antibody (Eculizumab) and C5aR1 antagonist (Anacopan) have been approved for clinical use, there are fewer clinical trials for C3, and most of them are in phase I, and there are no registered clinical trials for C3a or C3aR. In recent years, more and more studies have shown that the C3a/C3aR pathway may play a significant role in various kidney diseases.

Blocking complement with snake venom factors resulted in the inability to induce Heimann nephritis in rats, so complement activation is a necessary condition in its pathogenesis. The effectors of the complement hierarchy reaction include membrane attack complex (MAC), C3a, and C5a. In podocytes, antibodies in immune complexes activate the cascade reaction of the complement system, ultimately producing MAC. Previous studies have suggested that MAC is an essential molecule that causes renal injury, which destroys the glomerular filtration barrier and causes proteinuria by inducing sublytic injury to podocytes. However, inhibiting the formation of MAC in rats deficient in complement C6 did not completely prevent PMN rats from producing proteinuria, indicating that there are other non-MAC-independent mechanisms of podocyte injury.

In view of the insufficiency of existing therapeutic drugs, there is still a need in the art for drugs for treating primary membranous nephropathy.

Summary of the invention

The inventors analyzed the clinical data and found that C3a and C3aR were correlated with the clinical manifestations and prognosis of PMN. It is believed that the activation of the C3a/C3aR pathway has a pathogenic effect in PMN, as confirmed by the in vitro podocyte experiment and the in vivo Heimann nephritis rat model of the present invention. In addition, the inventors also proved that the C3aR antagonists SB290157 and JR14a can reduce the severity of PMN rat disease by blocking the C3a/C3aR pathway. At present, C3aR antagonists SB290157 and JR14a have been widely used in in vivo and in vitro experiments. Their antagonistic effect on C3aR is stable, and no serious lethal adverse reactions have occurred in animal experiments. Therefore, the inventors' research shows that PMN can be treated by intervening the C3a/C3aR pathway with C3aR antagonists.

In one aspect, the present invention provides a use of a C3a/C3aR pathway antagonist or a pharmaceutical composition comprising a C3a/C3aR pathway antagonist in the preparation of a medicament for treating or preventing membranous nephropathy in a subject.

In another aspect, the present invention provides a method of treating membranous nephropathy in a subject, comprising administering to the subject a C3a/C3aR pathway antagonist or a pharmaceutical composition comprising a C3a/C3aR pathway antagonist.

In one embodiment of the above aspects, the C3a/C3aR pathway antagonist is a C3aR antagonist.

In one embodiment of the above aspects, the C3aR antagonist is selected from the group consisting of N2-[(2,2-biphenylethoxy)acetyl]-L-arginine, 5-(bis(4-chlorophenyl)methyl)-3-methylthiophene-2-carbonyl)-l-arginine or a pharmaceutically acceptable salt thereof.

In one embodiment of the above aspects, the C3aR antagonist is N2-[(2,2-biphenylethoxy)acetyl]-L-arginine trifluoroacetate.

In one embodiment of the above aspects, the drug is an oral or parenteral drug.

In one embodiment of the above aspects, the parenteral drug is a drug that is administered subcutaneously, intradermally, intravenously, intramuscularly, intraarterially, or by infusion.

In one embodiment of the above aspects, the subject is a human or a mammal. In one embodiment, the mammal is a rodent, such as a rat.

In one embodiment of the above aspects, the membranous nephropathy is primary membranous nephropathy.

In one embodiment of the above aspects, the pharmaceutical composition comprises one or more of CD20 monoclonal antibody, calcineurin inhibitor drugs, glucocorticoids, mycophenolate mofetil, cyclophosphamide and angiotensin converting enzyme inhibitors (ACEI) or angiotensin receptor antagonists (ARB) drugs.

In one embodiment of the above aspects, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

In one embodiment of the above aspects, the drug is used to alleviate the clinical symptoms of membranous nephropathy, including reducing proteinuria, increasing serum albumin levels and/or reducing creatinine; and/or reducing the number of subepithelial immune complex deposits and/or foot process fusion in glomeruli in patients with membranous nephropathy, or reducing the degree of basement membrane thickening.

In one embodiment of the above aspects, the use is achieved by blocking or inhibiting the C3a/C3aR pathway. In one embodiment, the use is achieved by reducing the IgG antibodies specific to anti-Fx1a antibodies induced by sheep anti-Fx1a antibody serum in an animal model (Heimann nephritis rat model) (without affecting the total IgG level in the animal). The antagonists of the present invention can treat membranous nephropathy without affecting the basic immunity and natural immunity of the subject.

Compared with the prior art, the advantages of the present invention include:

(1) Fewer side effects: Hormones, existing immunosuppressants, and rituximab are characterized by a broad spectrum of interventions in the immune system. While treating diseases, they can cause patients to have low immune function, become more susceptible to infection, and even have the risk of increased tumor incidence. C3aR antagonists are highly specific and do not react with other molecules except C3aR. They do not affect the normal physiological functions of other complement system molecules or T and B lymphocytes, and have fewer side effects.

(2) It has a certain protective effect on other organs: In addition to being proven to have a therapeutic effect on PMN, C3aR antagonism has also been shown to have a protective effect on the heart (atherosclerosis), nervous system (Alzheimer's disease) and other systems. PMN is a chronic kidney disease that is common in the elderly. Blocking the C3a/C3aR pathway may protect other organs at the same time and improve the patient's quality of life.

(3) Allergic reactions are rare: The currently available rituximab is a human-mouse chimeric protein antibody, so it is easy to cause allergic reactions during use, so anti-allergic drugs such as hormones and diphenhydramine need to be added before use. However, the C3aR antagonists SB290157 and JR14a used in this article are both organic compounds, and the inventors speculate that allergic reactions are less common in patients when used in comparison with biological agents.

BRIEF DESCRIPTION OF THE DRAWINGS

The results, experiments and advantages obtainable by the present invention are summarized in the drawings and examples. The drawings and examples show preferred embodiments of the present invention, but it should be understood that the drawings and examples should not be considered as limiting the present invention.

Figure 1: Expression of C3aR in glomeruli of different diseases and healthy controls. Figure A shows immunohistochemical images. Figure B shows the relative C3aR expression in glomeruli of patients with various glomerular diseases and healthy controls. PMN: primary membranous nephropathy; DN: diabetic nephropathy; FSGS: focal segmental glomerulosclerosis; MCD: minimal change disease.

Figure 2: Expression of C3aR on podocytes (red fluorescence: C3aR; green fluorescence: podocyte marker protein podocin).

Figure 3: Correlation analysis between glomerular C3aR expression and clinical indicators of patients. A: Glomerular C3aR expression is positively correlated with urine protein; B: Glomerular C3aR expression is positively correlated with serum creatinine; C: Patients who achieved complete remission after treatment had lower plasma C3a levels.

Figure 4: Podocyte migration experiment. Upper figure: Line graph of podocyte migration distances in each group after stimulation for 12h, 24h, 36h, 48h, 60h and 72h; Lower figure: Scatter plot of podocyte migration distances in each group at 72h of stimulation.

Figure 5: Podocyte activity experiment: relative activity of podocytes in each group after 36h, 48h, and 72h of stimulation.

Figure 6: Expression of podocyte cytoskeleton proteins and cytokines mRNA under different stimulation conditions. A: Relative expression of C3aR mRNA under different stimulation conditions; B: Relative expression of PLA2R mRNA under different stimulation conditions; C: Relative expression of Bcl-2 mRNA under different stimulation conditions; D: Relative expression of synapotodin mRNA under different stimulation conditions; E: Relative expression of WNT3 mRNA under different stimulation conditions; F: Relative expression of β-catenin mRNA under different stimulation conditions.

Figure 7: C3aR, PLA2R and synapotodin protein levels in podocytes under different stimulation conditions. A: Western blot bands of C3aR, PLA2R, synapotodin and GAPDH proteins (reference protein); B: Scatter bar graph of relative expression levels of C3aR, PLA2R and synapotodin proteins.

Figure 8: Expression of PLA2R in podocytes. A: Relative IOD values of PLA2R immunofluorescence in podocytes of each group after stimulation; B: Western blot bands of C3aR, PLA2R, synapotodin and GAPDH protein (reference protein); C: Representative images of PLA2R immunofluorescence in each group.

Figure 9: Proteinuria levels in rats of each group. A, proteinuria versus time curve in the SB290157 treatment group; B, proteinuria versus time curve in the JR14a treatment group; C, proteinuria levels in rats of each group in the SB290157 experiment at week 7; and D, proteinuria levels in rats of each group in the JR14a experiment at week 7.

Figure 10: Amount of electron dense deposit, foot process width and GBM thickness in each group of rats. A-C are the results of SB290157 experiment; and D-E are the results of JR14a experiment.

Figure 11: Relative expression levels of specific anti-sheep IgG in the JR14a treatment group at weeks 2, 5, and 7.

Figure 12: Relative expression of specific anti-sheep IgG in the SB290157 treatment group at week 2, week 5, and week 7.

Figure 13: Changes in total IgG in each treatment group over time.

Figure 14: Relative expression levels of C3d, C4d, C5aR and MBL in rat glomeruli.

DETAILED DESCRIPTION

The inventors first discovered the pathogenic role of the C3a/C3aR pathway in PMN, and proposed and proved that PMN rats can be treated by using C3aR antagonists, which is a new way to treat PMN that has never been discovered and verified before. Specifically, the inventors first used immunohistochemistry to detect the expression of C3aR on the renal tissue (glomerulus) of PMN patients, and detected the level of C3a in the serum of PMN patients by ELISA. After analysis, it was found that the expression level of C3aR was significantly correlated with the severity of clinical symptoms (proteinuria, renal function) and prognosis of PMN patients. The inventors also conducted podocyte experiments, using PMN patient plasma and C3aR antagonists to prove that C3a in PMN patient plasma can interfere with podocyte function and induce podocyte apoptosis; and conducted PMN disease experiments in Heimann nephritis rats, respectively, by intraperitoneal injection of 30 mg/kg/d C3aR antagonist SB290157 or intragastric administration of 10 mg/kg/d C3aR antagonist JR14a to achieve the effect of blocking the C3a/C3aR pathway. Experimental C3aR antagonists have a history of more than 10 years, and are produced by major domestic and foreign biopharmaceutical companies. The inventors have confirmed that C3aR antagonists can treat PMN rats: C3aR antagonists have significant therapeutic effects on PMN rats, and are expected to be used in clinical trials. In the PHN disease model induced by anti-Fx1A antibody serum, the antagonists used in this article only inhibit antibodies specific to anti-Fx1A antibodies (such as sheep anti-Fx1A antibodies), and the total IgG level in rat plasma is not affected by C3aR blockers. No side effects such as infection were observed in all experimental rats.

C3a is a polypeptide containing about 77 amino acids. It is a potent inflammatory factor and can act extensively on immune and non-immune cells. C3a can regulate vasodilation, increase the permeability of small blood vessels, and induce contraction of smooth muscle. For cells with phagocytic function (neutrophils, macrophages), C3a has a strong chemotactic effect, causing phagocytes to migrate to damaged or inflamed areas of tissue. C3a can also promote the release of histamine by basophils and mast cells, triggering the oxidative burst phenomenon of neutrophils. C3a is also involved in tissue regeneration and fibrosis. In liver tissue regeneration, C3a can promote the production of cytokines and tumor necrosis factor α (TNF-α). C3a exerts a series of biological effects by binding to its specific receptor C3aR.

C3a receptor (C3aR) is a 7-transmembrane receptor belonging to the G protein-coupled family. In early studies, the expression of C3aR was detected on bone marrow-derived cells (such as monocytes, neutrophils, macrophages, T cells and mast cells), and it exhibited various physiological functions in tissue cells in different physiological and pathological states. In kidney tissue, both renal tubular epithelial cells and podocytes can express C3aR.

A C3a/C3aR signaling pathway antagonist refers to an agent that blocks the C3a/C3aR signaling pathway, which may be a C3a antagonist or inhibitor, or a C3aR antagonist or inhibitor. Preferably, the C3a/C3aR signaling pathway antagonist of the present invention is a C3aR antagonist or inhibitor. More preferably, the C3aR antagonist or inhibitor is SB290157 or JR14a.

The chemical name of SB290157 is N2-[(2,2-diphenylethoxy)acetyl]-L-arginine. SB290157 is a potent and selective C3a receptor antagonist. At present, the research and application of SB290157 are mainly in the following aspects: ①SB290157 has neuroprotective function and can be used as an adjuvant therapy for intravenous thrombolysis in thromboembolism, neuroprotective function in cerebral hemorrhage, Alzheimer's disease, small vessel disease and vascular dementia; ②SB290157 can reduce hypertension caused by Abelcet and has the application prospect of lowering blood pressure; ③SB290157 combined with opioid receptor agonists can produce a strong analgesic without tolerance; ④SB290157 may be a method for treating early age-related macular degeneration; ⑤SB290157 can inhibit diet-induced obesity, metabolic dysfunction and adipocyte and macrophage signal transduction; ⑥SB290157 has anti-inflammatory effects and has the application prospect of inhibiting arthritis, inhibiting late asthma reactions and airway hyperresponsiveness; ⑦SB290157 has the application prospect of treating lupus nephritis; ⑧SB290157 can be used as a drug to mobilize the transplantation of hematopoietic stem cells, etc. SB290157 can be used in the form of its salt, for example SB290157 trifluoroacetate (C24H29F3N4O6).

The chemical name of JR14a is 5-(Bis(4-chlorophenyl)methyl)-3-methylthiophene-2-carbonyl)-l-arginine (C25H26Cl2N4O3S). JR14a is a potent human complement C3a receptor thiophene antagonist. JR14a has higher selectivity for human C3a receptor than C5a receptor. JR14a can inhibit C3aR-mediated inflammation.

Membranous nephropathy (MN) is one of the most common primary glomerular diseases. Its pathological features are diffuse immune complex deposition under the epithelial cells of the glomerular basement membrane accompanied by diffuse thickening of the basement membrane. The main clinical manifestations are nephrotic syndrome (NS) or asymptomatic proteinuria. Membranous nephropathy can be primary or secondary to a variety of diseases, such as infection (hepatitis B and C viruses), systemic diseases (such as lupus erythematosus), drug treatment (such as gold, penicillamine, etc.) and malignant tumors. It is currently believed that membranous nephropathy is a glomerular damage mediated by autologous antibodies produced against antigenic components on the membrane of normal glomerular epithelial cells. Immune complexes are shed from the epithelial cell membrane to the epithelial cells of the basement membrane to form typical immune complex deposition. The deposited immune complexes activate complement, where C5b-9 is produced. The complement membrane attack complex causes proteinuria. The activated cytokines in the process of lesions lead to changes in the extracellular matrix components of the basement membrane, causing the basement membrane to thicken and further develop the lesions. Currently used drugs include angiotensin converting enzyme inhibitors, immunostimulants, glucocorticoids and cytotoxic drugs (such as cyclophosphamide (CTX) combined with glucocorticoids and chlorambucil combined with glucocorticoids). The C3aR antagonist of the present invention can be used to treat membranous nephropathy, especially primary membranous nephropathy, as demonstrated by effectively reducing the symptoms and renal damage of PHN rats. The C3aR antagonist of the present invention has many advantages over currently available drugs, such as not reacting with other molecules except C3aR, not affecting other molecules of the complement system or T and B lymphocytes to play normal physiological roles, and having fewer side effects; in addition to being proven to have PMN pathogenic effects, the C3a/C3aR pathway has been proven to have no significant pathogenic effects in the heart, nervous system and other systems. PMN is a chronic kidney disease that the elderly are prone to, and blocking the C3a/C3aR pathway may protect other organs at the same time, thereby improving the quality of life of patients; as a small molecule drug, it is not easy to cause allergic reactions in subjects.

Podocytes are key cells for selective filtration of the glomerular capillary wall, together with the glomerular basement membrane, glomerular endothelial cells with fenestrae, proteoglycans on the surface of endothelial cells and the lower zone of podocytes, they constitute the glomerular filtration barrier. More and more studies have shown that podocytes play an important role in the development of kidney diseases. Many acquired glomerular diseases with proteinuria as the main clinical manifestation (including diabetic nephropathy, membranous nephropathy, etc.) are accompanied by abnormalities in the morphology and function of podocytes.

"Treatment" is used herein to refer to therapeutic treatments that cure a disease, improve the clinical symptoms of a disease, improve the patient's prognosis, etc. In this article, PMN patients can be treated by blocking the C3a/C3aR pathway through injection or oral administration of C3aR antagonists to achieve one or more of the following effects: (1) Alleviate their clinical symptoms, including reducing proteinuria, increasing serum albumin levels, reducing creatinine, etc.; (2) Reduce the number of subepithelial immune complex deposits and/or foot process fusion, or reduce the degree of basement membrane thickening; (3) Improve the patient's long-term renal prognosis, prolong the time from onset to the need for renal replacement therapy, or eliminate the need for dialysis for life; (4) Reduce the number of chronic cardiovascular and cerebrovascular diseases caused by the disease itself or the use of hormones, immunosuppressants, and dialysis during the course of the disease; and (5) Avoid the side effects of conventional first-line treatment drugs, such as metabolic syndrome caused by hormones, immunosuppressants, and immunodeficiency.

"Pharmaceutically acceptable salt" means a physiologically or toxicologically tolerable salt, and includes pharmaceutically acceptable base addition salts and pharmaceutically acceptable acid addition salts when appropriate. For example, in the case where the compound contains one or more acidic groups (e.g., carboxyl groups), pharmaceutically acceptable base addition salts that can be formed include sodium salts, potassium salts, calcium salts, magnesium salts, and ammonium salts, or salts with organic amines such as diethylamine, N-methyl-glucamine, diethanolamine, or amino acids (e.g., lysine), etc.

The term "parenteral" includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarterial, or infusion forms.

The medicine or pharmaceutical composition of the present invention can be orally administered in any oral acceptable dosage form, including but not limited to capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. When tablets are used for oral administration, commonly used carriers include lactose and corn starch. Lubricants, such as magnesium stearate, are usually added. For oral administration in capsule form, available diluents include lactose and dry corn starch. When oral administration of water-based suspensions and/or emulsions, the active ingredient can be suspended or dissolved in the oil phase and combined with emulsifiers and/or suspending agents. If necessary, some sweeteners and/or aromatics and/or coloring agents can be added.

In order to further understand the present invention, the preferred embodiments of the present invention are described below in conjunction with examples, but it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the invention. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all deemed to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

Example

Example 1: C3aR is highly expressed in the glomeruli of patients with membranous nephropathy and is correlated with the severity of the disease

1. Materials and Methods

Mouse anti-human/rat/mouse C3aR monoclonal antibody Santa Cruz USA Rabbit anti-human podocin polyclonal antibody Invitrogen USA Goat anti-mouse IgG (H+l) secondary antibody Invitrogen USA Goat anti-rabbit IgG (H+l) secondary antibody Novus Biologicals USA Mouse two-step reagent Zhongshan Jinqiao China DAB colorimetric kit Zhongshan Jinqiao China Human Complement C3a ELISA Kit Qiaoyi Biology China

1.1 Research subjects and specimens

This study included 37 patients diagnosed with PMN by renal biopsy in the Department of Nephrology, Peking University First Hospital. Secondary MN and cases with other glomerular diseases, tubulointerstitial diseases, autoimmune diseases, tumors and infections were excluded. Ten DN, 10 MCD and 7 FSGS patients were included as disease controls, and 13 healthy kidney donors were included as healthy controls. Plasma specimens were collected on the day of renal biopsy from all PMN patients. Whole blood was collected using EDTA anticoagulant tubes and centrifuged at 2000 rpm/min for 15 minutes at 4°C. Plasma was collected and quickly frozen in a -80°C refrigerator after aliquoting.

This study strictly adhered to the Declaration of Helsinki and was approved by the Ethics Committee of Peking University First Hospital, and all tissue and blood specimens were obtained with written informed consent.

1.2 Clinical data collection

Clinical data of 37 PMN patients were collected, including basic information such as age and date of diagnosis, clinical data such as the most recent 24-hour urine protein quantification before renal puncture biopsy, plasma albumin, serum creatinine, eGFR, anti-PLA2R antibodies and renal pathology data, and changes in the condition after standardized treatment. All data and information were obtained through the medical records of inpatients at Peking University First Hospital, follow-up data of outpatients, or telephone inquiries.

1.3 Experimental methods

1.3.1 Detection of co-expression of C3aR and podocin in frozen renal biopsy specimens of PMN patients and healthy controls (indirect immunofluorescence method)

(1) Take out the slices stored at -40℃ or -20℃;

(2) Fixation: Frozen sections were fixed in precooled acetone at -20°C for 15 min.

(3) Air-dry the slices in a fume hood to evaporate the acetone.

(4) Wash with PBS for 3 min × 3 times;

(5) Prepare 3% BSA blocking solution in PBS buffer and filter through a 0.25 μm filter before use;

(6) Nonspecific antigen blocking: incubate with 3% BSA blocking solution at 37°C for 1 h;

(7) Incubation with primary antibodies: mouse anti-human C3aR monoclonal antibody (1:500 dilution) and rabbit anti-human podocin polyclonal antibody (1:100 dilution) at 4°C overnight and then rewarmed to room temperature for 45 min;

(8) Wash with PBS for 3 min × 3 times;

(9) Incubation with secondary antibodies: goat anti-mouse IgG (H+l) secondary antibody (1:500 dilution) and goat anti-rabbit IgG (H+l) secondary antibody (1:500 dilution) were incubated together at 37°C for 30 min in the dark;

(10) Wash with PBS for 3 min × 3 times;

(11) Sealing: Use anti-fluorescence quenching sealing medium containing DAPI to seal the slides;

(12) Film reading: Films were read in a dark room using a Leica fluorescence microscope.

1.3.2 C3aR detection in paraffin-embedded renal biopsy specimens of PMN, DN, FSGS, MCD and healthy controls (immunohistochemistry)

(1) Dewaxing to water: soaking in xylene for 15 min × 2 times, soaking in anhydrous ethanol for 5 s × 3 times, soaking in 95% ethanol for 5 s × 2 times, soaking in 80% ethanol for 5 s × 1 time, washing with tap water for 3 min × 3 times, washing with PBS for 3 min × 3 times;

(2) High temperature and high pressure repair: dilute 50× EDTA antigen repair solution with PBS buffer at 1:50, perform high temperature and high pressure repair in a pressure cooker for 3 min, and cool naturally to room temperature in a fume hood;

(3) Wash with PBS for 3 min × 3 times

(4) Block endogenous peroxidase: add 100 μl of hydrogen peroxide solution (reagent 1 in the mouse two-step kit purchased from Zhongshan Jinqiao) and incubate at room temperature in the dark for 20 min;

(5) Wash with PBS for 3 min × 3 times;

(6) Prepare 3% BSA blocking solution in PBS buffer and filter through a 0.25 μm filter before use;

(7) Nonspecific antigen blocking: incubate with 3% BSA blocking solution at 37°C for 1 h;

(8) Incubation with primary antibody: mouse anti-human C3aR monoclonal antibody (1:500 dilution), incubate at 4°C overnight and rewarm at room temperature for 45 min;

(9) Wash with PBS for 3 min × 3 times;

(10) Add 100 μl of reaction enhancement solution (reagent 2 in the mouse two-step kit) and incubate at room temperature for 20 min;

(11) Wash with PBS for 3 min x 5 times

(12) Add 100 μl of enzyme-labeled goat anti-mouse IgG polymer (reagent 3 in the mouse two-step kit) and incubate at room temperature for 20 min;

(13) Wash with PBS for 3 min × 5 times;

(14) DAB color development: DAB color development kit (purchased from Zhongshan Jinqiao) mixed reagent 1 and reagent 2 in a ratio of 1:20, added to the slices, and color developed at room temperature for 1-2 min. The color development was monitored under a microscope. When a light brown background appeared, the slices were immediately placed in PBS and rinsed thoroughly with tap water for 10 min.

(15) Hematoxylin staining: add hematoxylin stain solution and incubate at room temperature for 10 min;

(16) Rinse with tap water for 15 minutes until the water turns blue;

(17) Dehydration and transparency: dehydrate in 80%, 95% and anhydrous ethanol for 5 seconds respectively, and then soak in xylene for transparency for 15 minutes × 2 times;

(18) Sealing: Seal the slides with neutral gum and place them in a clean ventilation hood to air dry;

(19) Film reading: Leica optical microscope was used for film reading. At least 10 complete glomeruli were photographed continuously for each tissue.

(20) Quantitative measurement of glomerular C3aR staining intensity: The staining intensity was measured by integrated optical density (IOD) using Image Pro Plus 7.0 (Media Cybernetics, Maryland, USA).

1.3.3 Detection of plasma C3a levels in PMN patients (ELISA method)

(1) The kit (human complement C3a ELISA kit, purchased from Qiaoyi Biotechnology) should be equilibrated at room temperature for 20 minutes after being taken out of the refrigerator;

(2) dilute the 20× concentrated washing solution with deionized water at a ratio of 1:20;

(3) Dilute the 5× standard dilution with deionized water at a ratio of 1:5;

(4) Preparation of standards of different concentrations: Use 1× standard diluent to dilute the C3a standard according to the reagent reconstitution label to make the final concentration of C3a reach 2500 pg/ml. After standing at room temperature for 15-20 min, gently suspend until completely dissolved. Use standard diluent to dilute the standard in multiple ratios to 1250 pg/ml, 625 pg/ml, 312.5 pg/ml, 156.25 pg/ml, and 78.125 pg/ml. The standard diluent is used as the zero concentration;

(5) The patient's whole blood was anticoagulated with EDTA, centrifuged at 2000 rpm/min for 15 min, and quickly frozen in a -80°C refrigerator after aliquoting. The frozen plasma was taken out of the refrigerator, thawed at 4°C to avoid repeated freezing and thawing, and diluted 15,000 times;

(6) Add the diluted samples or standards of different concentrations to the coated 96-well plate at 100 μl/well, replicate three wells for each sample, seal the wells with a sealing film, and incubate at room temperature for 120 min;

(7) Add 300 μl/well of washing solution and wash the plate 5 times. After the last wash, pat the plate dry on thick absorbent paper.

(8) Add biotinylated antibody working solution (100 μl/well), seal the reaction wells with sealing tape, and incubate at room temperature for 60 min.

(9) Wash the plate 5 times, patting it dry on thick absorbent paper after the last wash;

(10) Add avidin-linked HRP enzyme working solution (100 μl/well), seal the reaction wells with sealing tape, and incubate at room temperature in the dark for 20 min;

(11) Wash the plate 5 times, patting it dry on thick absorbent paper after the last wash;

(12) Add the colorimetric reagent TMB (100 μl/well) and incubate at room temperature for 15 min in the dark;

(13) Add stop solution (50 μl/well), mix well, and immediately use a microplate reader to detect, set the OD value wavelength to 405 nm and read the value;

(14) Use EIA software to draw a standard curve, with the standard concentration as the horizontal axis and the OD value as the vertical axis. Use a smooth line to connect the coordinate points of each standard. The C3a concentration of the specimen can be found on the standard curve through the OD value of the specimen. The measured concentration is multiplied by the sample dilution factor (15000) to obtain the actual C3a concentration of the plasma sample.

2. Data Analysis

SPSS 23.0 (IBM, New York, USA) was used for statistical analysis. For normally distributed data, the results were expressed as mean ± standard deviation (SD). For non-normally distributed data, the results were expressed as median (interquartile range, IQR). T-test and Mann-Whitney U test were used to evaluate the differences in quantitative and semiquantitative data. Depending on the distribution, one-way analysis of variance or Kruskal-Wallis test was used to compare the differences between groups for continuous variables. P < 0.001 was considered statistically significant, P < 0.01 was considered statistically significant, and P < 0.05 was considered statistically significant.

3. Results

3.1 Expression of C3aR in glomeruli of various glomerular diseases and healthy controls

The IOD of the immunohistochemistry results (Figure 1A) was measured by Image-pro plus software, and the relative expression of C3aR in the glomerulus was quantitatively expressed. The results showed that the expression of C3aR in the glomeruli of 37 PMN patients was 0.6 (0.6, 0.7), which was significantly higher than that of DN [0.6 (0.6, 0.7) vs. 0.4 (0.3, 0.5), P < 0.001], FSGS [0.6 (0.6, 0.7) vs. 0.5 (0.5, 0.6), P < 0.001], MCD [0.6 (0.6, 0.7) vs. 0.3 (0.3, 0.4), P < 0.001] and healthy control group [0.6 (0.6, 0.7) vs. 0.2 (0.2, 0.3), P < 0.001] (Figure 1B). In addition, the expression of C3aR in FSGS glomeruli was 0.5 (0.5, 0.6), which was higher than that in DN [0.5 (0.5, 0.6) vs. 0.4 (0.3, 0.5), P = 0.013], and significantly higher than that in MCD [0.5 (0.5, 0.6) vs. 0.3 (0.3, 0.4), P < 0.001] and healthy controls [0.5 (0.5, 0.6) vs. 0.2 (0.2, 0.3), P < 0.001]. The expression of C3aR on glomeruli in MCD was 0.3 (0.3, 0.4), which was significantly higher than that in healthy controls [0.3 (0.3, 0.4) vs. 0.2 (0.2, 0.3), P = 0.005].

3.2 Expression of C3aR on glomerular podocytes in PMN patients

C3aR immunofluorescence staining of frozen PMN renal tissue specimens showed bright fluorescence under a high-power fluorescence microscope, while the fluorescence of the glomeruli of normal control renal tissue was almost invisible. Podocin is a podocyte skeletal protein and is often used as a podocyte marker protein. There is no difference in expression between PMN glomeruli and normal glomeruli. Co-staining of C3aR and podocin showed that the fluorescence of C3aR and podocin on the glomeruli of PMN patients overlapped, indicating that podocytes expressed C3aR; while in normal control renal tissue, C3aR was mainly expressed in the tubular interstitium, and podocin was expressed on podocytes, and there was no fluorescence overlap between the two (Figure 2).

3.3 Clinical characteristics, pathological manifestations and treatment response of PMN patients

This study included 37 PMN patients, including 26 males and 11 females, with a median age of 56 (46.5, 62.0) years. The main clinical features were urine protein 6.1 (3.3, 10.1) g/24h, plasma albumin concentration 26.0 (23.0, 31.0) g/l, serum creatinine 78.6 (67.3, 117.8) μmol/l, and eGFR 92.2 (59.6, 100.3) ml/min/1.73m2. Among the 37 PMN patients, 25 (67.6%) patients had positive circulating anti-PLA2R antibodies (＞20 U/ml), and the median antibody level was 150.0 (22.0, 437.5) U/ml. The ELISA method was used to detect C3a in the plasma specimens on the day of renal biopsy, and the results showed that its median level was 1817.7 (1142.9, 6721.4) ng/ml.

The renal biopsy pathological results of all patients were provided by the Pathology Department of Nephrology, Peking University First Hospital, and the results showed that 33 patients (89.2%) had positive glomerular PLA2R staining, with a median staining intensity of 1+. Granular deposition of IgG and C3 was observed in all 37 patients, with a median staining intensity of 2+ and 3+, respectively.

3.4 Expression of glomerular C3aR in PMN patients and its correlation with clinical features and prognosis

The results of correlation analysis showed that the glomerular C3aR level of PMN patients was positively correlated with 24-hour urine protein quantification (r=0.569, P<0.001) and serum creatinine (r=0.642, P<0.001), which was statistically significant; and negatively correlated with plasma albumin (r=-0.3719, P=0.023), which was statistically significant (Figure 3, A and B). The expression level of C3aR was not correlated with anti-PLA2R antibodies (P=0.463) and plasma C3a (P=0.806).

In terms of renal pathology, the expression of C3aR was not correlated with glomerular PLA2R (P=0.467), IgG deposition (P=0.299) and C3 deposition (P=0.333).

After conventional treatment recommended by the KDIGO guidelines (15 patients received oral hormone therapy, 13 patients received oral cyclosporine/tacrolimus therapy, 4 patients received intravenous cyclophosphamide therapy, 12 patients received intravenous rituximab therapy, the above treatments included the same patient receiving two or more drug treatments simultaneously or successively, and 12 patients only received ACEI/ARB), 33 patients (89.2%) achieved remission, of which 13 patients (35.1%) achieved complete remission (CR) and 20 patients (54.1%) achieved partial remission (PR). During follow-up, 6 patients relapsed after remission, accounting for 18.2% of the patients who achieved remission. The expression of C3aR in renal biopsy tissues of PMN patients who achieved remission was lower than that of patients who did not achieve remission at the onset of the disease (P=0.021), which was statistically significant (Figure 3, Panel C). Among patients whose disease remitted after treatment, there was no difference in C3aR levels between CR and PR (P=0.537), and there was no difference in C3aR levels between patients who relapsed after remission and those who did not relapse (P=0.194).

Example 2: Podocyte activity and expression levels in patients with membranous nephropathy.

This example mainly explores the changes in the ability of podocytes to migrate after being stimulated by migration experiments, explores the activity of podocytes at different time points under different stimulation conditions by MTT activity detection, and explores the effect of C3a in the plasma of PMN patients on podocytes from the aspects of cell function and apoptosis. At the same time, this example explores the changes in podocyte cytoskeleton proteins and various cytokines at the mRNA level and protein level respectively by qRT-PCR, Western blot and cell fluorescence technology.

1. Materials and Methods

The conditionally immortalized human podocytes used in this experiment were kindly provided by Professor Jochen Reiser (Rush University Medical Center, Chicago, USA). The following reagents are all commercial kits. The assay methods in the examples were all performed according to the instructions of the commercial kits.

1.1 Collection and preparation of plasma from PMN patients

(1) Plasma samples were collected from 5 PMN patients who underwent renal puncture biopsy and were diagnosed with PMN in the Department of Nephrology of Peking University First Hospital and who did not receive immunosuppressive treatment. The whole blood was collected using sodium heparin anticoagulation tubes and EDTA anticoagulation tubes, respectively, and then placed in a low-temperature environment of 4°C, centrifuged at 2000 rpm/min for 15 minutes, and plasma was collected. After being divided and quickly frozen in a -80°C refrigerator. Plasma samples were collected from 5 healthy control volunteers, and the processing and storage methods were the same as above. Sodium heparin anticoagulation plasma was used for cell stimulation, and EDTA anticoagulation plasma was used to detect C3a concentration.

(2) All plasma samples were filtered using a 0.22 μm filter;

(3) Stimulation solution preparation plan:

PMN patient plasma group:

Patient plasma 10%

Penicillin, Streptomycin 1%

RPMI 1640 medium 89%

PMN patient plasma + C3aR antagonist group (divided into 4 concentration gradients):

Patient plasma 10%

SB290157 (C3aR antagonist) Concentration gradient: 200nM, 400nM, 2000nM, 4000nM

Penicillin, Streptomycin 1%

RPMI 1640 medium 89%

PMN patient plasma + JR14a antagonist group (divided into 4 concentration gradients):

Patient plasma 10%

JR14a (C3aR antagonist) Concentration gradient: 200nM, 400nM, 2000nM, 4000nM

Penicillin, Streptomycin 1%

RPMI 1640 medium 89%

PMN patient plasma + recombinant human C3a protein:

Healthy control plasma

Healthy control plasma 10%

Penicillin, Streptomycin 1%

RPMI 1640 medium 89%

Positive control group

Negative control group

Fetal bovine serum 5%

Penicillin, Streptomycin 1%

RPMI 1640 medium 94%

1.2 Podocyte differentiation and starvation

(1) Podocytes proliferate at 33°C. When the cells have grown to 80%-95% of the bottom area of the culture flask, they are passaged at a ratio of 1:5-6, added with 37°C culture medium, and placed in a 37°C 5% CO2 cell culture incubator for differentiation.

(2) After 10-14 days of differentiation, podocyte differentiation is complete and experiments can be performed;

(3) Before stimulating the cells, synchronize the cells by discarding the culture medium, washing them once with PBS, and starving them for 6 h with RPMI 1640 culture medium without fetal bovine serum;

(4) Discard the synchronization medium and add stimulation solution.

1.3 MTT podocyte activity assay

(1) Equilibrate all reagents stored in a -20°C freezer to room temperature;

(2) Take the cells stimulated for 36 h, 48 h, and 72 h to the clean bench between tissues for manipulation;

(2) Aspirate the culture medium and collect it in a 15 ml centrifuge tube;

(3) Wash three times with pre-cooled 4°C PBS and collect in a centrifuge tube;

(4) Add an appropriate amount of EDTA-free trypsin and place in a 37°C incubator for 1 to 2 minutes. Observe the cell detachment under a microscope. When the cells have basically detached from the bottom of the culture flask, add negative culture medium to terminate the digestion and collect the suspension in a centrifuge tube.

(5) Wash once with 4°C PBS and collect in a centrifuge tube;

(6) Centrifugation at 1200 g for 5 min;

(7) Discard the supernatant and add an appropriate amount of RPMI 1640 medium to resuspend the cells to a cell density of 1-5×10 ⁶ /ml;

(8) Add 50 μl of RP1640 medium containing cells to each well of a 96-well plate, and repeat three times. Because the medium contains phenol red, it is necessary to add background wells (negative RPMI 1640 medium 50 μl/well);

(9) Add 50 μl of MTT reagent/well and culture in an interstitial cell culture incubator at 37°C for 3 h;

(10) Add 150 μl/well of MTT solvent and incubate at 37°C for 15 min in the dark;

(11) Immediately use a microplate reader to detect, set the OD value wavelength to 590 nm and read the value;

(12) The absorbance is proportional to the active cells. Relative cell activity (absorbance) = average of the repeated readings of each sample - average reading of the background wells.

1.4 Detection of mRNA expression of C3aR and other important factors after podocyte stimulation (real-time fluorescence quantitative PCR, i.e., qRT-PCR)

Total RNA was extracted from podocytes after 24h and 48h stimulation, and the expression of C3aR, PLA2R, synapotodin, vinculin, actin-α4, BCL-2, b-catenin, WNT1, WNT2, WNT3, PIK3CA, AKT1, GSK3B, NF-κB, TNF-α, IL-1β, IL-6 and IL-10 at the mRNA level was quantitatively detected by qRT-PCR. GAPDH was used as the internal reference gene.

1.5 Gene expression determination

Total RNA was extracted from cells using the Tiangen Cell Total RNA Extraction Kit DP430, reverse transcribed into cDNA, and gene expression was determined by QRT-PCR.

(1) Obtain the target gene ID from the NCBI website (https://www.ncbi.nlm.nih.gov/), query the primer sequence from the primerbank website, and verify the primer quality in a preliminary experiment. The forward and reverse primers for all target genes are shown in the table below. Dissolve the primers in RNase-free double distilled water according to the instructions on the primer package.

(2) Add the following reagents in sequence to prepare the PCR reaction system (10 μl system):

(3) Run the PCR reaction program: Place the 96-well plate on the PCR instrument and perform the QRT-PCR reaction according to the following conditions:

Standard reaction mode (primer Tm ≥ 60°C)

Melting curve conditions

(4) Calculate the expression level of the target gene:

① Output data in Excel format;

② Calculate the mean CT value of each target gene in each group, negative group and GAPDH;

③ Calculate ΔCT: ΔCT = CT value of each target gene - CT value of the internal reference gene (GAPDH);

④ Calculate ΔΔCT: ΔΔCT = ΔCT of sample group - ΔCT of negative group;

⑤ Calculate the expression level of the target gene: gene expression level = POWER (2, -ΔΔCT).

Table 1 Primer sequence information for PCR amplification of human C3aR and other important factors

1.6 Detection of the expression of C3aR and other proteins after podocyte stimulation (Western blot)

After 48 hours of stimulation, total protein in podocytes was extracted, and the expression of C3aR at the protein level was semi-quantitatively detected by Western blot technology. GAPDH was used as the internal reference protein.

1.7 Detection of PLA2R in human podocytes cultured in vitro (indirect immunofluorescence assay)

(1) Discard the culture medium, add 4°C PBS to wash three times, discard the PBS and place the culture plate at room temperature;

(2) Fixing cells: Add an appropriate amount of 4% paraformaldehyde to each well of the cell culture plate to fix the cells;

(3) Wash with PBS for 3 min × 3 times;

(4) Prepare 3% BSA blocking solution in PBS buffer and filter through a 0.25 μm filter before use;

(5) Nonspecific antigen blocking: incubate in 3% BSA blocking solution at 37°C for 1 h and then aspirate the blocking solution;

(6) Incubation with primary antibody: mouse anti-human PLA2R monoclonal antibody (1:200 dilution), incubate at 4°C overnight and rewarm at room temperature for 45 min;

(7) Wash with PBS for 3 min × 3 times;

(8) Incubation with secondary antibody: goat anti-mouse IgG (H+l) secondary antibody (1:500 dilution) incubated at 37°C for 30 min in the dark;

(9) Wash with PBS for 3 min × 3 times;

(10) Sealing: Use anti-fluorescence quenching sealing medium containing DAPI to seal the slides;

(11) Film reading: Films were read in a dark room using a Leica fluorescence microscope.

2. Results

2.1 Podocyte migration assay

After stimulation with PMN patient plasma, PMN patient plasma + recombinant human C3a protein, and recombinant human C3a protein, the podocyte migration function was reduced compared with the healthy control group and the negative control group. After the addition of C3aR antagonists (SB290157 and JR14a), the migration ability was basically restored to the level of the healthy control group. The addition of 2 μg/ml C3a protein to PMN patient plasma induced more severe damage to podocyte migration function than the plasma group, indicating that PMN plasma and C3a protein have an additive effect (Figure 4, upper panel).

At 72 hours of stimulation, the podocyte migration distance in the PMN patient plasma group was 61.7 (25.0, 111.1) μm, which was significantly lower than that in the healthy control group [61.7 (25.0, 111.1) μm vs. 179.1 (170.2, 185.6) μm, P = 0.001]. After adding different concentrations of C3aR antagonists (SB290157 or JR14a) to the patient plasma, the podocyte migration distance increased to 177.2 (156.1, 196.9) μm, which was significantly higher than that in the patient plasma group (P < 0.001), and basically recovered to the level of the healthy control group [177.2 (156.1, 196.9) μm vs. 179.1 (170.2, 185.6) μm, P = 0.848]. After adding 2 μg/ml recombinant human C3a protein to the patient's plasma, the podocyte migration function further decreased. At 72 hours, the migration distance was 1.1 (-25.0, 65.0) μm, which was lower than that of the patient's plasma group [61.7 (25.0, 111.1) μm vs. 1.1 (-25.0, 65.0) μm, P = 0.146], but not statistically significant; it was significantly lower than that of the healthy control group [179.1 (170.2, 185.6) μm vs. 1.1 (-25.0, 65.0) μm, P < 0.001]. (Figure 4, lower panel)

2.2 MTT cell viability assay

In the MTT experiment, the OD value is proportional to the number of active cells. Since the total number of cells in each group is the same, the OD value can be used to represent the cell activity. At 36 hours of stimulation, the OD values of the PMN patient plasma group, the group with different concentrations of C3aR antagonists added to the patient plasma, the group with 2 μg/ml recombinant human C3a protein added to the PMN patient plasma, and the healthy control group were 1.7 (1.2, 1.8), 1.4 (1.2, 1.6), 1.5 (1.7, 1.1), and 2.2 (1.5, 2.3), respectively, and there was no statistical significance among the groups (all P＞0.05). At 48 hours after stimulation, the OD values of the PMN patient plasma group, the group with different concentrations of C3aR antagonists (SB290157 or JR14a) added to the patient plasma, the group with 2 μg/ml recombinant human C3a protein added to the PMN patient plasma, and the healthy control group were 1.2 (1.1, 1.2), 1.6 (1.3, 2.2), 1.3 (1.3, 1.3), and 1.4 (1.2, 1.5), respectively, with no statistically significant difference among the groups (all P＞0.05). At 72h of stimulation, the OD values of the PMN patient plasma group, the group with different concentrations of C3aR antagonist added to the patient plasma, the group with 2μg/ml recombinant human C3a protein added to the PMN patient plasma, and the healthy control group were 0.3 (0.2, 0.3), 0.4 (0.2, 0.6), 0.1 (0.1, 0.1), and 0.4 (0.3, 0.4), respectively. The podocyte activity in the PMN patient plasma group was significantly lower than that in the healthy control group (P=0.007). After the addition of 3aR antagonist SB290157 (MCE, China, HY-101502A) or C3aR antagonist JR14a (MCE, China, HY-138161), the cell activity was higher than that of the plasma group (P = 0.232), but there was no statistical significance, and the cell activity level was equivalent to that of the healthy control group (P = 0.984); after the addition of recombinant human C3a protein to the plasma, the cell activity was further reduced compared with the plasma group, with significant statistical significance (P = 0.003). Figure 5 shows the relative activity of podocytes at 36h, 48h and 72h of stimulation under various stimulation conditions.

2.3 Changes in mRNA levels of podocyte cytoskeleton proteins and cytokines

After 48 h of PMN patient plasma stimulation, the expression of C3aR mRNA in podocytes was upregulated compared with that in the healthy control group [1.1 (0.8, 1.3) vs. 0.6 (0.6, 0.7), P = 0.011]. After the addition of different concentrations of C3aR antagonists (SB290157 or JR14a), the expression level was significantly decreased [1.1 (0.8, 1.3) vs. 0.6 (0.4, 0.8), P = 0.001] (Figure 6A).

After 48 hours of PMN patient plasma stimulation, the expression of PLA2R mRNA in podocytes was significantly upregulated compared with that in the healthy control group [17.3 (17.1, 19.8) vs. 0.9 (0.6, 1.1), P < 0.001]. After the addition of antagonists, the expression level was reduced [17.3 (17.1, 19.8) vs. 1.2 (0.4, 2.2), P = 0.027] (Figure 6B).

After 48 h of PMN patient plasma stimulation, the expression of Bcl-2 mRNA in podocytes was downregulated compared with that in healthy controls, but without statistical significance [0.9 (0.4, 0.9) vs. 1.1 (0.4, 1.6), P = 0.332]. The addition of higher concentrations of antagonists (2000 nM and 4000 nM SB290157, 100 nM and 200 nM After JR14a), its expression level increased [0.9 (0.4, 0.9) vs. 2.2 (0.3, 2.6), P = 0.038], and the Bcl-2 mRNA level in the antagonist group was equivalent to that in the healthy control group [2.2 (0.3, 2.6) vs. 1.1 (0.4, 1.6), P = 0.950]; the Bcl-2 mRNA expression level in the PMN patient plasma group after adding 2 μg/ml recombinant human C3a protein was further downregulated compared with the plasma group [0.4 (0.2, 0.5) vs. 0.9 (0.4, 0.9), P = 0.037] (Figure 6, C).

After 48 hours of PMN patient plasma stimulation, the mRNA expression of synapotodin, a podocyte skeleton protein, was downregulated compared with that of the healthy control group [1.8 (0.8, 2.1) vs. 7.0 (4.2, 8.5), P = 0.020]. After the addition of antagonists, the expression level increased [1.8 (0.8, 2.1) vs. 5.7 (4.2, 8.6), P = 0.020]. The synapotodin level in the antagonist group was equivalent to that in the healthy control group [5.7 (4.2, 8.6) vs. 7.0 (4.2, 8.5), P = 0.863]. When 2 μg/ml recombinant human C3a protein was added to the plasma of PMN patients, the expression of synapotodin mRNA in the podocyte skeleton protein was downregulated compared with that in the healthy control group [1.8 (0.8, 2.1) vs. 5.7 (4.2, 8.6), P = 0.020]. The mRNA expression level was further downregulated compared with the plasma group [1.8 (0.8, 2.1) vs. 0.6 (0.0, 1.1), P = 0.105], but there was no statistical significance (Figure 6 D).

PMN patient plasma induced activation of the podocyte WNT/β-catenin pathway. After 48 h of PMN patient plasma stimulation, the expression of WNT3 mRNA in podocytes was significantly increased compared with that in the healthy control group [1.2 (0.9, 1.2) vs. 0.1 (0.1, 0.1), P = 0.008], and the expression was reduced after the addition of antagonists [1.2 (0.9, 1.2) vs. 0.6 (0.4, 0.9), P = 0.035] (Figure 6E). The changes in β-catenin mRNA expression were consistent with those of WNT3. The expression level in the plasma group was higher than that in the healthy control group [0.8 (0.8, 0.8) vs. 0.4 (0.4, 0.5), P = 0.037]. After the addition of the antagonist, the expression level decreased [0.8 (0.8, 0.8) vs. 0.6 (0.4, 0.7), P = 0.069], but there was no statistical significance (Figure 6F).

In addition to the above factors, the changes of vinculin, WNT1, WNT2, PIK3CA, AKT1, GSK3β, NF-κB, TNF-α, IL-1β, IL-6 and IL-10 mRNA among the groups were also detected, and no changing trend was observed.

2.4 Changes in protein levels in podocytes

After 48 hours of PMN patient plasma stimulation, Western blot results (Figure 7A) showed that the C3aR protein content in podocytes was significantly increased compared with the healthy control group and the negative control group [C3aR/GAPDH IOD: 1.2 (1.1, 1.4) vs. 0.2 (0.1, 0.3), P < 0.001)]; after adding different concentrations of C3aR antagonists SB290157 or JR14a, the C3aR protein level decreased [1.2 (1.1, 1.4) vs. 0.7 (0.3, 1.0), P = 0.259)], but there was no statistical significance; the C3aR protein content in the antagonist JR14a group was reduced [1.2 (1.1, 1.4) vs. 0.3 (0.2, 0.5), P = 0.020)] (Figure 7B).

After 48 h of PMN patient plasma stimulation, Western blot results showed that the PLA2R protein content in podocytes was significantly increased compared with the healthy control group and the negative control group [PLA2R/GAPDH IOD: 2.0 (1.7, 2.2) vs. 0.0 (0.0, 0.1), P < 0.001)]. After adding different concentrations of C3aR antagonist, the PLA2R protein content was decreased [2.0 (1.7, 2.2) vs. 1.0 (0.8, 1.5), P = 0.012)] (Figure 8, Panel B).

After 48 hours of PMN patient plasma stimulation, Western blot results showed that the content of podocyte skeletal protein synaptopodin was significantly lower than that of the healthy control group and the negative control group [synaptopodin/GAPDH IOD: 0.0 (0.0, 0.1) vs. 2.2 (2.7, 1.1), P < 0.001)]. After adding different concentrations of C3aR antagonists, the synaptopodin protein content increased [0.0 (0.0, 0.1) vs. 1.1 (0.4, 2.7), P = 0.021)] (Figure 8, Panel B).

2.5 Results of podocyte PLA2R immunofluorescence staining

After 48 h of PMN patient plasma stimulation, the results of podocyte immunofluorescence showed that the relative IOD value of PLA2R was higher than that of the healthy control group [9.0 (7.6, 9.7) vs. 3.0 (0.8, 3.1), P = 0.030]. After adding different concentrations of C3aR antagonists (SB290157 or JR14a), the IOD value was significantly decreased [9.0 (7.6, 9.7) vs. 6.2 (3.8, 7.2), P = 0.007]. The relative IOD value of PLA2R in the antagonist group was still higher than that in the healthy control group, but there was no statistical significance [6.2 (3.8, 7.2) vs. 3.0 (0.8, 3.1), P = 0.068] (Figure 8, Panel C).

The results of this example show that PMN patient plasma induced in vitro culture of human podocytes C3aR and PLA2R upregulation, anti-apoptotic factor Bcl-2 and skeleton protein synaptopodin downregulation, and activated the Wnt/β-catenin pathway, resulting in decreased podocyte migration function and decreased cell activity. After adding C3aR-specific antagonists SB290157 and JR14a to the patient's plasma, the C3a/C3aR pathway was antagonized, and the expression levels of C3aR, skeleton protein synapotodin, and anti-apoptotic factor Bcl-2 were basically restored to the level of the healthy control group, and the expression of PLA2R was partially reduced, but still higher than that of the healthy control group; the Wnt/β-catenin pathway was inhibited; the podocyte migration function and cell activity were basically restored to the level of the healthy control group. Based on this, it is inferred that C3a in the plasma of PMN patients stimulates the upregulation of C3aR expression on the podocyte membrane, activates the C3a/C3aR pathway, induces the activation of the Wnt/β-catenin pathway, and induces the upregulation of PLA2R expression, the downregulation of the anti-apoptotic factor Bcl-2 and the skeleton protein synaptopodin, which ultimately leads to podocyte structural damage, decreased migration function, and decreased cell activity. The inventors believe that C3a in the plasma of PMN patients can activate the Wnt/β-catenin pathway by acting with C3aR, upregulate the expression of C3aR and PLA2R, downregulate the anti-apoptotic factor Bcl-2 and the skeleton protein synapotodin, leading to decreased podocyte migration function and decreased cell activity. After adding C3aR-specific antagonists to the plasma of PMN patients, the above effects can be blocked and podocyte damage can be alleviated.

Example 3: C3aR antagonist treatment of Heimann nephritis (PHN) rats

The most widely used PMN animal model is the active or passive Heymann nephritis (HN) rat model constructed by injecting exogenous proteins. This example adopts the PHN model and uses C3aR antagonists to block the C3a/C3aR pathway to explore the therapeutic effect of C3aR antagonists on PMN and its mechanism of action. Heymann nephritis rats were treated with intraperitoneal injection of C3aR antagonist SB290157 30mg/kg/d or oral administration of C3aR antagonist JR14a 10mg/kg/d.

1. Materials and Methods

1.1 Research subjects

Thirty 5-week-old SPF male Sprague-Dawley rats weighing 130-150 g were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., with the laboratory animal production license number: SCXK (Beijing) 2016-0006.

All animal experiments involved in this study were approved by the Animal Ethics Committee of Peking University First Hospital, and the Experimental Animal Welfare Ethics Review Acceptance Number is 202073.

Experimental Materials

1.2 Experimental methods

1.2.1 Experimental design of HN rat model

SB290157: 30 5-week-old male Sprague-Dawley rats were randomly divided into 3 groups, namely model group (18 rats), early treatment group (6 rats), and negative control group (6 rats). The model group and early treatment group were given a single tail vein injection of sheep anti-rat Fx1a antibody serum (sheep anti-rat Fx1A serum PTX-002S, Ptobetex, USA) 0.8ml/100g body weight to construct PHN. The time of serum injection was defined as day 0. The negative control group was injected with the same dose of normal saline in the same way on day 0. In the early treatment group, C3aR antagonist SB290157 was intraperitoneally injected at a fixed time point every day from day 0 until death. Blood and 24-hour urine specimens were collected regularly. On the 21st day, 18 rats in the model group were sampled stratified according to urine protein levels, and 8 rats were randomly selected as the late treatment group. Starting from the 22nd day, the late treatment group received intraperitoneal injections of C3aR antagonist at a fixed time point every day, 30 mg/kg body weight, until they were killed.

JR14a: Early treatment group, C3aR antagonist JR14a was gavaged at a fixed time point every day from day 0, 10 mg/kg/d body weight, until sacrifice. Blood and 24h urine samples were collected regularly. On day 21, 6 rats were selected as the late treatment group according to the stratified sampling of urine protein levels from the 14 rats in the model group. Starting from day 22, the late treatment group was gavaged with C3aR antagonist JR14a at a fixed time point every day, 10 mg/kg/d body weight, until sacrifice.

Successful model construction was defined as follows: 24-hour proteinuria ≥ 100 mg on day 21.

1.2.2 Plasma specimens

The plasma samples of rats were collected one day before the model was established (day -1). No blood was collected within 2 weeks after the injection of anti-Fx1a antibody to prevent drug loss. After that, blood was collected once every 7 days (Day 14, 21, 28, 35, 42, 49). After the rats were anesthetized by intraperitoneal injection of 5% pentobarbital 1.0 ml/kg, blood was collected from the inner canthus after the rats lost consciousness. Whole blood was collected using EDTA and sodium heparin anticoagulation blood collection tubes, respectively, and centrifuged at 4°C 2000 rpm/min for 15 minutes. The upper plasma was collected, aliquoted and frozen in a -80°C refrigerator.

1.2.3 Urine specimen

The 24-hour urine samples of rats were collected one day before the model was established (day -1), and then collected once a week (days 7, 14, 21, 28, 35, 42, and 49). Using rat metabolic cages, each rat was individually housed in a metabolic cage with sufficient feed and drinking water for 24 hours. During the collection period, all urine samples of the rats were placed in centrifuge tubes and centrifuged at 4°C 1500 rpm/min for 15 minutes. The urine volume was recorded, and the supernatant was collected, packaged, and frozen in a -40°C refrigerator.

1.2.4 Kidney specimens

(1) On day 49, rats were anesthetized with 5% pentobarbital (1 ml/kg body weight) intraperitoneally. After the rats lost consciousness, the abdominal cavity was opened and blood was collected through the abdominal aorta using a venipuncture needle and a 20 ml syringe. The rats died after blood loss and the venipuncture needle was left in place.

(2) Bluntly dissect the left kidney of the rat, cut out three pieces of renal cortical tissue with a thickness of about 3 to 5 mm, and fix them in 4% paraformaldehyde for subsequent preparation of paraffin tissue embedding and sectioning; put them in a 1.5 ml EP tube and immediately freeze them in liquid nitrogen for subsequent preparation of frozen tissue embedding and sectioning; put them in electron microscopy solution for fixation and save them for subsequent preparation of electron microscopy specimens; mince the remaining renal tissue, put them in a 1.5 ml EP tube and immediately freeze them in liquid nitrogen for later use; use hemostatic forceps to clamp the left renal artery and vein;

(3) Physiological saline was perfused into the rat's blood vessels through a venous puncture needle placed in the abdominal aorta until the right kidney became white and swollen. The right kidney of the rat after perfusion was bluntly dissected, chopped, placed in a 1.5 ml EP tube, and immediately frozen in liquid nitrogen for later use.

1.2.5 24-hour proteinuria in rats

(1) Collect 24-hour urine of rats using metabolic cages and record urine volume;

(2) Use a pipette to take 100 μl of clarified urine into a biochemical cup;

(3) The test was carried out using the biuret method using an automatic biochemical analyzer. This part of the experiment was commissioned to be completed by a test technician.

1.2.6 Detection of rat glomerular microstructural changes (electron microscopy)

(1) Sample preparation: The rats were killed and the kidneys were removed within 3 min. A 2 mm × 2 mm piece of cortical tissue as thin as possible was taken. The tissue was immediately placed in electron microscopy fixative for 2 h and then stored at 4°C to avoid freezing.

(2) Wuhan Saiweier Biotechnology Co., Ltd. was commissioned to prepare electron microscopic specimens and perform transmission electron microscopic scanning, with 5 random scanning electron microscopic photos at each magnification for each rat kidney specimen;

(3) Semi-quantitative measurement and analysis of the following items:

①The number of subepithelial electron-dense deposits;

②Basement membrane thickness;

③ Foot process width

1.2.7 Detection of rat anti-sheep anti-Fx1A serum IgG concentration in rat plasma (ELISA method)

(1) Reagent preparation:

0.05M bicarbonate buffer (pH = 9.6)

NaHCO ₃ 2.95 g

Na ₂ CO ₃ 1.08g

ddH ₂ O 900ml

(2) Antigen coating: Antigen-coated wells: Dilute sheep anti-Fx1A serum in carbonate buffer (pH = 9.6) at a ratio of 1:300 to coat half of the ELISA plate; Non-antigen-coated wells: Dilute 1% BSA in carbonate buffer to coat the other half of the ELISA plate; Coating solution 100 μl/well, 4°C overnight;

(3) Washing: Wash the plate three times with 0.1% PBST (1 ml Tween-20 added to 1000 ml PBS), 300 μl/well, 90 s/time, and pat dry on absorbent paper after each wash;

(4) Blocking: Add 1% BSA solution (diluted with PBST) to each well of the ELISA plate, 100 μl/well, and incubate at 37°C for 1 h;

(5) Pat the ELISA plate dry, dilute the plasma sample to be tested with 1% BSA solution at a ratio of 1:50, 100 μl/well, and gently shake at 37°C for 1 h at a shaking speed of 100 rpm;

(6) Washing: Wash the plate three times with PBST and pat dry;

(7) Incubation with secondary antibody: Alkaline phosphatase-labeled rabbit anti-rat IgG was diluted with 1% BSA solution at a ratio of 1:5000, added to the ELISA plate at 100 μl/well, and incubated at 37°C for 1 h;

(8) Washing: Wash the plate three times with PBST and pat dry;

(9) Add alkaline phosphatase colorimetric solution, 100 μl/well, and incubate in the dark;

(10) Immediately use a microplate reader to detect, set the OD value wavelength to 405 nm and read continuously;

(11) Result calculation: A negative control without adding plasma samples should be set up for each plate. The OD value of each sample = OD of antigen-coated well - OD of non-antigen-coated well - OD of negative well; where OD of negative well = OD of negative antigen-coated well - OD of negative non-antigen-coated well;

(12) Threshold: The plasma of 6 rats in the negative control group was tested. The OD threshold for antibody positivity was the mean of the normal control OD value plus 3 times the standard deviation.

1.2.8 Detection of total IgG concentration in rat plasma (ELISA method)

(1) After taking the kit out of the refrigerator, it should be equilibrated at room temperature for 20 minutes;

(2) dilute the concentrated washing solution with deionized water at a ratio of 1:19;

(3) Preparation of standards of different concentrations: centrifuge the lyophilized powder of IgG standard at 10,000 rpm/min for 1 min, dilute with standard diluent according to the reagent reconstitution label, let stand at room temperature for 15 to 20 min, then gently suspend until completely dissolved, and dilute the standard with standard diluent in a multiple-ratio gradient, with the standard diluent as the zero concentration;

(5) The whole blood of rats was anticoagulated with EDTA, centrifuged at 2000 rpm/min for 15 min, and quickly frozen in a −80°C refrigerator after aliquoting. The frozen plasma was taken out of the refrigerator, thawed at 4°C to avoid repeated freezing and thawing, and diluted 10,000 times;

(6) Add the diluted samples or standards of different concentrations to the coated 96-well plate at 100 μl/well, and duplicate each sample in 3 wells. Seal the reaction wells with a sealing film and incubate at 37°C for 90 min.

(7) Shake off the samples and standards and pat dry on thick absorbent paper;

(8) Add biotinylated antibody working solution (100 μl/well), seal the reaction wells with sealing tape, and incubate at 37°C for 60 min.

(9) Wash the plate 5 times, patting it dry on thick absorbent paper after the last wash;

(10) Add avidin-linked HRP enzyme working solution (100 μl/well), seal the reaction wells with sealing tape, and incubate at room temperature for 30 min in the dark;

(11) Wash the plate 5 times, patting it dry on thick absorbent paper after the last wash;

(12) Add the colorimetric reagent TMB (100 μl/well) and incubate at room temperature in the dark for 15-30 min;

(13) Add stop solution (50 μl/well), mix well, and immediately use a microplate reader to detect, set the OD value wavelength to 405 nm and read the value;

(14) Use EIA software to draw a standard curve, with the standard concentration as the horizontal axis and the OD value as the vertical axis. Use a smooth line to connect the coordinate points of each standard. The total IgG concentration of the specimen can be found on the standard curve through the OD value of the specimen. The measured concentration is multiplied by the sample dilution factor (3) to obtain the true total IgG concentration of the plasma sample.

1.2.9 Detection of C3a concentration in rat plasma (ELISA method)

(1) The kit (rat C3a ELISA kit, Novus biologicals, USA) should be equilibrated at room temperature for 20 min after being taken out of the refrigerator;

(2) dilute the concentrated washing solution with deionized water at a ratio of 1:24;

(3) Preparation of standards of different concentrations: centrifuge the lyophilized powder of C3a standard at 10000 rpm/min for 1 min, dilute with standard diluent according to the reagent reconstitution label to make the final concentration of C3a reach 40 ng/ml, let stand at room temperature for 15-20 min, then gently suspend until completely dissolved, dilute the standard with standard diluent in multiple ratios to 20 ng/ml, 10 ng/ml, 5 ng/ml, 2.5 ng/ml, and 1.25 ng/ml, and use the standard diluent as the zero concentration;

(5) The whole blood of rats was anticoagulated with EDTA, centrifuged at 2000 rpm/min for 15 min, and quickly frozen in a −80°C refrigerator after aliquoting. The frozen plasma was taken out of the refrigerator, thawed at 4°C, avoiding repeated freezing and thawing, and diluted 3 times;

(6) Add the diluted samples or standards of different concentrations to the coated 96-well plate at 100 μl/well, and duplicate each sample in 3 wells. Seal the reaction wells with a sealing film and incubate at 37°C for 90 min.

(7) Shake off the samples and standards and pat dry on thick absorbent paper;

(8) Add biotinylated antibody working solution (100 μl/well), seal the reaction wells with sealing tape, and incubate at 37°C for 60 min;

(9) Wash the plate 5 times, patting it dry on thick absorbent paper after the last wash;

(10) Add avidin-linked HRP enzyme working solution (100 μl/well), seal the reaction wells with sealing tape, and incubate at room temperature for 30 min in the dark;

(11) Wash the plate 5 times, patting it dry on thick absorbent paper after the last wash;

(12) Add the colorimetric reagent TMB (100 μl/well) and incubate at room temperature in the dark for 15-30 min;

(13) Add stop solution (50 μl/well), mix well, and immediately use a microplate reader to detect, set the OD value wavelength to 405 nm and read the value;

(14) Use EIA software to draw a standard curve, with the standard concentration as the horizontal axis and the OD value as the vertical axis. Use a smooth line to connect the coordinate points of each standard. The C3a concentration of the sample can be found on the standard curve through the OD value of the sample. The measured concentration is multiplied by the sample dilution factor (3) to obtain the actual C3a concentration of the plasma sample.

1.2.10 Detection of IgG, complement molecules and immune cell marker protein levels in rat glomeruli

Detection of rat IgG, sheep IgG and complement molecules including C3aR, MAC, C3d, C3, C4d, C1q, factor B, MBL and C5aR in rat kidney paraffin or frozen sections.

1.2.11 Detection of rat IgG, sheep IgG and C3C in glomeruli of rat kidney frozen sections (direct immunofluorescence method)

(1) Take out the slices stored at -40℃ or -20℃;

(2) Fix tissue: Place the slices in pre-cooled acetone at -20°C for 15 min.

(3) Air-dry the slices in a fume hood to evaporate the acetone.

(4) Wash with PBS for 3 min × 3 times;

(5) Prepare 3% BSA blocking solution in PBS buffer and filter through a 0.25 μm filter before use;

(6) Nonspecific antigen blocking: incubate with 3% BSA blocking solution at 37°C for 1 h and then shake off;

(7) Incubation antibodies: FITC-labeled goat anti-rat IgG monoclonal antibody (1:50 dilution)/alexa flour 488-labeled rabbit anti-sheep IgG antibody (1:150 dilution)/FITC fluorescent anti-C3c antibody (1:500 dilution), incubate at 37°C in the dark for 1 h;

(8) Wash with PBS for 3 min × 3 times;

(9) Sealing: Use anti-fluorescence quenching sealing medium containing DAPI to seal the slides;

(10) Film reading: Films were read in a dark room using a Leica fluorescence microscope.

1.2.12 Rat kidney paraffin section specimen glomerulus C3aR is the same as in Example 1. Incubation with primary antibody: mouse anti-rat C5aR1 monoclonal antibody (1:100 dilution)/rabbit anti-rat C1q polyclonal antibody (1:100 dilution).

1.2.13 Detection of C3aR, C5b-9, C3d, C4d, factor B, MBL, CD68 (macrophages), Foxp3 (regulatory T cells), CD4 (helper T cells) and CD8 (cytotoxic T cells) in glomerular paraffin sections of rat kidneys (immunohistochemistry) The experimental procedures were the same as those in Example 1.

Primary antibody incubation: mouse anti-rat C3aR monoclonal antibody (Santa Cruz, USA) (1:500 dilution, EDTA solution high temperature and high pressure for 3 min repair) / mouse anti-rat C5b-9 monoclonal antibody (HycultBiotech, China) (1:50 dilution, 0.4 mg/ml proteinase K incubated at 37°C for 5 min repair) / goat anti-rat C3d monoclonal antibody (R&D, USA) (1:20 dilution, 0.4 mg/ml proteinase K 37℃ incubation for 5 min for repair)/rabbit anti-rat C4d monoclonal antibody (CST, USA) (1:100 dilution, EDTA solution high temperature and high pressure for 3 min for repair)/rabbit anti-rat factor B monoclonal antibody (Abcam, USA) (1:200 dilution, citric acid solution high temperature and high pressure for 3 min for repair)/rabbit anti-rat MBL monoclonal antibody (Abcam, USA) (1:200 dilution, EDTA microwave high temperature for 2 min + thawing for 8 min for repair)/mouse anti-rat CD68 monoclonal antibody (BIO-RAD, USA) (1:100 dilution, pepsin incubation at 37℃ for 30 min for repair)/mouse anti-rat Foxp3 monoclonal antibody (Abcam, USA) (1:1000 dilution, EDTA solution high temperature and high pressure for 3 min for repair)/rabbit anti-rat CD4 monoclonal antibody (CST, USA) (1:400 dilution, citric acid solution high temperature and high pressure for 3 min for repair)/mouse anti-rat CD8 monoclonal antibody (Santa Cruz, USA) (1:100 dilution, citric acid solution high temperature and high pressure for 3 min for repair).

2. Results

2.1 Construction of PHN disease model using sheep anti-Fx1A antibody serum

When constructing the PHN rat disease model, the disease control group, early treatment group and late treatment group were injected with the same dose of sheep anti-Fx1A antibody serum, and the negative control group was injected with the same dose of normal saline.

The level of sheep IgG in rat glomeruli was detected by immunofluorescence method. The results showed that the deposition of sheep IgG in the disease control group, early treatment group and late treatment group was higher than that in the negative control group, which was statistically significant (P<0.001). There was no statistical difference among the disease group, early treatment group and late treatment group (P values were 0.108, 0.239 and 0.875, respectively). This shows that the amount of antibody serum injected into the three groups of rats when constructing the model was equivalent, and there was no statistical difference in the level of sheep IgG that effectively entered the rats and was deposited on the glomeruli. The severity of the disease model was consistent among the groups, and the subsequent treatment effect of the C3aR antagonist was not affected by the severity of the disease model among the groups.

2.2 C3aR antagonists can effectively reduce symptoms and renal damage in PHN rats

2.2.1 C3aR antagonists can effectively reduce urine protein levels in rats with PHN

PHN rats are mainly manifested by a significant increase in urine protein. In this study, 24-hour urine was collected from rats to determine the severity of the disease from clinical manifestations by testing the urine protein level of rats. At the same time, the levels of plasma creatinine, urea nitrogen, and total cholesterol in rats were tested to explore the changes in blood biochemical indicators such as renal function and blood lipids in PHN rats, C3aR antagonists SB290157 and JR14a early and late treatment groups, and negative control group rats.

The PHN model was established in SD rats by a single intravenous injection of sheep anti-Fx1A antibody serum. At week 3, all rats developed proteinuria, which gradually worsened. At week 7, compared with the negative control rats, the disease control rats developed severe proteinuria [SB290157: 299.1 (237.2, 367.0) vs. 94.0 (35.2, 119.5) mg/d, P < 0.001; JR14a: 484.9 (460.7, 512.6) vs. 195.1 (179.2, 221.5) mg/d, P < 0.001], while serum creatinine, blood urea nitrogen and total cholesterol were within the normal range (P > 0.05).

The early treatment group was given C3aR antagonist from day 0, and the proteinuria of rats was significantly reduced at week 7 compared with the disease control group [SB290157: 299.1 (237.2, 367.0) vs. 120.6 (76.0, 161.0) mg/d, P<0.001; JR14a: 484.9 (460.7, 512.6) vs. 282.6 (247.3, 306.0) mg/d, P<0.001]. At week 3, all rats in the untreated model group reached the successful standard of HN model construction (proteinuria> 100 mg/24h), and were randomly divided into the disease control group and the late treatment group. There was no difference in proteinuria between the two groups. The late treatment group began to be given C3aR antagonist at week 3. The proteinuria level in the late-stage treatment group was lower than that in the disease control group [SB290157: 238.6 (166.7, 346.5) vs. 196.3 (119.1, 289.8) mg/d, P = 0.0.039; JR14a: 513.1 (324.6, 571.6) vs. 255.3 (308.8, 345.0) mg/d, P < 0.001] (Figure 9).

2.2.2 C3aR antagonists significantly reduce glomerular damage in rats with PHN

Similar to the glomerular lesions in PMN patients, the renal lesions in PHN rats are mainly manifested as subepithelial immune complex deposition, foot process fusion, and GBM thickening. Therefore, this experiment performed electron microscopy on the kidney specimens of experimental rats and analyzed the severity of the disease from the following three perspectives.

2.2.3 Decreased number of subepithelial immune complex deposits

The results of this experiment showed that the number of immune complex deposits under the glomerular epithelium of rats in the PHN disease group was significantly higher than that in the negative control group (SB290157: P < 0.001; JR14a: P < 0.001). After early or late treatment, the number of immune complex deposits was significantly reduced (early treatment group: SB290157: P < 0.001; JR14a: P < 0.001; late treatment group: SB290157: P < 0.001; JR14a: P < 0.001) (Figure 10 A and D).

2.2.4 Reduced foot process fusion

The results of this experiment showed that the width of the glomerular foot process of the rats in the PHN disease group was the largest, significantly wider than that of the negative control group (SB290157: P < 0.001; JR14a: P < 0.001), indicating that the foot process fusion of the kidneys of the diseased rats was serious. The width of the foot process was reduced in both the early treatment group and the late treatment group (early treatment group: SB290157: P = 0.012; JR14a: P < 0.001; late treatment group: SB290157: P = 0.002; JR14a: P = 0.001) (Figure 10 B and E).

2.2.5 Reduction of basement membrane thickening

The results of this experiment showed that the GBM thickness of the PHN disease group was the largest and significantly wider than that of the negative control group (SB290157: P = 0.005; JR14a: P < 0.001), indicating that the GBM of the diseased rat kidney was thickened. After treatment with C3aR antagonists, the thickening of GBM in the early treatment group and the late treatment group was reduced, among which the treatment effect of JR14a was more obvious (early treatment group: SB290157: P = 0.160; JR14a: P < 0.001; late treatment group: SB290157: P = 0.998; JR14a: P = 0.002) (Figure 10 C and F).

Electron microscopic results showed that the renal lesions of PHN rats were consistent with those of PMN patients, showing electron-dense deposition, foot process fusion, and basement membrane thickening. Treatment with C3aR antagonists SB290157 and JR14a effectively alleviated renal damage in rats with the disease, and the effect of early treatment was more significant than that of late treatment (Figure 10).

3. C3aR antagonists inhibit circulating C3a and glomerular C3aR levels in PHN rats

Similar to human primary MN, the C3a/C3aR pathway was activated in PHN rats. At week 7, compared with the negative control group, the plasma C3a [SB290157: 42.3 (36.1, 50.5) vs. 11.3 (3.5, 14.1) ng/ml, P < 0.001; JR14a: 33.6 (30.1, 36.3) vs. 5.6 (4.9, 5.8) mg/d, P < 0.001] and glomerular C3aR [SB290157: P < 0.001; JR14a: P = 0.050] of the disease group rats were significantly increased.

C3aR antagonists can inhibit the C3a/C3aR pathway. Compared with the disease control group, the plasma C3a level in the treatment group was reduced [early treatment SB290157: 42.3 (36.1, 50.5) vs. 25.5 (18.6, 29.6) ng/ml, P = 0.001; late treatment SB290157: 42.3 (36.1, 50.5) vs. 20.5 (16.6, 28.3) ng/ml, P < 0.001; early treatment JR14a: 33.6 (30.1, 36.3) vs. 18.1 (14.5, 24.2) mg/d, P = 0.001; early treatment JR14a: 33.2) vs. 32.2] (21.3, 37.9) mg/d, P = 0.476]. C3aR staining was reduced in the treatment groups (early treatment SB290157: P < 0.001; late treatment SB290157: P < 0.001; early treatment JR14a: P = 0.032; early treatment JR14a: P = 0.221).

4. C3aR antagonists inhibit the production of specific anti-sheep IgG and complement activation in the circulation of PHN rats

After injection of sheep anti-Fx1A antibody serum, the experimental rats underwent passive immune response and produced anti-sheep IgG antibodies. The specific antibodies entered the subepithelial tissue through GBM to form immune complexes, activated the complement system, and ultimately led to kidney damage. The levels of specific anti-sheep IgG and total IgG in the circulation of rats were detected by ELISA at 2, 5, and 7 weeks after passive immunization. The results showed that the negative control group rats basically did not produce anti-sheep IgG, and the anti-sheep IgG levels in the disease control group, early treatment group, and late treatment group were significantly higher than those in the negative control group (Figures 11-12). Throughout the experiment, the level of anti-sheep IgG antibodies in the plasma of rats in the disease control group was stable (P>0.05). There was no statistical difference in the total IgG level among the groups (P>0.05) (Figure 13).

In the treatment groups, sheep anti-Fx1A antibodies on days 14 and 35 were comparable to those in the PHN disease group (P>0.005). On day 49, specific IgG levels were significantly reduced (early treatment SB290157: P=0.015; late treatment SB290157: P<0.001; early treatment JR14a: P=0.009; late treatment JR14a: P=0.067). Subsequently, due to the decrease in specific IgG, the overexpression of C1q (P=0.037; P=0.190; P=0.023; P=0.011), C5b-9 (P=0.013; P=0.004; P=0.049; P=0.045) and factor B (P=0.007; P=0.011; P<0.001; P=0.007) was also inhibited. The total IgG level was not affected. Antagonizing the C3a/C3aR pathway affects the function of DC cells, Treg cells, and B cells, and reduces the production of specific IgG. However, the total IgG level in rat plasma was not affected by the C3aR blocker, and no side effects such as infection were observed in all experimental rats. It is speculated that the C3aR antagonist only affects the production of specific IgG induced by passive immunity and does not affect the natural immune process. After using the C3aR antagonist, the glomerular C5aR, C3d, C4d, and MBL of the diseased rats were measured. After treatment with the C3aR antagonist, there was no significant change in the levels of C5aR, C3d, C4d, and MBL in the rat glomeruli, as shown in Figure 14.

5. Changes of glomerular infiltrating immune cells in PHN rats after C3aR antagonist treatment

After treatment with C3aR antagonist, there was no significant change in the levels of Foxp2 (regulatory T cell marker protein), CD68 (macrophage marker protein), CD4 (helper T cell marker protein) and CD8 (cytotoxic T cell marker protein) in rat glomeruli, indicating that there was no statistically significant change in the levels of regulatory T cells, macrophages, helper T cells and cytotoxic T cells.

Animal experiments on PHN rats have shown that C3aR antagonist treatment significantly reduces the level of proteinuria and the degree of renal pathological damage in PHN rats, effectively inhibits the expression of C3aR in the renal cortex of PHN rats, alleviates podocyte damage, and reduces the production and renal deposition of specific IgG in the circulation of PHN rats.

It should be understood that although the invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate rather than limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the appended claims.

Sequence Listing

<110> Peking University

<120> Use of C3a/C3aR pathway antagonists in the treatment of primary membranous nephropathy

<130> C22P1819

<160> 38

<170> PatentIn version 3.5

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<223> IL-1β forward primer

<400> 31

gccagtgaaa tgatggctta tt 22

<210> 32

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> IL-1β reverse primer

<400> 32

aggagcactt catctgttta gg 22

<210> 33

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> IL-6 forward primer

<400> 33

cactggtctt ttggagtttg ag 22

<210> 34

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> IL-6 reverse primer

<400> 34

ggacttttgt actcatctgc ac 22

<210> 35

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> IL-10 forward primer

<400> 35

gttgttaaag gagtccttgc tg 22

<210> 36

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> IL-10 reverse primer

<400> 36

ttcacaggga agaaatcgat ga 22

<210> 37

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> GAPDH forward primer

<400> 37

aatgcctcct gcaccaccaa 20

<210> 38

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> GAPDH reverse primer

<400> 38

accatgagtc cttccacgat acc 23

Claims (8)

1. Use of a c3a/C3aR pathway antagonist or a pharmaceutical composition comprising a C3a/C3aR pathway antagonist for the manufacture of a medicament for treating or preventing membranous nephropathy in a subject, wherein the C3a/C3aR pathway antagonist is a specific C3aR antagonist, the specific C3aR antagonist selected from the group consisting of: n2- [ (2, 2-biphenylethoxy) acetyl ] -L-arginine, 5- (bis (4-chlorophenyl) methyl) -3-methylthiophene-2-carbonyl) -L-arginine, or a pharmaceutically acceptable salt thereof.
2. 2. The use of claim 1, wherein the C3aR antagonist is N2- [ (2, 2-biphenylethoxy) acetyl ] -L-arginine trifluoroacetate.
3. 3. The use according to any one of claims 1-2, wherein the medicament is an oral or parenteral medicament.
4. 4. The use according to any one of claims 1-2, wherein the medicament for parenteral administration is a medicament for subcutaneous, intradermal, intravenous, intramuscular, intraarterial or infusion administration.
5. 5. The use of any one of claims 1-2, wherein the subject is a human or a mammal.
6. 6. The use according to any one of claims 1-2, wherein membranous nephropathy is primary membranous nephropathy.
7. 7. The use according to any one of claims 1-2, wherein the pharmaceutical composition comprises one or more of CD20 mab, calmodulin inhibitors, glucocorticoids, mycophenolate mofetil, cyclophosphamide, angiotensin converting enzyme inhibitors or angiotensin receptor antagonists.
8. 8. The use according to any one of claims 1-2, wherein the medicament is for alleviating clinical symptoms of membranous nephropathy, comprising lowering proteinuria, increasing serum albumin levels and/or lowering creatinine; and/or reduce the number of glomerular epithelial immune complex deposits and/or podophylloic fusion, or reduce the extent of basement membrane thickening in patients with membranous nephropathy.