Cyclophosphamide ameliorates membranous nephropathy by upregulating miR-223 expression,promoting M2 macrophage polarization and inhibiting inflammation

Abstract

BACKGROUND:

OBJECTIVE:

This study aims to investigate the role of the immune and inflammatory modulator miR-223 in the immunosuppressive and anti-inflammatory effects of cyclophosphamide (CTX) on membranous nephropathy (MN).

METHODS:

miR-223 mimetics or inhibitors was used to regulate miR-223 levels. LPS induced inflammatory cell model and cell polarization. CTX was used to treat Lipopolysaccharides (LPS) induced inflammatory response and polarization. Cationic bovine serum albumin (c-BSA) induced BALB/c mouse MN model, while CTX was used to treat c-BSA induced MN.

RESULTS:

The miR-223 level in LPS induced inflammatory model cells was lower than that in control cells. The levels of inflammatory factors in LPS $+$ miR-223 mimetics and CTX $+$ miR-223i cells were lower than those in LPS and miR-223i cells. The protein levels of LPS $+$ miR-223 mimic, CTX $+$ miR-223i macrophage M2 phenotype markers Arginase-1 (Arg1), transforming growth factor $\beta$ 1 (TGF- $\beta$ 1), anti-inflammatory factors interleukin-4 (IL4) and interleukin-13 (IL13) were significantly higher than those of LPS and miR-223i. The effect of CTX was confirmed in a BALB/c mouse MN model induced by cationic bovine serum albumin (c-BSA).

CONCLUSION:

CTX upregulates the expression of miR-223, promotes polarization of M2 macrophages, alleviates the inflammatory response and renal injury of MN.

Keywords

Cyclophosphamide membranous nephropathy macrophage miR-223 inflammatory response

1. Introduction

Membranous nephropathy (MN), also known as membranous glomerulonephritis, is a leading cause of adult nephrotic syndrome. The main pathological features encompass the deposition of immune complexes within the glomerular basement membrane epithelial cells, thickening of the basement membrane, and fusion of the foot process. With damage to podocyte integrity, proteins are excreted in the urine. Severe proteinuria can result in hypoalbuminemia, asymptomatic bacteremia, and a hypercoagulable state. For persistent proteinuria, about 40–50% of patients develop renal failure within 10 years [1]. In some studies, the spontaneous remission rate of untreated MN reaches 20–30%, and the renal survival rate within 10 years was 60–80% [2]. Despite showing “benign” characteristics, MN remains the second or third leading cause of renal failure among the primary glomerulonephritis types in the United States and Europe [3]. Among patients with persistent nephrotic syndrome, 40–50% of individuals will develop renal failure within a span of 10 years. Additionally, these patients are at a relatively heightened risk for thromboembolism and potentially life-threatening cardiovascular events [2, 4, 5].

The pathogenesis of MN as an autoimmune disease remains unclear. The disease can involve close collaboration between genetic and environmental factors [6]. Together, these factors could play a key role in immune tolerance, complement activation, glomerular inflammation, and loss of the glomerular cell adaptation process, and thereby in the occurrence and development of MN [7]. The development of MN is mainly related to the inflammatory response and kidney injury. Some studies suggest that urinary protein can stimulate the production of pro-inflammatory and pro-fibrotic cytokines, chemokines, and growth factors (C-C motif chemokine 5 (CCL5) and monocyte chemotactic protein-1 (MCP-1)), induce a pro-inflammatory and pro-fibrotic environment in the kidney, aggravate damage to renal tubules, and accelerate the progression of nephropathy [8, 9]. In this study, the nuclear factor kappa-B (NF- $\kappa$ B) transcription factor may have played a leading role. Indeed, NF- $\kappa$ B is activated in patients with immune-mediated glomerular injury and high proteinuria [4, 5].

Inflammation is primarily regulated by microRNAs (miRNAs) [10]. miRNAs can either attenuate inflammation, promote the repair of tissue damage, or aggravate inflammation [11]. miR-223, a newly discovered miRNA, regulates NF- $\kappa$ B activity in several tissues [12, 13]. miR-223 inhibits NF- $\kappa$ B subunit p65 phosphorylation and nuclear translocation, thereby inhibiting NF- $\kappa$ B activity. This negative regulation of NF- $\kappa$ B by miR-223 promotes the polarization of M2 macrophages [14, 15], leading to anti-inflammatory and protective effects on glomerular cells. Therefore, therapeutic strategies aimed at targeting miR-223 and promoting M2 macrophages may be of interest for treating MN. Cyclophosphamide (CTX) is a commonly used immunosuppressant in clinical practice, often used in combination with glucocorticoids, to effectively reduce proteinuria and kidney injury in patients with primary MN [16, 17]. This study aimed to investigate whether the immunosuppressive effect of CTX in MN involves miR-223-induced macrophage M2 polarization.

2. Materials and methods

2.1 Cell lines

The mouse macrophage line Raw264.7 was purchased from Peking Union Medical College Cell Bank. They were cultured in Dulbecco’s modification of Eagle’s medium Dulbecco (DMEM) medium containing 10% fatal bovine serun (FBS) (Gibco, USA, 10270-106-FCM) and 100 U/ml penicillin/streptomycin (Beyotime, C0222), and placed in a fully wet incubator containing 5% CO₂ at 37^∘C.

2.2 Laboratory animals

In this study, 20 healthy adult male SPF BALB/c mice weighing 20 $\pm$ 2 g and aged 6 weeks were adopted, which were purchased from Shanghai Ligen Biotechnology Co., Ltd. Adaptive feeding was carried out in SPF barrier of experimental animal center of our hospital (temperature 22–25^∘C, humidity 50–70%, 12-hour light and dark cycle, drying, ventilation, free diet, and drinking water) for 1 week, which was approved by Experimental Animal Ethics Committee of Qiqihar Medical University (OMU-AECC-2022-105).

2.3 Construction of animal model

The MN model was induced using cationic bovine serum albumin (C-BSA, Beijing North TZ-BIOTECH, 77165) in 15 mice randomly selected from 20 mice [18], and the remaining five mice served as normal controls (NC). Mice in the model group were given 0.2 mg C-BSA (2 mg/mL) and the same volume of Freund’s complete adjuvant (FCA) (Sigma, F5881). The mice were pre-immunized by multiple subcutaneous injections in the bilateral armpits and groins, whereas those in the normal group were administered the same volume of FCA subcutaneously. After a two-week pre-immunization period, the model group received immunization with C-BSA at a dosage of 13 mg/kg three times per week with one-day intervals, while the normal group was administered normal saline concurrently for six weeks. Twelve mice survived and exhibited abnormal levels of urinary protein after formal immunization.

Twelve mice were randomly divided into the MN model group (MN, $n=$ 6) and CTX treatment group (MN $+$ CTX, $n=$ 6). Random grouping method for experimental animals: The computer software is utilized to generate random numbers and groups for experimental animals, ensuring an unbiased randomization process where each animal has an equal opportunity to be assigned to any group. CTX was dissolved in normal saline, the treatment group was given a single intraperitoneal injection of 30 mg/kg [19], and the model group was administered an equal volume of normal saline. AgomiR-223 (Guangzhou Ribobio, 5’-UGUCAGUUUGUCAAAUACCCCA-3’) was used to control miR-223 levels in MN mice. AgomiR-223 was prepared according to the manufacturer’s instructions, and intraperitoneal injection of AgomiR-223 (5 nmol) was performed every two days for a total of seven days [20, 21]. The mice were euthanized after each treatment. To achieve this, the mice are placed in closed containers and exposed to carbon dioxide and other gases for suffocation.

2.4 Construction of in vitro research model

The cells were divided into the normal (Nor), CTX treatment (CTX), miR-223 mimic (miR-223 mimic), miR-223 inhibitor inhibition (miR-223i), CTX and miR-223 mimic (CTX $+$ miR-223 mimic), CTX and miR-223 inhibitor (CTX $+$ miR-223i), LPS (LPS), LPS, and miR-223 mimic (LPS $+$ miR-223 mimic). LPS (Sigma, L2630) was added to the culture medium at a concentration of 5 mg/L to induce an inflammatory response as a control. The miR-223 mimic and inhibitor were designed and synthesized by Guangzhou Ribobio Co., Ltd.; miR-223 mimic: 5’-UGUCAGUUUGUCAAAUACCCA-3’, 5’-GGGUAUUGACAAACUGACAUU-3’; miR-223 inhibitor: 5’-CTTCCTCGTGCTTTACGGTATCG-3’. According to the manufacturer’s instructions, 1.25 $\mu$ l 20 $\mu$ M miRNA mimic was obtained after dilution with 30 $\mu$ l 1X riboFECT™CP Buffer. 3 $\mu$ l riboFECT™CP Reagent was added to the diluent, gently blown, mixed, and incubated at room temperature for 0–15 min. The mixture was then added to the protocell medium at a concentration of 50 nM.

2.5 Test of renal function in mice

After CTX administration for 7 days, mice in each group were anesthetized with inhaled anesthetics, and then blood was collected through the abdominal aorta, and serum was quickly centrifuged using blood urea nitrogen (BUN), serum creatinine (Scr), triglyceride (TG), and total cholesterol (TC) test kits (Xiamen Lunchangshuo Biotechnology Co., Ltd.) in accordance with the manufacturer’s instructions for the detection of renal function indicators [22]. Urine samples were obtained from mice in each group. Renal tubular injury markers [23], such as Kidney injury molecule 1 (KIM-1), N Acetyl Glucosamine (NAG), Neutrophil gelatinase-associated lipocalin (NGAL), and Retinol-Binding Protein (RBP) (Shanghai Sieger Biotechnology Co., Ltd.), were detected using an Enzyme-linked immunosorbent assay (ELISA) kit, and the differences in each group were compared after Ucr standardization [9].

2.6 Gene expression detection by qPCR

Total RNA was extracted from mouse kidney tissue and RAW264.7 cells using TRIZOL reagent (Sigma CGP4014) and RNeasy mini kit (QIAGEN,74104). cDNA was synthesized using the Omniscript RT kit (QIAGEN,205111) and amplified by qPCR using SYBR Green (QIAGEN, 204245). The expression of RT-qPCR genes was quantitatively analyzed using the CFX96 (Bio-Rad) system. The reaction system for RT-PCR was 10 $\mu$ L:1 $\mu$ L cDNA product, 5 $\mu$ L SYBR Green, 0.2 $\mu$ L forward and reverse primers (10 $\mu$ M), 3.6 $\mu$ L ddH2O. amplification conditions: 95^∘C 10 min, 95^∘C 2 s, 60^∘C 20 s, 70^∘C 10 s, 40 cycles. With mGAPDH as a reference, the relative expression level of the data was calculated using the $\Delta\Delta$ Ct method.

The primers were synthesized by Shanghai Biotechnology: miR-223-3p F:5-CCGCCCGTGTCAGTTT GTCAAAT-3, R:5-GTGCAGGGTCCGAGGT-3; GAPDH F:5-GGCTCTCTGCTCCTCCCTGTT-3’, R:5-GCGGGATCTCGCTCCTGGAAG-3; Arg1 F:5-AGCACTGAGAAGCTGTC-3, R:5-TACGTCTCGCAA GCCAATGT-3; NF-kB F:5’-TGGATTGAAGACGCGGTTCT-3’, R:5’-TGTTGATACTC CAGAGACC-3’; MCP-1 F:5’-TATTGTCCACTGACCC-3’, R:5’-CTTCACCCAACTCCTAACCT-3; iNOS F:5’-CT ATCAGGAAAAATGCAGGAGAT-3’, R:5’-GAGCACGCTGAGTACCTCATT-3’; CCL5 F:5’-AGGTAA AACTAAGGATGTCAGC-3’, R:5’-CTCCGGAAATTCGAGTCTTCT-3’.

2.7 Protein expression detection by Western blotting

Western blot analysis was used to determine the protein expression. Total protein was extracted using lysis buffer containing protease and phosphatase inhibitors (Beyotime P2316M) and quantified using a BCA kit (Beyotime, E112-01/02). The membrane was separated by SDS-PAGE gel electrophoresis and transferred to PVDF membrane (Millipore, IPVH00010), then incubated for 12 h in a 4^∘C-environment using a specific antibody. The membrane was incubated for 2 h with a secondary immunoglobulin G (IgG) antibody bound to horseradish peroxidase (HRP). The target protein was detected using enhanced chemiluminescence (Thermo Fisher Scientific, USA) and the band strength was quantified and standardized according to the load control. The antibodies used were purchased from Abcam (Cambridge, UK) and CST. Arg1 (CST, 89872), TGF $\beta$ -1 (Abcam, ab215715), GAPDH (Abcam, ab9485), goat anti-rabbit IgG H&L (DyLight^® 488), and pre-adsorbed secondary antibody (Abcam, ab96899) were used and stored according to the manufacturer’s instructions.

2.8 Immunofluorescence

Macrophages were isolated from the peritoneal cavities of mice in each group. Aseptic PBS-H (PBS containing 10 U/ml heparin and 10% calf serum) precooled in 3.5 ml ice was injected along the midline of the abdomen. The abdomen was gently massaged for 5 min, the abdominal wall was cut, the exudate was sucked out with a straw, and the abdominal cavity was washed with precooled PBS-H of the same capacity 2–3 times. The exudate was combined in a centrifuge tube, centrifuged at 1500 rpm for 10 min, and the supernatant was removed. Cells were washed with precooled RPMI-1640 (Sigma, R8758) culture medium for 3 times, centrifuged at 4^∘C for 1500 rpm for 10 min each time, and the supernatant was removed. The cells were suspended in an appropriate amount of precooled RPMI-1640 medium and counted using trypan blue staining. Macrophages were mixed in a medium containing 30% calf serum (RPMI-1640) at a concentration of 2 $\times$ 10⁶–4 $\times$ 10⁶/ml. The cell suspension was inoculated into a pre-slipped Petri dish at a density of 2 $\times$ 10⁵–4 $\times$ 10⁵ macrophages per square centimeter. The were cultured in a saturated water vapor carbon dioxide incubator with 5% CO₂ at 37^∘C for 4 h. The dish was shaken and the unadhered cells were removed. The cells were washed with PBS three times, and a follow-up immunofluorescence operation was carried out.

Cell slides were fixed with paraformaldehyde for 30 min after cleaning PBS, penetrated with TRITON X-100 for 15 min after rinsing PBS, and sealed for 1 h after cleaning PBS; Macrophages were incubated with CD38 (0979R, Yaji Biotechnology) and Egr2 (8368R, Yaji Biotechnology) at 4^∘C for 12 h. After returning to room temperature, the primary antibody was removed from the PBS, and the corresponding secondary antibodies (ab6791, ab150132, Abcam) were added and incubated for 2 h in the dark. After washing with PBS, DAPI liquid seal tablets were used to prevent fluorescence quenching and the images were observed under a fluorescence microscope.

2.9 ELISA

IL-1, IL-6, TNF- $\alpha$ , IL-4, IL-13 specific ELISA kit (Uscn Life Science, USA) were used according to the manufacturer’s instructions. The absorbance of the samples was quantified using a microplate reader (Biotek, Vermont, USA) to detect differences in expression in each group.

2.10 Statistical analysis

SPSS software (version 22.0) was used for the data analysis. The continuous variables were tested for normality. Continuous data obeying normal distribution are described as mean $\pm$ standard deviation (M $\pm$ SD). An independent sample $t$ -test was used for comparisons between the two groups. Single-factor analysis of variance was used for comparisons between multiple groups. $P<$ 0.05. Cell experiments were conducted three times in parallel, and animal experiments were conducted three times in parallel.

3. Results

3.1 CTX could alleviate the inflammatory response of macrophages caused by inhibition of miR-223 activity

miR-223 levels in RAW264.7 cells were modulated using either mimic or inhibitor. qPCR validated miR-223 regulation using mimetics and inhibitors (Fig. 1A). Cells in which miR-223 was increased or inhibited were further treated with LPS to reproduce a cell inflammation model, or with CTX. The results showed that the miR-223 level in the LPS was significantly lower than that in the normal ( $P<$ 0.01) (Fig. 1B). Enzyme-linked immunosorbent assay (ELISA) and qPCR were used to detect the expression levels of inflammation-related factors in each group. The results showed that the levels of IL-1, IL-6, TNF- $\alpha$ , and iNOS in cells where miR-223 was inhibited were significantly higher than those in the normal, and there was no statistical difference between them in the LPS group, indicating that suppression of miR-223 activity could cause an inflammatory response. Simultaneously, the miR-223 mimic significantly decreased the levels of IL-1, IL-6, TNF- $\alpha$ , and iNOS in response to LPS ( $P<$ 0.01). However, the expression levels of inflammatory factors decreased significantly after CTX treatment in cells in which miR-223 expression was inhibited ( $P<$ 0.01). This result indicated that CTX was capable of alleviating the inflammatory response of macrophages caused by the inhibition of miR-223 (Fig. 1C–F).

Figure 1.

CTX could reduce the level of inflammatory factors in cells. A. Expression levels of miR-223 transfected in vitro were determined by qPCR. The levels of miR-223 in each group (Nor, LPS) were detected using qPCR. The levels of inflammatory factors (Nor, LPS, LPS $+$ miR-223 mimic, miR-223i, and CTX $+$ miR-223i) were detected using qPCR and ELISA. ^{*P <} 0.05, ^**P < 0.01, ^***P < 0.001.

Figure 2.

CTX could promote the up-regulation of macrophage M2 markers. Note: The expression of Arg1 and TGF- $\beta$ 1 as M2 phenotypic markers was detected by A–C. WB; D. Immunofluorescence detection of cell (Nor, LPS, LPS $+$ miR-223 mimic, miR-223i, CTX $+$ miR-223i) M1/M2 phenotype distribution and difference analysis; The expression of anti-inflammatory cytokines IL-4 and IL-13 was detected by E–F. ELISA. ^{*P <} 0.05, ^**P < 0.01, ^***P < 0.001.

3.2 CTX up-regulated miR-223 and promoted macrophage polarization to M2 to exert anti-inflammatory effect

The focus of this study lies in the polarization of macrophages towards an anti-inflammatory phenotype, based on their functional and phenotypic characteristics. It has been found that the overexpression of miR-223 reduces the levels of pro-inflammatory cytokines such as IL-6 and iNOS in the supernatant of RAW264.7 cells, and weakens the trend of M1 polarization induced by LPS [24, 25]. The protein expression levels of the M2 phenotypic markers Arg1 and TGF- $\beta$ 1 were assessed in each group. Western blot results showed that when miR-223 levels were reduced or inhibited (LPS group and miR-223i group), the protein levels of Arg1 and TGF- $\beta$ 1 were significantly lower than those in the normal group ( $P<$ 0.001); however, the protein levels of M2 markers Arg1 and TGF- $\beta$ 1 increased significantly after up-regulating the expression of miR-223 in inflammatory models (LPS $+$ miR-223 mimic vs. LPS) or CTX (CTX $+$ miR-223i vs. miR-223i) (Fig. 2A–C). Simultaneously, this study employed CD38 and Egr2 as specific markers for M1 and M2 cells [26], respectively, and observed a discernible disparity in the ratio of M1 to M2 cells through immunofluorescence analysis. The results showed that the proportion of M2 macrophages was higher in the group overexpressing miR-223 (LPS $+$ miR-223 mimic, CTX $+$ miR-223i) (Fig. 2D). The expression of anti-inflammatory factors was assessed using ELISA in each group. The results revealed an increase in the levels of anti-inflammatory cytokines IL4 and IL13 in the LPS $+$ miR-223 mimic and CTX $+$ miR-223i groups (Fig. 2E and F).

3.3 CTX up-regulated miR-223 and promoted M2 polarization of macrophages in MN model mice

The results of in vitro experiments showed that CTX can promote the transformation of macrophages to an anti-inflammatory phenotype (M2) by upregulating miR-223. To verify whether the above results are valid for the whole organism, we developed MN model mice. To investigate the role of miR-223 in the effects of CTX, MN mice were treated with CTX and miR-223 agomir. The qPCR results showed that the expression of miR-223 in the MN mouse model was significantly lower than that in the normal group ( $P<$ 0.001). After CTX, the expression of miR-223 in the MN group significantly increased (compared to MN) (Fig. 3A), confirming that CTX upregulated miR-223. Therefore, we examined the phenotypic distribution of macrophages in the peripheral blood of mice in each group. CD38 and Egr2 were used to specifically label M1 and M2 cells, respectively [26]. Immunofluorescence results showed that the proportion of M2 macrophages in MN mice was relatively low, whereas the proportion of M2 macrophages in the CTX and miR-223 agomir treatment groups increased (Fig. 3B and C). The results showed that CTX promoted M2 polarization of macrophages by up-regulating the expression of miR-223.

Figure 3.

Up-regulation of CTX could change the phenotypic distribution of macrophages in peripheral blood of mice. Note: A. qPCR was used to detect the expression of miR-223 in the peripheral blood of mice in each group (Nor, MN, MN $+$ CTX, MN $+$ agomir); B. Immunofluorescence was used to detect the distribution of M1 and M2 macrophages in each group. The proportion difference between the M1 and M2 types in the cells of each group was counted. ^{*P <} 0.05, ^**P < 0.01, ^***P < 0.001.

3.4 CTX alleviated the inflammatory level of MN mice

Regulation of M2 polarization of macrophages by CTX led us to speculate on the anti-inflammatory role of CTX in MN animal models. Kidney tissue samples were obtained from mice in each group, and inflammation-related factors were detected. ELISA was used to detect the expression of inflammatory pathway-related factors in the model and treatment groups. The results showed that the levels of IL1, IL6, and TNF- $\alpha$ in the CTX administration group decreased significantly compared with those in the model group (Fig. 4A–C), and qPCR results showed that CTX significantly decreased the expression of immunocyte chemokines CCL5, MCP-1, and OPN (vs. MN, $P<$ 0.01) (Fig. 4D–F). The results showed that CTX administration could reduce the inflammatory response in MN and further damage to renal tissue.

Figure 4.

CTX could reduce the level of inflammatory factors in peripheral blood of mice. Note: A–C. ELISA was used to detect the levels of IL-1, IL-6-TNF-a in each group (Nor, MN, MN $+$ CTX); The pro-inflammatory chemokines monocyte chemoattractant protein 1 (MCP-1), CCL5 and OPN were detected by D–F. qPCR. ^{*P <} 0.05, ^**P < 0.01, ^***P < 0.001.

3.5 CTX improved renal function in MN mice

To further evaluate the protective effect of CTX in MN, we measured the tissue injury markers N-acetyl- $\beta$ -D-glucosidase (NAG), retinol-binding protein (RBP), KIM-1, neutrophil gelatinase-associated lipocalin (NGAL), serum urea nitrogen (BUN), creatinine (Scr), triglyceride (TG), and total cholesterol (TC) in each group of mice. The results showed that the values of NAG, RBP, KIM-1 and NGAL in urine samples of mice in model group were significantly higher than those in normal group ( $P<$ 0.01), and all the data were significantly lower than those in model group after 7 days of CTX administration ( $P<$ 0.05), indicating that the damage to renal tubules was alleviated after CTX administration (Fig. 5A–D); the values of BUN, Scr, TG and TC in the blood samples of mice in the model group were significantly higher than those in the normal group ( $P<$ 0.01). After 7 days of CTX administration, all data were significantly lower than those in the model group ( $P<$ 0.05), indicating that the renal function of the administration group was improved by CTX (Fig. 5E–H).

Figure 5.

CTX could reduce the level of renal injury markers. Note: Urine NAG, NGAL, KIM-1, and RBP were detected by A–D ELISA, and the results were compared by standardization of urinary creatinine (Ucr). ELISA was used to detect the expression of BUN, Scr, TG, and TC in the serum samples of mice. ^{*P <} 0.05, ^**P < 0.01, ^***P < 0.001.

Figure 6.

Schematic diagram of CTX up-regulating miR-223 to promote the transformation of M1 macrophages to M2 phenotype.

4. Discussion

MN is the most common cause of NS among adults. The etiopathogenesis of this disease includes persistent proteinuria in one-third of patients and chronic kidney failure in one-third of patients. The typical pathological feature of MN is the appearance of a large amount of urinary protein, which results from impairment of the filtration barrier of the kidney. Subsequently, continuous protein leakage further damages glomerular function and leads to glomerulosclerosis, mesangial hyperplasia, and kidney damage. Therefore, all patients with MN should receive supportive treatment to reduce proteinuria [27]. The clinical use of CTX combined with glucocorticoids can effectively alleviate the clinical symptoms of MN patients, reduce their 24-hour urinary protein levels, and improve renal function. At present, general supportive treatment for all patients with MN includes limiting sodium and protein in the diet, controlling blood pressure, minimizing proteinuria by inhibiting the renin-angiotensin system, treating dyslipidemia, and anticoagulation for specific patients. MN is an autoimmune disorder. Current guidelines recommend a combination of CTX and glucocorticoids for treatment. CTX, a cytotoxic immunosuppressant commonly employed in post-tumor surgery immunotherapy, is often utilized in this context. The gonadal and toxic effects of this treatment have been scientifically proven, with a significant impact on fertility. In the case of infertility in young patients, it is generally recommended to avoid drug interventions. If immunosuppressants are used chronically, they should be administered in small doses to minimize potential side effects. It is important to note that high doses can lead to serious adverse reactions and inadequate treatment duration may result in recurrence. Additionally, careful attention must be paid to dose accumulation.

Cationic bovine serum albumin (BSA) was used to induce MN in mice and CTX was used to treat them. After 7 days of treatment, renal function was assessed in the MN mice. The results showed that blood urea nitrogen (BUN), serum creatinine (Scr), triglyceride (TG), and total cholesterol (TC) were significantly reduced, IL1, IL6, and TNF- $\alpha$ related to promoting inflammatory response in serum were significantly reduced, whereas L4, IL13, and TGF- $\beta$ 1 were increased, indicating that the inflammatory response in the kidney was inhibited. This study has shown that miR-223 expression was significantly decreased in the MN group, and miR-223 levels returned to normal levels after CTX treatment. In order to further study the immunosuppressive mechanism of CTX in the treatment of MN, we used miR-223 mimic and inhibitor to construct an in vitro study model in RAW264.7 cells and treated it with CTX. The results showed that CTX upregulated the expression of miR-223 in RAW264.7, inhibited the inflammatory response, and promoted M2 polarization of macrophages. Wan et al. found that immune infiltration by B cells, T cells, and macrophages plays an important role in MN [28]. Studies have shown that macrophage infiltration is positively correlated with C3 deposition and glomerular function loss and can continuously induce complement activation, resulting in renal tubular injury in patients with MN [7, 29]. At the same time, our results showed that the M1 polarization characteristics of the MN model group were high, while macrophages tended to transform to M2 after CTX administration (Fig. 6).

Wang et al. found that CTX can upregulate the expression level of miR-223 in myeloid cells in lung cancer [30]. The same results were observed in both mice and the RAW264.7 cell line, suggesting that the regulatory mechanism of CTX on miR-223 is not specific to a particular cell type. miR-223 was first identified as a regulator of bone marrow formation and differentiation in 2003, and has various regulatory functions in the immune response. The abnormal expression of miR-223 is associated with the occurrence and development of various infectious diseases by affecting neutrophil infiltration, macrophage function, dendritic cell maturation, and inflammatory body activation [31]. Ying et al. found that miR-223, as a downstream factor of the PPAR- $\gamma$ -dependent pathway, can regulate the activation of M2 macrophages in adipose tissue [32]. In addition, Kim et al. confirmed that the downregulation of miR-223 in macrophages can promote inflammatory responses in adipose tissue [33]. Similarly, high levels of miR-223 inhibit the expression of the TNF receptor-related factor TRAF6 in the myocardium, downregulate the NF- $\kappa$ B signaling pathway, and alleviate myocarditis-related injury. miR-223 plays a key role in autoimmune diseases such as enteritis and rheumatoid arthritis. miR-223 regulates rheumatoid arthritis (RA) progression by increasing the sensitivity of macrophages to pro-inflammatory cytokines and inhibiting their response to anti-inflammatory signals [34]. Wu et al. showed that the overexpression of miR-223-3p can inhibit IL-17RD and alleviate the injury induced by RA. In addition, animal experiments have shown that icariin can inhibit NLRP3 by upregulating miR-223-3p expression, reducing apoptosis of RA joint fibroblast-like synoviocytes (RA-FLSC), and alleviating tissue damage [35].

The role of M2 macrophage subtypes in nephropathy remains understudied, despite their crucial involvement in renal injury and repair. M2 macrophages can be divided into three subtypes: M2a, M2b, and M2c. M2a and M2b are the main subtypes in the renal tissue, the distribution of M2a macrophages is primarily observed in the tubulointerstitium, while M2c macrophages are predominantly found in the glomerulus. Studies have shown that immunosuppressive therapy can increase the number of M2c macrophages [36] and inhibit M2c in animal models to effectively alleviate tubular atrophy, interstitial dilatation, and proteinuria in adriamycin nephropathy, and concluded that M2c may protect against kidney injury [37]. Tindings of this study suggest that CTX can enhance the M2 polarization of macrophages by upregulating the expression of miR-223, thereby ameliorating kidney injury and dysfunction induced by MN.

In conclusion, CTX increased the expression of miR-223 and promoted the polarization of macrophages to the M2 cell type, thus alleviating the inflammatory response and renal injury in MN.

This study has some limitations. Firstly, there is a lack of data on the treatment effectiveness of clinical patients. Therefore, if possible, In the future, we will continue to explore the long-term effects of cyclophosphamide treatment on miR-223 expression and M2 macrophage polarization, as well as the correlation between miR-223 expression levels and disease severity in patients with membranous nephropathy. Provide reference value for the treatment of membranous nephropathy in clinical practice.

Author contributions

CY and QM conceived and designed the study, and drafted the manuscript. CY, QM, and YS collected, analyzed, and interpreted the experimental data. NZ and LP revised the manuscript for intellectual content. All authors have read and approved the final manuscript.

Funding

This study is funded by the Joint Guidance Project of Qiqihar Science and Technology Plan (LSFGG-2023012) and Innovation Incentive Project of Qiqihar Science and Technology Plan (CSFGG-2021160).

Data availability

The datasets used and/or analyzed during the present study are available at https://doi.org/10.7910/DVN/ GBOVX6.

Ethics approval and consent to participate

The study was approved by the Ethics Committee of The Third Affiliated Hospital of Qiqihar Medical University.

Footnotes

Acknowledgments

The authors have no acknowledgments.

Conflict of interest

The authors declare that they have no conflict of interest.

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