The Synthetic Melanocortin Agonist NDP‐MSH Ameliorates THSD7A‐Associated Membranous Nephropathy in an Active Immunization Mouse Model

ABSTRACT

Melanocortin‐based therapies have demonstrated protective effects in experimental membranous nephropathy (MN). However, existing evidence is derived exclusively from passive immunization models that lack direct involvement of human MN‐associated autoimmunity, limiting translational relevance. A clinically relevant model is essential to more precisely define melanocortin efficacy and mechanisms in MN. To address this gap, we established an active immunization model of THSD7A‐associated MN by immunizing mice with recombinant THSD7A antigen. This clinically relevant and accessible model recapitulated cardinal features of human MN, including insidious onset, progressive proteinuria, and characteristic histopathology such as subepithelial immune deposition, complement fixation along glomerular tufts, glomerular basement membrane thickening, and podocyte injury with foot process effacement and loss of homeostatic markers. Rescue treatment with the pan‐melanocortin receptor agonist NDP‐MSH significantly reduced proteinuria and mitigated glomerular damage and podocyte injury. This was accompanied by a marked reduction in circulating anti‐THSD7A autoantibody levels, reduced glomerular immune deposition, and diminished complement activation. Mechanistically, ex vivo treatment of primed B cells isolated from diseased mice demonstrated that NDP‐MSH directly inhibited plasma cell differentiation and autoantibody production. This suppression was accompanied by increased expression of microphthalmia‐associated transcription factor (MITF) and downregulation of interferon regulatory factor 4 (IRF4), indicating activation of the MITF/IRF4 signaling axis, a pathway implicated in negative regulation of B cell differentiation. Collectively, these findings extend the therapeutic potential of melanocortin signaling to a preclinical model that closely mirrors human MN and provide mechanistic insight into its immunomodulatory actions. These results support further investigation of targeted melanocortin‐based therapies for MN.

Active immunization with recombinant THSD7A in mice induces autoreactive B cell activation and differentiation into CD138⁺ antibody‐secreting cells, leading to the production of anti‐THSD7A autoantibodies, glomerular immune injury and proteinuria, characteristic of membranous nephropathy. NDP‐MSH treatment modulates the MITF/IRF4 axis in primed B cells, thereby suppressing plasma cell differentiation, reducing autoantibody production, immune complex deposition, and complement activation, ultimately ameliorating glomerular injury.

1. Introduction

The melanocortin system is a highly conserved neuropeptide hormone network in mammals that plays an essential role in maintaining systemic homeostasis and overall health. This system consists of the endogenous ligands α‐, β‐, and γ‐melanocyte–stimulating hormones (MSH) and adrenocorticotropic hormone (ACTH), which signal through five G protein–coupled melanocortin receptors (MC1R–MC5R) [1]. Collectively, melanocortinergic signaling regulates a wide spectrum of physiological processes [2, 3, 4], including pigmentation, steroidogenesis, appetite control and energy homeostasis, parenchymal cytoprotection, sexual and reproductive function, endocrine secretion, cardiovascular and hemodynamic regulation, neurological processes, as well as anti‐inflammatory and immunomodulatory responses.

Burgeoning evidence indicates that melanocortins exert important protective effects in the kidney [5, 6, 7]. Natural and synthetic melanocortin peptides have demonstrated therapeutic efficacy in a variety of glomerular diseases in human patients or preclinical models, including minimal change disease (MCD) [8], focal segmental glomerulosclerosis (FSGS) [9, 10], lupus nephritis (LN) [11], IgA nephropathy (IgAN) [12], and membranous nephropathy (MN) [13, 14, 15]. Among these glomerular diseases, MN, one of the most common causes of nephrotic syndrome in nondiabetic adults, appears to exhibit the most robust response to melanocortin‐based therapy. Indeed, clinical studies have shown that treatment with natural [15] or synthetic ACTH [16] can induce partial or complete remission of proteinuria in patients with MN with an efficacy comparable to or superior to the standard Ponticelli regimen [17].

Notably, the therapeutic benefit of ACTH in MN is unlikely to be mediated primarily through steroidogenesis. In support of this, ACTH has still demonstrated efficacy even in patients who are refractory to conventional steroid‐containing regimens, suggesting steroid‐independent mechanisms of action [18, 19]. Consistent with this concept, nonsteroidogenic melanocortins, such as α‐MSH [20] and its potent synthetic analog [Nle⁴, DPhe⁷]‐α‐MSH (NDP‐MSH) [13], have also shown marked protective effects against proteinuria and glomerular injury in experimental models of MN. However, the precise mechanisms underlying these salutary effects remain incompletely understood. Emerging studies suggest that melanocortin therapy may confer renoprotection possibly through a combination of direct podocyte cytoprotective actions [21] and systemic immunomodulation [13, 22]. Nevertheless, these conclusions remain tentative, as existing evidence is exclusively derived from passive immunization models that lack direct relevance to human MN. For example, a number of melanocortin peptides, including α‐MSH, NDP‐MSH, and MS05, have been shown to ameliorate albuminuria and MN in passive Heymann nephritis (PHN), a classic rat model of MN [14, 20]. However, the target antigens in PHN [23] do not correspond to the autoantigens implicated in human MN, limiting the translational relevance of these findings [24].

Human MN is a prototypical immune complex–mediated autoimmune glomerulonephritis, and over the past two decades, multiple podocyte autoantigens have been identified in patients with primary MN. Among these, phospholipase A2 receptor 1 (PLA2R1) [25] and thrombospondin type‐1 domain–containing 7A (THSD7A) [26] together account for more than 80% of primary MN cases. In addition, several newer, less frequent antigens, including semaphorin 3B [27], protocadherin 7 [28], neural epidermal growth factor–like 1 (NELL1) [29], and exostosin 1/2 (EXT1/EXT2) [30], have also been identified, further refining disease stratification and informing therapeutic decision‐making.

The development of preclinical MN models that faithfully recapitulate human disease has been challenging. PLA2R is not expressed in many commonly used experimental animals [24], including rodents, and this precludes the generation of PLA2R‐associated MN models in wild type mice or rats. In contrast, THSD7A is highly expressed in rodent podocytes [31], making it an attractive target for modeling human‐relevant MN. Recently, our group developed a heterologous mouse model of THSD7A‐associated MN and tested the efficacy of melanocortin therapy, for the first time, in this model implicating the human MN antigen [13, 22]. Despite this important advance, a key limitation is its passive nature of the heterologous mouse model, relying on the administration of xenogeneic anti‐mouse THSD7A antibody. This approach leads to rapid disease onset and acute glomerular injury, which contrasts sharply with the insidious onset and slow progression characteristic of human MN, a disease driven by sustained autoantibody production due to B cell autoimmunity.

To more precisely define the therapeutic efficacy of melanocortins and elucidate the underlying mechanisms in MN, it is therefore essential to employ an active immunization model that mirrors the autoimmune pathogenesis of MN. Recently, Seifert et al. [32] reported the development of an active immunization murine model of THSD7A‐associated MN that closely recapitulates key features of human MN. However, the recombinant THSD7A antigens used in that study were generated in‐house and are not commercially available, limiting broader application of this model.

To provide a foundation for mechanistic elucidation and therapeutic innovation in THSD7A‐associated MN, there is a critical need to establish a pragmatic and widely accessible active immunization mouse model using commercially available THSD7A recombinant protein antigens. To address this gap, the present study aimed to develop an active immunization mouse model of THSD7A‐associated MN using a commercially sourced THSD7A antigen. As a further demonstration of the utility of this model, we evaluated the therapeutic efficacy of the nonsteroidogenic pan‐melanocortin receptor agonist NDP‐MSH, which has recently been approved for the treatment of porphyria. This approach enables a rigorous assessment of melanocortin therapy in a human‐relevant autoimmune MN model and provides a deepened insight for steroidogenic‐independent mechanisms of melanocortin‐mediated renoprotection.

2. Materials and Methods

2.1. Animal Study

Animal experiments were performed in the Department of Laboratory Animal Resources (DLAR) at the University of Toledo following approval from the Institutional Animal Care and Use Committee (IACUC) of the University of Toledo (protocol number 400176). These experiments adhered to the regulations set forth by the U.S. Department of Agriculture and complied with the guidelines established by the National Institutes of Health for the humane care and use of laboratory animals. All mice were housed in the DLAR at the University of Toledo and provided with food and water ad libitum.

To establish a murine model of MN, wild‐type (WT) C57BL/6 male mice were subjected to a total of four immunizations. Briefly, mice were initially immunized by subcutaneous injection with 20 μg of recombinant mouse THSD7A protein (RPU52773; Biomatik, Kitchener, Ontario, Canada) emulsified in an equal volume of complete Freund's adjuvant (CFA). This was followed by three booster immunizations, each consisting of 20 μg THSD7A emulsified in an equal volume of incomplete Freund's adjuvant (IFA), administered at 2‐week intervals. Control mice received equivalent volumes of CFA or IFA emulsified with PBS.

Blood samples were collected from the facial vein using a Goldenrod 3 mm lancet to puncture the facial vein before the first immunization and at weeks 3, 7, and 10 following immunization.

To test the efficacy of the nonsteroidogenic pan‐MCR agonist NDP‐MSH [22], immunized mice were randomly assigned to receive daily subcutaneous injections of NDP‐MSH (custom‐synthesized peptide from GL Biochem Ltd., Boston, MA, USA) at an equal dose (0.7 μmol/kg body weight per day) or an equal volume of vehicle (PBS) from weeks 7 to 9.

Mice were euthanized at week 10 after prime immunization. Urine, blood, and kidney specimens were collected.

To assess the effects of the NDP‐MSH on T cells, mice were randomly assigned to receive daily subcutaneous injections of NDP‐MSH at an equal dose (0.7 μmol/kg body weight per day) or an equal volume of PBS vehicle for 3 weeks. At the end of treatment, mice were euthanized, and spleens were harvested for flow cytometric analysis of CD3⁺ cells.

2.2. Urine Protein Analyses

Protein composition of urine samples was assessed by SDS–PAGE followed by Coomassie Brilliant Blue staining (Sigma‐Aldrich). Urinary albumin levels were quantified using a mouse albumin enzyme‐linked immunosorbent assay (ELISA) kit (Bethyl Laboratories, Montgomery, TX, USA), whereas urinary creatinine concentrations were determined with a creatinine assay kit (BioAssay Systems, Hayward, CA, USA). The extent of proteinuria was expressed as the urinary albumin‐to‐creatinine ratio (uACR).

2.3. Transmission Electron Microscopy

As previously described [13], kidney cortical tissues were cut into small pieces (approximately 1 mm³), fixed in 2.5% glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA, USA), and processed for transmission electron microscopy using standard procedures. Ultrathin sections mounted on grids were examined using a Talos L120C transmission electron microscope (Thermo Fisher Scientific, Waltham, MA, USA).

2.4. Isolation of Glomeruli

Glomeruli were isolated as previously described [14, 22]. Briefly, following euthanasia, mouse kidneys were perfused with PBS via the left ventricle until complete blanching was observed. The left renal pedicle was then ligated, and the left kidney was excised and processed for subsequent histological analyses. The right kidney was further perfused with PBS containing iron oxide solution (iron (III) oxide; Sigma, 310050) using a syringe pump and subsequently harvested for glomerular isolation.

2.5. Western Immunoblot Analysis

Isolated glomeruli were homogenized and cultured cells were lysed with RIPA lysis buffer supplemented with phosphatase/protease inhibitors as described previously [33]. Samples with equal amounts of proteins were separated by SDS–polyacrylamide gel and transferred onto PVDF membranes. The membranes were sequentially probed with the primary antibodies or mouse serum at 4°C overnight and corresponding horseradish peroxidase‐coupled (HRP) secondary antibodies for chemiluminescence detected using GBOX system (Syngene, Cambridge, UK). A complete list of antibodies used in this study is provided in Table S1. For immunoblot analysis, band intensities were scanned and quantified as integrated pixel density using ImageJ software.

2.6. Preparation, Culture, and Flow Cytometry Analysis of Splenocytes

After euthanasia, spleens were harvested from mice and passed through a 70‐μm cell strainer to prepare single‐cell suspensions. After lysis of red blood cells, splenocytes were prepared. For flow cytometry analysis, splenocytes were fixed with 4% paraformaldehyde for 30 min at 4°C. Cells were then washed with Dulbecco's phosphate‐buffered saline and stained with a Alexa Fluor 488‐conjugated anti‐CD138 antibody (Santa Cruz Biotechnology) or FITC‐conjugated anti‐CD3 antibody (Biolegend). Flow cytometric acquisition was performed on a flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA), and data were analyzed using FlowJo software. To culture splenocytes, freshly isolated cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum, 2‐mercaptoethanol, streptomycin, and penicillin and incubated at 37°C in a humidified atmosphere with 5% CO₂. Cells were cultured with or without NDP‐MSH (10⁻⁷ M) for the indicated duration before being harvested for subsequent analyses [14]. Total splenocyte numbers were determined prior to staining, and absolute numbers of CD138⁺ cells were calculated by multiplying the percentage of CD138⁺ cells obtained by flow cytometry by the total number of splenocytes isolated from each spleen.

2.7. Immunofluorescence Staining

Cryosections of kidney specimens or cultured cells were fixed, permeabilized, and incubated with primary antibodies against the indicated molecules, followed by staining with Alexa Fluor 488‐ or 594‐conjugated secondary antibodies and counterstaining with 4,6‐diamidino‐2‐phenylindole (DAPI; Abcam). Images were acquired using a Nikon Eclipse Ni‐U microscope (Nikon, Tokyo, Japan) or a Leica TCS SP5 multiphoton laser scanning confocal microscope (Leica Microsystems Inc., Buffalo Grove, IL, USA).

2.8. Blood Pressure Measurements

Blood pressure (BP) was measured via a CODA noninvasive blood pressure system (Kent Scientific, Torrington, CT, USA). All mice were acclimated to the system for at least 1 week before initiating blood pressure measurement to avoid undue stress and experimental artifact [34, 35]. For each measurement, an experimental session consisting of five acclimation cycles followed by 10 measurement cycles was conducted. Only those measurement cycles that met the acceptance criteria set by the CODA software were retained for analysis.

2.9. Enzyme‐Linked Immunosorbent Assay (ELISA)

Circulating levels of anti‐THSD7A IgG in mouse serum were measured by ELISA. Briefly, microplates were coated with 100 ng of recombinant mouse THSD7A protein and then incubated with diluted serum samples after blocking with 1% bovine serum albumin (BSA). After three washing steps with PBS containing 0.05% Tween‐20, the plates were incubated with horseradish peroxidase‐conjugated anti‐mouse IgG. Color development was achieved using tetramethylbenzidine, and reactions were terminated by the addition of 100 μL of 1 M sulfuric acid per well. Absorbance was measured using a microplate spectrophotometer (Cytation 5; BioTek Instruments, Winooski, VT, USA) [14].

2.10. Statistical Analysis

Data were presented as mean ± SD (standard deviation). All data were statistically analyzed using SPSS 22.0 software. Student's t‐test was used for comparisons between two groups. One‐way ANOVA followed by Tukey's post hoc test was applied for multiple group comparisons. Two‐way ANOVA was used where appropriate to assess the effects of two independent variables and their interaction. A value of p < 0.05 was considered statistically significant.

3. Results

3.1. Active Immunization With Recombinant THSD7A Induces a Murine Model of Membranous Nephropathy That Recapitulates Key Features of Human Disease

To establish a clinically relevant model of THSD7A‐associated MN, mice were actively immunized with a commercially available recombinant mouse THSD7A protein. Mice received a prime immunization and three subsequent booster immunizations with either THSD7A antigen (THSD7A‐immunized) or PBS (control), and were then followed at 3, 7, and 10 weeks after the initial immunization (Figure 1A).

FIGURE 1.

Active immunization with recombinant mouse THSD7A protein induces membranous nephropathy in mice. (A) Schematic diagram depicts the animal experimental design. (B) Representative immunoblot analysis of glomeruli isolated from wild‐type mice using serum collected from control (1:100) or THSD7A‐immunized mice (1:100) 4 weeks after final immunization, with a commercial anti‐THSD7A antibody (1:1500) serving as a positive control. (C) Anti‐THSD7A antibody titers as measured by ELISA. (D) Urine samples pooled from control or THSD7A‐immunized mice were subjected to SDS–PAGE, with bovine serum albumin (BSA) serving as a standard control. The band for albumin is indicated by an arrowhead. (E) Urinary albumin excretion was assessed by urinary albumin‐to‐creatinine ratios (uACR). *p < 0.05 vs control (n = 6). (F) Kidney cryosections were subjected to immunofluorescence staining for mouse IgG (red) and DAPI (blue). Representative micrographs are shown (scale bar = 25 μm). (G) Representative EM micrographs show podocyte foot process effacement (yellow arrowheads), subepithelial electron‐dense deposits (red arrowheads) and marked GBM thickening (scale bar = 2 μm).

To confirm that the induced autoantibodies recognized and targeted native glomerular THSD7A, immunoblotting of mouse glomerular lysates was performed using serum from immunized mice as the primary probing antibody. Serum from THSD7A‐immunized mice detected a specific band corresponding to THSD7A, consistent with results obtained using a commercial anti‐THSD7A antibody as a positive control, whereas serum from control mice showed no reactivity (Figure 1B). As shown in Figure 1C, ELISA analysis demonstrated a progressive and robust increase in circulating anti‐THSD7A IgG levels in THSD7A‐immunized mice, with titers peaking between weeks 7 and 10. In contrast, anti‐THSD7A IgG was undetectable in control mice at all time points.

Concomitant with the rise in circulating autoantibody levels, THSD7A‐immunized mice developed massive proteinuria, with albumin identified as the predominant urinary protein by urine protein electrophoresis (Figure 1D). Quantitative uACR analysis revealed that urinary albumin excretion progressively increased in parallel with serum anti‐THSD7A IgG titers, indicating a close association between autoantibody production and glomerular injury (Figure 1E). This was concomitant with prominent mouse IgG deposition in a granular, pseudo‐linear pattern in glomeruli along the glomerular capillary wall in THSD7A‐immunized mice, as shown by fluorescence immunohistochemistry (Figure 1F). Moreover, ultrastructural analysis by transmission electron microscopy revealed extensive subepithelial electron‐dense immune deposits, accompanied by diffuse podocyte foot process effacement and substantial glomerular basement membrane (GBM) thickening, hallmark features of MN (Figure 1G). In contrast, control mice exhibited normal glomerular architecture without immune deposition. Collectively, these findings confirm that active immunization with recombinant THSD7A induces a robust murine model of MN that closely mirrors the immunopathological and ultrastructural characteristics of human THSD7A‐associated disease.

3.2. Delayed NDP‐MSH Treatment Attenuates Albuminuria and Glomerular Injury in THSD7A‐Associated MN

Having established a clinically relevant model, we next evaluated the therapeutic efficacy of melanocortin therapy using the synthetic melanocortin receptor agonist NDP‐MSH (Figure 2A). To enhance translational relevance, treatment was initiated at week 7 after the prime immunization, a time point at which disease was fully established and characterized by marked albuminuria, thereby mimicking the clinical scenarios of MN at diagnosis.

FIGURE 2.

Melanocortin therapy improves proteinuria and mitigates podocytopathy in a THSD7A‐associated active immunization mouse model of membranous nephropathy. (A) Schematic diagram depicts the animal experimental design. (B) Animals were treated as elaborated in A. Urine samples pooled from treated mice were subjected to SDS–PAGE, with bovine serum albumin (BSA) serving as a standard control. The band for albumin is indicated by an arrowhead. (C) Urinary albumin excretion was assessed by uACR. *p < 0.05 (n = 6). (D) Representative EM micrographs show podocyte foot process effacement (yellow arrowheads) and subepithelial electron‐dense deposits (red arrowheads) (scale bar = 2 μm).

After 2 weeks of treatment, NDP‐MSH–treated mice exhibited a statistically significant reduction in albuminuria compared with PBS‐treated controls, with an approximately 42.3% decrease, as demonstrated by urine protein electrophoresis (Figure 2B) and quantified by the urinary albumin‐to‐creatinine ratio (Figure 2C). Notably, this partial remission of proteinuria was accompanied by a marked improvement in glomerular histopathology, as assessed by electron microscopy, including pronounced attenuation of GBM thickening, substantial resolution of podocyte foot process effacement, and a significant reduction in subepithelial electron‐dense immune deposits in NDP‐MSH–treated mice (Figure 2D).

The apparent discordant relationship between the robust histopathological improvement and the modest anti‐proteinuric effect suggests that certain corollary activities of NDP‐MSH may partially counteract its renoprotective benefits. Given that hypertension is a well‐established risk factor for albuminuria, and that α‐MSH and its synthetic analogs have been associated with hypertensive effects [36], systemic blood pressure was assessed using the CODA programmable noninvasive tail‐cuff sphygmomanometer. Shown in Figure S1A, NDP‐MSH treatment resulted in significant elevations in mean arterial pressure (MAP), with increases of approximately 15 mmHg, accompanied by a concomitant increase in heart rate (Figure S1B).

Collectively, these findings indicate that delayed melanocortin therapy effectively ameliorates established glomerular injury in THSD7A‐associated MN, despite the presence of a hypertensive effect.

3.3. NDP‐MSH Preserves Podocyte Integrity and Suppresses Podocyte Injury

Given the central role of podocyte injury in the pathogenesis of proteinuria and MN, we next examined whether NDP‐MSH exerted direct protective effects on podocytes. Immunofluorescence staining of cryosections of kidney specimens was performed for podocyte homeostatic markers, including podocalyxin and podocin, as well as the podocyte injury marker desmin. As shown in Figure 3A, THSD7A‐immunized mice displayed a marked loss of podocalyxin and podocin expression within glomeruli, accompanied by increased desmin expression in podocin‐positive glomerular cells (Figure 3B), consistent with severe podocytopathy. In contrast, delayed treatment with NDP‐MSH largely restored glomerular expression of podocalyxin and podocin while significantly suppressing desmin expression, indicating preservation of podocyte structure and function. These morphological findings were corroborated by immunoblot analysis of isolated glomeruli (Figure 3C), combined with densitometric quantification, confirming restoration of podocyte homeostatic proteins and suppression of injury‐associated markers, including desmin and EGR1, in NDP‐MSH–treated mice (Figure 3D). At this disease stage, expression of the podocyte nuclear marker WT1 remained comparable across groups, suggesting minimal podocyte loss and indicating that the observed injury reflects early, potentially reversible podocyte dysfunction rather than podocytopenia.

FIGURE 3.

Melanocortin therapy ameliorates podocyte injury in an active immunization mouse model of THSD7A‐associated membranous nephropathy. (A) Immunofluorescence staining of kidney specimens for podocalyxin (PODXL). Scale bar = 25 μm. (B) Dual‐color immunofluorescence staining of kidney specimens for podocin (green) and desmin (red). Scale bar = 25 μm. (C) Representative immunoblots showing immunoblot analysis of isolated glomeruli homogenates for indicated proteins. (D) Quantification of protein expression by densitometric analyses of immunoblots, expressed as relative levels normalized to β‐actin. Statistical significance was determined by one‐way ANOVA. *p < 0.05 (n = 6); ns, no significance.

3.4. NDP‐MSH Reduces Glomerular Immune Complex Deposition and Complement Activation

The NDP‐MSH‐reduced glomerular immune deposits observed above by electron microscopy suggest a potential effect on immune‐mediated glomerular injury. To verify this finding, kidney sections were stained for mouse IgG and the membrane attack complex C5b–9. As shown in Figure 4A, THSD7A‐immunized mice exhibited intense granular pseudo‐linear deposition of mouse IgG along the glomerular capillary walls, consistent with an autoimmune pathogenic mechanism. This was accompanied by prominent C5b–9 deposition along the glomerular tuft, which colocalized with areas of diminished podocin staining, indicating complement‐mediated podocyte injury. NDP‐MSH treatment substantially reduced both IgG and C5b–9 deposition, while restoring podocin expression (Figure 4B). Quantitative fluorometric analysis confirmed a significant reduction in immune deposition and complement activation after NDP‐MSH treatment (Figure 4C). These findings reveal that melanocortin therapy mitigates immune complex–mediated glomerular injury in THSD7A‐associated MN.

FIGURE 4.

NDP‐MSH treatment reduces immune deposition and attenuates complement activation in glomeruli in an active immunization mouse model THSD7A‐associated membranous nephropathy. (A) Kidney cryosections were subjected to immunofluorescence staining for mouse IgG (red) and DAPI (blue). Scale bar = 25 μm. (B) Dual‐color immunofluorescence staining of kidney specimens for podocin (green) and C5‐b9 (red). Scale bar = 25 μm. (C) Computerized morphometric analysis of mean fluorescence intensity (MFI) for IgG, podocin or C5‐b9 staining in glomeruli. Statistical significance was determined by one‐way ANOVA. *p < 0.05 (n = 3).

3.5. NDP‐MSH Suppresses Plasma Cell Differentiation and Reduces Anti‐THSD7A Autoantibody Production

To determine whether the renal protective effects of NDP‐MSH stem from a systemic immunomodulation, splenocytes were isolated from experimental groups. Flow cytometric analysis of isolated splenocytes demonstrated that NDP‐MSH treatment, compared with vehicle treatment, significantly reduced the frequency of CD138‐positive antibody‐secreting cells, including plasma cells and plasmablasts (Figure 5A). Measurement of anti‐THSD7A IgG in culture supernatants revealed robust antibody production by splenocytes derived from THSD7A‐immunized mice, whereas splenocytes from NDP‐MSH–treated mice produced significantly lower levels of anti‐THSD7A IgG (Figure 5B). To test effects on other immunocytes, wild‐type mice were treated with NDP‐MSH for 3 weeks. Flow cytometric analysis showed no significant change in the proportion of CD3⁺ T cells (Figure S2), although it remains unknown whether NDP‐MSH modulates antigen presenting cell or T cell activity. These findings indicate that melanocortin therapy might suppress pathogenic autoantibody production, at least in part, by limiting plasma cell differentiation.

FIGURE 5.

Melanocortin therapy suppresses autoreactive humoral immune response in an active immunization mouse model THSD7A‐associated membranous nephropathy. (A) Animals were treated as elaborated in Figure 2A. Splenocytes were isolated from THSD7A‐immunized mice after NDP‐MSH or vehicle treatment and were subjected to flow cytometric analysis of CD138⁺ antibody‐secreting cells, with corresponding absolute cell counts shown. Statistical significance was determined by Student's t‐test. *p < 0.05 (n = 3). (B) Splenocytes isolated from THSD7A‐immunized mice treated with PBS or NDP‐MSH were collected and cultured ex vivo for 4 days. Cell culture supernatants were collected for the measurement of IgG levels, which were further corrected for the number of viable cells. *p < 0.05 (n = 3).

3.6. NDP‐MSH Activates the MITF/IRF4 Signaling Axis to Inhibit B‐Cell Differentiation Into Plasma Cells

Finally, we investigated whether NDP‐MSH exerts a direct effect on regulating B cell differentiation to plasma cells. Splenocytes isolated from THSD7A‐immunized mice were cultured ex vivo and treated with PBS or NDP‐MSH for 48 h (Figure 6A).

FIGURE 6.

NDP‐MSH treatment directly suppresses plasma cell differentiation in primed B cells isolated from mice with active immunization THSD7A‐associated membranous nephropathy. (A) Schematic diagram depicts the experimental design. Splenocytes were isolated from THSD7A‐immunized mice and were cultured ex vivo in the presence or absence of NDP‐MSH for 48 h. (B) Representative immunoblots showing immunoblot analysis of cells for indicated proteins and quantification of protein expression by densitometric analyses of immunoblots, expressed as relative levels normalized to GAPDH. Statistical significance was determined by Student's t‐test. *p < 0.05 (n = 4). (C) Dual‐color immunofluorescence staining of splenocytes for CD19 (green) and IRF4 (red). Scale bar = 10 μm. (D) Representative immunoblot analysis of CD138 in splenocytes from THSD7A‐immunized mice treated with NDP‐MSH or vehicle, with corresponding densitometric quantification. Statistical significance was determined by Student's t‐test. *p < 0.05 (n = 4).

Immunoblot analyses demonstrated that NDP‐MSH significantly upregulated MITF expression while concurrently suppressing IRF4 expression (Figure 6B), indicating activation of the MITF/IRF4 signaling axis, a pathway well implicated in negative regulation of B cell differentiation [37]. Importantly, within CD19‐positive B cells shown by fluorescent immunocytochemistry, NDP‐MSH treatment resulted in marked suppression of IRF4, a transcription factor essential for plasma cell differentiation (Figure 6C). Consistent with this finding, CD138 expression was significantly reduced in NDP‐MSH–treated splenocytes, confirming inhibition of B cell differentiation into antibody‐secreting plasma cells, as shown by immunoblot analysis (Figure 6D).

4. Discussion

Through establishing an active immunization mouse model of THSD7A‐associated MN, this study demonstrates that delayed intervention with NDP‐MSH significantly ameliorates proteinuria and renal histopathology characteristic of MN. Importantly, the therapeutic benefit was observed after disease onset, underscoring the potential translational relevance of melanocortin‐based therapy for established MN in clinical settings. Mechanistically, the renoprotective effects of NDP‐MSH were primarily attributable to its immunomodulatory actions on the autoimmune response, leading to suppression of anti‐THSD7A autoantibody production and reduced glomerular immune complex deposition. Specifically, NDP‐MSH treatment inhibited the differentiation of B lymphocytes into antibody‐secreting plasma cells in mice with THSD7A‐associated MN. To the best of our knowledge, this study is the first to evaluate melanocortin therapy in an active immunization model of MN that involves a clinically relevant human MN autoantigen, thereby providing a more faithful representation of human disease immunopathogenesis (Figure 7).

FIGURE 7.

Schematic diagram illustrates the proposed working model for the renoprotective effect of NDP‐MSH in an active immunization mouse model of THSD7A‐associated membranous nephropathy. Active immunization of mice with commercially available recombinant THSD7A protein elicits an autoimmune response against the self‐antigen THSD7A, and causes antigen presenting cell (APC), T cell and B cell activation, thereby generating autoreactive B cells. Under tight regulation by the MITF/IRF4 signaling axis—a pathway critical for B cell differentiation—these autoreactive B cells differentiate into CD138⁺ antibody‐secreting cells, including plasmablasts and plasma cells, and produce abundant anti‐THSD7A autoantibodies. These autoantibodies target podocytes and induce glomerular injury and proteinuria, establishing an active immunization mouse model that faithfully recapitulates the cardinal features of human membranous nephropathy (MN). This study demonstrates that NDP‐MSH upregulates microphthalmia‐associated transcription factor (MITF) and concomitantly suppresses interferon regulatory factor 4 (IRF4) expression in primed B cells derived from mice with THSD7A‐associated MN. This modulation of the MITF/IRF4 axis inhibits plasma cell differentiation, resulting in reduced numbers of CD138⁺ antibody‐secreting cells, diminished glomerular immune complex deposition and complement activation and amelioration of membranous nephropathy. It remains unclear whether NDP‐MSH directly modulates APC or T‐cell activity (indicated by “?”). Despite its pronounced histological renoprotective effects, NDP‐MSH simultaneously exerts a hypertensive effect, which may partially offset its anti‐proteinuric and renoprotective benefits mediated through immunomodulation.

Melanocortin therapy, exemplified by NDP‐MSH, has previously been shown to confer protection against MN in experimental models [9, 13]. However, prior investigations were limited to passive or heterologous models of MN [38], which differ fundamentally from active immunization models in terms of immunopathogenic mechanisms, disease kinetics, and initiation of pathogenic responses. Passive MN models rely on glomerular response and immune reactions to xenogeneic nephritogenic antibodies and are characterized by a rapid heterologous phase with abrupt onset of proteinuria, followed by an autologous phase driven by host immune responses against the foreign antibodies [39]. In contrast, the active immunization model used in the present study is dominated exclusively by an autoimmune phase, involving sustained APC, T cell and B cell activation, and antigen‐specific humoral immune responses that generate pathogenic autoantibodies against intrinsic podocyte antigens such as THSD7A (Figure 7). Consequently, disease progression in this model is gradual and more closely mirrors the natural history of human MN.

Despite these fundamental differences in immunopathogenesis, NDP‐MSH treatment in the active immunization model was reminiscent of its effect in passive models of MN and attenuated glomerular injury and podocyte damage [22]. This was evidenced by improved ultrastructural features, including reduced podocyte foot process effacement and GBM thickening, as well as preserved expression of key podocyte homeostatic proteins such as podocin. While this study demonstrated potential immunomodulatory and renoprotective effects of NDP‐MSH, the melanocortin receptors mediating these effects remain elusive. Recent studies suggest that MC1R may modulate immune responses by regulating the activity of T lymphocytes, B lymphocytes, macrophages, and antigen‐presenting cells [14]. In addition, activation of MC5R may confer direct podocyte‐protective effects [13], thereby protecting glomeruli against injury induced by immune reactions or nephrotoxins. Since NDP‐MSH is capable of activating all melanocortin receptors except MC2R, it is plausible that coordinated or redundant signaling through more than one melanocortin receptor contributes to the beneficial effects observed in this study, possibly via immunomodulation of APC, T and B cells.

In addition, it remains uncertain how delayed melanocortin receptor activation improves recovery in mice with established THSD7A‐associated MN. One possibility is that the reduced damage observed following NDP‐MSH treatment simply reflects attenuation of cumulative injury after therapy initiation, as evidenced in this study by reduced accumulation of nephritogenic autoantibodies and complement activation in glomeruli, thereby lowering the overall disease burden. Alternatively, NDP‐MSH may also mitigate injury that has already been established. Previous studies have shown that α‐MSH can promote tissue repair and remodeling through anti‐inflammatory and anti‐fibrotic effects [6, 40, 41]. However, these possibilities cannot be definitively distinguished based on the current data and require further mechanistic investigation.

Supporting these in vivo findings, ex vivo experiments demonstrated that NDP‐MSH treatment of primed B cells isolated from diseased mice directly suppressed plasma cell differentiation and antibody production. Recent studies have implicated melanocortin receptor 1 (MC1R) signaling in the regulation of B cell activation through the MC1R–cAMP–MITF–IRF4 axis [14]. Consistent with this mechanism, as a potent agonist of MC1R, NDP‐MSH treatment in our study was associated with increased MITF expression and concomitant downregulation of IRF4 in ex vivo–treated B cells. These findings suggest that MC1R signaling may contribute to the immunomodulatory effects of NDP‐MSH in the active immunization model of THSD7A‐associated MN. Nevertheless, further mechanistic studies are warranted to delineate the relative contributions of various melanocortin receptor subtypes in mediating the renoprotective effect. Such insights will be essential for the rational design and optimization of melanocortin‐based therapeutics with enhanced efficacy and specificity for MN.

Although the present study demonstrates a clear therapeutic benefit of NDP‐MSH in an active immune‐mediated model of MN, it is important to recognize that NDP‐MSH may not represent the optimal or ideal melanocortin compound for clinical translation. As a superpotent and long‐acting synthetic analog of α‐MSH, NDP‐MSH acts as a pan‐agonist across all known melanocortin receptors except MC2R [13, 42, 43]. While such broad receptor activation is advantageous for proof‐of‐concept studies, it may also elicit undesired systemic effects that may limit therapeutic utility. Indeed, in previous studies in acute models of podocytopathy elicited by Adriamycin [44] or puromycin aminonucleoside [8], which cause podocyte toxic injury and heavy proteinuria with minimal involvement of systemic immunopathogenesis, treatment with α‐MSH [44] or NDP‐MSH [8] barely ameliorated albuminuria despite a trend toward improvement in podocyte injury. In our study, NDP‐MSH treatment was associated with elevated blood pressure and heart rate. This observation is consistent with prior reports demonstrating that α‐MSH or its analogs exert potent hypertensive effects in both animal models and humans [36, 45, 46, 47, 48], likely mediated through activation of MC3R in the cardiovascular system or MC3R/MC4R pathways within the central nervous system [49]. A limitation of the present study is that baseline blood pressure was not assessed in this THSD7A‐associated MN model, which precludes definitive exclusion of potential hemodynamic confounding, although experimental MN is not typically associated with systemic hypertension [50]. Moreover, blood pressure was measured using the tail‐cuff method. Although widely used and noninvasive, this method may be influenced by restraint‐related stress and may not fully reflect basal blood pressure [51]. More accurate methods, such as radiotelemetry, should be considered in future studies. In addition, although no signs of distress or abnormal behavior were observed during routine monitoring, the potential contribution of nociceptive stress to the observed hemodynamic changes cannot be fully excluded, particularly given the role of MC4R in pain perception and sympathetic activation [52]. More comprehensive and quantitative assessments of pain‐related responses will be valuable in future studies to determine whether the observed hemodynamic alterations reflect nociceptive stress or direct cardiovascular effects of melanocortin receptor activation. Given that hypertension is a well‐established risk factor for proteinuria, this pressor effect may partially offset the anti‐proteinuric and renoprotective benefits of NDP‐MSH that are attributable to its immunomodulatory actions.

Accordingly, it is plausible that selectively targeting renoprotective melanocortin receptor subtypes while minimizing MC3R and MC4R activation may facilitate the development of agents with enhanced renoprotective efficacy and improved safety profiles. The rational design and systematic evaluation of receptor‐selective melanocortin agonists therefore represent a promising and strategically important direction for future therapeutic development in MN.

In summary, this study establishes a pragmatic and readily accessible active immunization mouse model of MN using commercially available recombinant THSD7A protein antigens, providing a valuable platform for mechanistic and therapeutic investigations. Using this clinically relevant model, we demonstrate that NDP‐MSH exerts renoprotective effects in MN, at least in part, through suppression of B cell activation and pathogenic humoral immune responses, leading to reduced production of nephritogenic autoantibodies against podocyte THSD7A. These findings expand the understanding of melanocortin immunomodulation in glomerular disease and support further exploration of targeted melanocortin‐based therapies for membranous nephropathy.

Author Contributions

Mingzhuo Zhang: data curation, formal analysis, methodology, writing – original draft. Mengxuan Chen: data curation, writing – review and editing. Yan Ge: methodology. William Gunning: methodology, resources. Rujun Gong: funding acquisition, project administration, supervision, writing – review and editing. All authors approved the final version of the paper.

Funding

R.G. was supported in part by the University of Toledo incentive funds and the U.S. National Institutes of Health grant DK133203 and DK114006.

Conflicts of Interest

R.G. has consulted for Reata, Mallinckrodt, and ANI Pharmaceuticals on topics unrelated to this study. All other authors declared no competing interests. All authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflicts of interest.

Supporting information

Figure S1: NDP‐MSH treatment elevates blood pressure in mice, accompanied by a sustained increase in heart rate.

Figure S2: NDP‐MSH treatment does not alter T cell proportions in splenocytes.

Table S1: List of antibodies used in this study.

Data Availability Statement

The original contributions presented in the study are included in the article/Supporting Information. Further inquiries can be directed to the corresponding authors.