IgG Glycosylation-Dependent CLEC7A Signaling Drives Podocyte Dysfunction in Lupus Nephritis

Abstract

Background:

Lupus nephritis (LN) is a severe complication of systemic lupus erythematosus (SLE) that can lead to end-stage kidney disease and increased mortality. Immunoglobulin G (IgG) from LN patients displays abnormal glycosylation, contributing to podocyte injury. CLEC7A (C-type lectin domain family 7 member A) is a transmembrane lectin receptor that recognizes fucose on IgG. This study investigates the role of lectin-glycan interactions in LN-related podocyte dysfunction.

Methods:

IgG isolated from patients with active lupus nephritis and healthy controls was exposed to differentiated human podocytes to assess gene and protein responses using qPCR, western blotting, immunofluorescence, phalloidin staining, calcium flux, and wound-healing assays. Human kidney biopsies and MRL/lpr mouse kidneys were examined to validate CLEC7A–SYK pathway activation and the glycan-dependent effects of LN-IgG.

Results:

Human podocytes exposed to the CLEC7A ligand curdlan or IgG from LN patients, but not from healthy individuals or SLE patients without LN, showed increased CLEC7A expression, impaired motility, disrupted cytoskeleton, and reduced nephrin expression. Deglycosylation of LN-IgG blocked CLEC7A binding and preserved podocyte function. Mechanistically, curdlan and LN IgG increased calcium flux and activated spleen tyrosine kinase (SYK). MRL/lpr mice with nephritis exhibited elevated CLEC7A and SYK expression in podocytes compared to controls. At the translational level, we show that podocytes in kidney biopsies from patients with active LN exhibit increased expression of SYK and CLEC7A compared to healthy controls. Pharmacological SYK inhibition or CLEC7A silencing effectively prevented podocyte injury.

Conclusions:

We reveal a novel IgG lectin-glycan-CLEC7A-SYK axis involved in the pathogenesis of LN, which can be targeted therapeutically in patients with LN.

Graphical Abstract

Introduction

Lupus nephritis (LN) is a severe and potentially life-threatening complication of systemic lupus erythematosus (SLE) (1). Approximately 50% of individuals with SLE develop LN, which is associated with a strikingly elevated mortality rate, approximately 26 times higher than in SLE patients without renal involvement (2). This increased mortality far exceeds the risk associated with other major causes of death among patients with SLE, such as cancer or cardiovascular disease (3). Each episode of lupus nephritis contributes to cumulative nephron loss, thereby accelerating progression toward end-stage renal disease (ESRD) (4). A central mechanism underlying this progression is glomerular injury driven by immune complex deposition in distinct regions of the glomerulus, which initiates inflammatory and structural damage (5). Among the earliest cellular manifestations of this injury is podocyte damage, a well-established hallmark of LN that directly contributes to proteinuria and disease progression (6–8).

Immunoglobulin G (IgG) is the predominant immunoglobulin in human serum, and modifications to the glycan chains attached to its fragment crystallizable (Fc) region have been shown to significantly modulate its biological activity (9). Distinct glycan patterns have been shown to modulate the inflammatory potential of IgG (10, 11). In our previous studies, we identified that aberrant IgG glycosylation plays a pivotal role in driving podocyte injury in LN (6, 8). Specifically, we found that fucose on IgG leads to podocyte injury while terminal galactosylation confers a protective effect (5,7).

Core fucosylation, a major modification of N-glycans, has been implicated in immune regulation, notably through its modulation of antibody-dependent cell-mediated cytotoxicity (ADCC) (12). C-type lectin receptors (CLEC) are a large family of carbohydrate-binding proteins that recognize specific sugar structures, particularly those containing carbohydrate motifs like mannose, fucose, or galactose. CLEC7A (also known as Dectin-1), can recognize β-glucans found in fungal cell walls and has an extracellular carbohydrate-recognition domain (CRD) and a cytoplasmic domain containing a partial immunoreceptor tyrosine-based activation motif (ITAM) (13). CLEC7A is also the only known mammalian receptor that specifically recognizes fucose on IgG (14).

In the present study, we tested the hypothesis that abnormally glycosylated LN-derived IgG triggers CLEC7A-mediated signaling pathway leading to podocyte damage.

Materials & Methods

Immortalized human podocyte cell line:

The immortalized human podocyte cell line was cultured as previously described (6). Briefly, cells were cultured with RPMI-1640 with 10% FBS, insulin, transferrin, and selenium. These cells proliferate at 33°C and differentiate into mature podocytes 7–10 days after transfer to 37°C due to the temperature-sensitive SV40-T gene and a telomerase gene. Ten days after being transferred to 37°C, cells were treated with IgG (10 μg/mL) from SLE patients with active LN, patients with SLE and no kidney involvement, or healthy controls. Cells were collected 24–72 hours after stimulation and analyzed by western blotting.

Human subjects:

Study participants included adults with a diagnosis of SLE, with or without nephritis, who were recruited from Tulane Hospital, Nephrology and Hypertension clinics in New Orleans under an Institutional Review Board approved protocol (IRB Study Number: 2021–1409). Healthy individuals were also enrolled in accordance with the same protocol. Active LN was defined based on clinical and laboratory criteria, including proteinuria and/or biopsy-confirmed disease. Exclusion criteria included terminal illness and vulnerable populations such as pregnant women, prisoners, and cognitively impaired individuals; no vulnerable subjects were included. Recruitment was conducted through voluntary participation without the use of personal contacts. Written informed consent was obtained from all participants prior to enrollment. Blood samples were collected, and serum was isolated for immunoglobulin purification. The sample size of this study was 5 LN patients, 5 SLE patients and 5 healthy individuals.

Human kidney biopsy samples were obtained from patients with LN and control human kidney tissue were derived from living donor protocol biopsies. Biopsy samples were collected by the Department of Medicine at Tulane University School of Medicine and procured through Arkana Laboratories.

Mice:

C57BL/6 and MRL/lpr mice were purchased from the Jackson Laboratory (Bar Harbor, ME). All mice (N=6) with an MRL/lpr background for experiments were females, and males were excluded. Animals were sacrificed at 12–16 weeks of age. This was an exploratory pilot study. We used N = 6 per group (no exclusions) as is common for pilot work to estimate variance and effect sizes for future, fully powered studies.

IgG isolation and tagging:

Immunoglobulins were isolated from human serum samples using Melon^™ Gel IgG Spin Purification Kit (Thermo Scientific^™; Catalog 45206), following manufacturer instructions. FITC tagging was performed by using FluoroTag^™ FITC Conjugation Kit (Sigma Aldrich; Catalog FITC1–1KT).

Quantitative PCR:

We performed real-time PCR assays as described earlier (15). Briefly, total RNA was isolated from cultured podocytes using the RNeasy Plus Mini Kit (QIAGEN, catalog 74136). Purified total RNA (0.2–1 μg) was used to prepare cDNA through high-capacity RNA to c-DNA kit (Applied Biosystems, catalog 4368814). Candidate gene expressions were estimated using the fluorescent dye SYBR Green methodology and CFX96 Touch Real-Time PCR (Bio-Rad). Bioinformatically validated primer sets (QuantiTect Primer Assays, QIAGEN, or IDT) were purchased for use in SYBR Green-based RT-PCR. Real-time fluorescence from SYBR Green (Applied Biosystems) was measured by the Bio-Rad CFX Manager 3.1 System Software. Gene expression was normalized to the endogenous controls (β-actin or GAPDH) and comparative CT values were estimated. Relative gene expression was calculated through 2–(ΔΔCT) method and expressed in arbitrary units (a.u.) relative to paired controls.

Scratch/wound healing:

Human podocytes were plated in six-well plates and treated after reaching 70–80% confluency. Scratches were created using a sterile 200 μl pipette tip. Following scratch formation, cells were immediately treated with vehicle and curdlan for 48 hrs. In separate experiments after scratch formation, cells were exposed to no IgG, LN-IgG or LNDG-IgG for 72 hrs. Areas covered by cells were quantified and subjected to statistical analysis. Areas covered by cells were imaged between 0 and 72 h for quantification.

Immunofluorescence/ Phalloidin staining:

Frozen kidney biopsies from individuals with LN/ controls and paraffin embedded mice (WT/ MRL-lpr) kidney sections were immunofluorescent stained for SYNPO, CLEC7A and SYK. Immunofluorescence staining was done for CLEC7A, Nephrin, and Phalloidin in human podocyte cell line after being exposed to healthy, LN, or deglycosylated LN IgG. For visualization of cytoskeleton of podocytes, we stained cells using phalloidin DyLight 488 (catalog 21833) staining through regular immunocytochemistry methods (similarly for other antibodies too) as described earlier (16). Briefly, human podocytes were exposed to healthy, LN, or deglycosylated LN IgGs, and cells were washed with PBS at the end of incubation and fixed with 4% PFA. Fixed cells were washed 3 times with PBS, and permeabilized cells with Triton X-100 (0.1%) and BSA (2%) in PBS. We blocked cells in normal goat serum (Cell Signaling Technology, catalogue 5425S) plus 0.1% Triton X-100 (PBST) for 1 h. Cells were stained with Phalloidin DyLight 488 (green; catalog 21833) to visualize cytoskeleton and Alexa Fluor^® 555 (red; catalog A27039) to visualize CLEC7A. Stained cells were mounted with coverslips using UltraCruz Aqueous Mounting Medium (Santa Cruz Biotechnology, catalogue sc-24941). We captured images on original magnification, 20X, or 40X using a fluorescent microscope (M5000, Thermo Fisher Scientific; Pittsburgh, PA, USA and Nikon ECLIPSE Ti2). We used ImageJ software to quantitate and analyze IF images.

Western blotting:

Protein was isolated from cells and quantified through Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, catalog 23225). Immunoblotting was performed to detect the expression of proteins with antibodies (CLEC7A, PSYK525, TSYK). Equal amounts of proteins (50–80 μg) were separated through NuPAGE 4%–12% Bis-Tris Gel (Thermo Fisher Scientific, catalog NP0322BOX) electrophoresis and transferred onto a nitrocellulose membrane (LI-COR Biotechnology, catalog 92631092). The membrane was blocked with PBS, TBS, or 5% milk in Tris-Buffered Saline-Tween (0.05%) solution based on primary antibody-specific recommendations of the manufacturer. After overnight incubation with primary antibody, blots were incubated with LI-COR secondary antibody (Odyssey IRDye 680RD, catalog 92668070, or 800CW, catalog 92632211) for 1 hour. Western blots were visualized by Odyssey CLx Imaging System (LI-COR Biotechnology) using near-infrared fluorescence capture. Western blot image analysis was performed using Image Studio software (LI-COR Biotechnology) to obtain integrated intensities. Data was analyzed after normalizing with endogenous control protein (β-actin or GAPDH).

Pull down assay:

We used Pierce co-immunoprecipitation kit (catalog 26149; Thermo Scientific) for pull down assay and followed manufacturer’s protocol. Briefly, human podocytes after exposing with LN-IgG for 24hrs were lysed using ice cold lysis buffer and cell lysate was pre-cleared through control agarose resin slurry to remove any cell debris. SYK antibody was immobilized in a spin column and cell lysate was allowed to bind with SYK antibody for overnight at 4°C. Unbound sample was washed, and bound protein was eluted using elution buffer. The pull-down product was subjected to the Western blot validation and separated by SDS-PAGE.

siRNA transfection:

To knock down the expression of a CLEC7A, we used commercially available siRNA (Dharmacon^™ siGENOME SMARTpool Human CLEC7A siRNA, Catalog M-021476-02-0005). As a control for transfection, Universal negative control #1 siRNA (Sigma-Aldrich, catalog SIC001) was used, which consists of a scrambled sequence. To transfect human podocyte cells with CLEC7A siRNA, we used Mirus TransIT-X2^® Dynamic Delivery System (Mirus Bio LLC, Madison, WI, USA, catalogue MIR 6003) as described earlier (16).

Fluo 4 Calcium Flux:

Podocytes were cultured and differentiated in 96 well plates to near confluence. Fluo-4, AM (Invitrogen, Catalog# F14201) stock (1mM) was dissolved in the cell media and a final concentration of 4 μM of Fluo-4 was used in the experiments. Cells were loaded with Fluo-4 for 30 mins and then IgGs (10 μg/ml) were added to the cells for different time points up to 60 mins at 10-min time intervals. Cells were washed with PBS before fluorescence measurements. Fluorescence was measured using a BioTek Synergy^™ HT Multi-Detection Microplate Reader (BioTek^®, Winooski, VT, USA) through excitation at 494 nm and emission at 506 nm wavelength.

Statistics:

Statistical differences were calculated via two tailed t-test or 1-way ANOVA with Tukey’s post-hoc test. P values were considered significant if they were below 0.05. *P < 0.05, **P<0.01, ***P<0.001. We used the ARRIVE checklist when writing our report (17).

Results

Podocytes express CLEC7A which is upregulated in LN.

To investigate the expression of CLEC7A in podocytes, we performed immunofluorescence staining on kidney tissue samples from individuals with LN and healthy controls. Our results revealed that CLEC7A expression was significantly elevated in the glomeruli of 7 LN patients with Class III or IV disease compared to 5 healthy controls (Figure 1A). Next, we conducted immunostaining on a human podocyte cell line, confirming the presence of CLEC7A in healthy podocytes (Figure 1B). When exposed to LN IgG (n=4), we found podocytes exhibited a marked upregulation of CLEC7A transcription (Figure 1C), and protein (Figure 1D) expressions compared to those exposed to healthy IgG (n=5). These findings are consistent with prior transcriptomic analyses, which have shown that CLEC7A is overexpressed in the glomeruli of LN patients, ranking amongst the top 2% of the most highly upregulated genes in LN glomeruli (18).

Figure 1: CLEC7A is expressed in human podocytes.

(A) Immunofluorescence of human kidney biopsy tissue sections shows upregulation of CLEC7A (Red) in podocytes of patients with LN; (B) Immunocytochemistry for CLEC7A (Red) shows it is expressed in immortalized human podocyte cell line and LN IgG exposure upregulates CLEC7A gene expression than non-LN SLE serum exposure, t-test P<0.05, 40x (C) CLEC7A gene expression (fold change) in immortalized human podocyte cell line exposed with Healthy IgG vs LN IgG, t test P=0.003, N=5 and (D) CLEC7A protein expression (GAPDH normalized) in immortalized human podocyte cell line exposed with Healthy IgG vs LN IgG, t-test P<0.05, N=5.

Activation of CLEC7A by agonist curdlan leads to podocyte injury.

CLEC7A, while widely expressed, may not function in all cell types (19). To assess whether CLEC7A expressed in podocytes can signal and induce podocyte injury, we utilized curdlan, a known β−1,3-glucan agonist of CLEC7A (20). Exposure to curdlan resulted in significant rearrangement of the podocyte cytoskeleton, as evidenced by a reduction in actin filament density and total cell fluorescence for phalloidin (Figure 2A), along with decreased motility in cultured podocytes, as demonstrated by the wound healing assay (Figure 2B). Since podocytes are terminally differentiated, wound closure in this context reflects podocyte migration, rather than a combination of proliferation and migration typically observed in general wound healing assays. A notable decrease in the relative migration rate of curdlan-exposed podocytes was observed at 48 hours (P<0.01; Figure 2B).

Figure 2: CLEC7A activation causes podocyte injury.

(A) Phalloidin staining shows disorganized cytoskeleton (less actin filaments) in cells exposed to curdlan; (B) Exposure to curdlan decreases motility (migration rate) of differentiated podocytes; P<0.01 at 48hrs, N=4; (C) Calcium flux surged in podocytes exposed with curdlan than untreated podocytes, N=5; (D) Expression of nephrin decreases in curdlan activated human podocytes, t-test P value <0.05, N=5.

We next investigated calcium flux in podocytes, given that CLEC7A is a member of the Ca²⁺-dependent sugar-binding C-type lectins, and calcium flux plays a critical role in regulating podocyte shape and structure. Following curdlan exposure, we observed a significant increase in calcium flux in curdlan-treated podocytes compared to untreated controls (Figure 2C). Nephrin, a critical podocyte marker, is essential for maintaining the integrity of the glomerular filtration barrier, and its deficiency results in severe proteinuria (21). Previous studies have demonstrated that nephrin transcription is reduced in lupus nephritis (22). Curdlan-exposed podocytes exhibited significantly lower nephrin mRNA expression compared to untreated controls (Figure 2D; t-test, P<0.05, N=5). Collectively, these results suggest that CLEC7A activation by curdlan induces cytoskeletal disruption, impaired migration, elevated calcium flux, and downregulation of nephrin in podocytes, leading to podocyte dysfunction.

Deglycosylation of LN IgG Prevents Podocyte Injury by Disrupting CLEC7A-Glycan Binding.

Glycans attached to IgG can bind to the carbohydrate recognition domain (CRD) of CLEC7A. To determine whether IgG glycans bind CLEC7A in cultured podocytes, we conjugated IgG with a fluorescent dye (green) and performed immunofluorescence studies, probing for CLEC7A with Alexa Fluor 555 (red) antibody. The colocalization of fluorescently tagged IgG (green) and CLEC7A (red) resulted in yellow fluorescence. The boxed area highlights the colocalization of IgG and CLEC7A in a single cell (Figure 3A). We found LN IgG and healthy IgG colocalized significantly to CLEC7A while deglycosylation of IgG via PNGase treatment disrupted this interaction (Figure 3A; One way ANNOVA P<0.05). To increase resolution of the type of glycan that may modulate this binding and signaling we treated IgG with fucosidase and galactosidase (Supplementary Fig 1). Fucosidase treated IgG (green) did not colocalize with CLEC7A (red) suggesting that the presence of fucose is necessary for binding to CLEC7A corroborating findings from our previous study suggesting that fucose on IgG leads to podocyte injury (6). We also treated IgG with galactosidase and exposed podocytes to this IgG. Galactosidase treated IgG colocalized with CLEC7A to a larger extent that that untreated IgG. These data suggest that while fucose on IgG is necessary for CLEC7A interaction with IgG, the presence of galactose can modulate this binding. This is consistent with our data from our prior findings suggesting that galactose on IgG can prevent podocyte injury while fucose on IgG is pathogenic to podocytes (6).

Figure 3: Glycans are essential for binding IgG to CLEC7A and removing them by enzyme PNGase F prevents podocyte injury.

(A) Colocalization of Florescent tagged IgGs (B: Green) and CLEC7A (C: Red) shows mutual binding (D: Yellow); Deglycosylation of IgGs by PNGase F treatment disrupted this binding; 40x, boxed area zoomed; Quantification of co-localization by ImageJ, One way ANNOVA P<0.05; (B) Phalloidin staining shows less actin filaments in LN IgG exposed podocytes than untreated and deglycosylated LN IgG restored cytoskeleton, boxed images zoomed at right to show actin filaments; (C) Wound healing assay shows decreased motility in LN IgG exposed podocytes in comparison to untreated and deglycosylated IgG restores motility; (D) Fluo 4 calcium flux intensity by flow cytometry shows that LN-IgG treatment surges calcium flux and de-glycosylation normalizes this effect up to the levels of Healthy IgG; (E, F) LN-IgG downregulates Nephrin gene expression but deglycosylation of LN-IgG normalizes nephrin expression, One way ANNOVA P<0.05, N=5.

We next investigated whether IgG glycan-CLEC7A binding could trigger podocyte injury. Phalloidin staining revealed that LN derived IgG significantly disrupted actin filaments in human podocytes compared to untreated controls, while deglycosylation restored the normal cytoskeleton (Figure 3B). Additionally, exposure to LN IgG reduced podocyte motility, similar to curdlan exposure. Exposure to deglycosylated LN IgG significantly improved podocyte migration at 72 hours at levels similar to those of controls (t-test, P < 0.0001, N = 4) (Figure 3C). In line with our hypothesis, LN IgG downregulated nephrin mRNA (Figure 3E) and protein (Figure 3F) expression whereas deglycosylation restored normal gene and protein nephrin expression.

Furthermore, consistent with the effects of curdlan, flow cytometry analysis using Fluo-4 showed that treatment with LN IgG elevated calcium flux, while deglycosylation normalized this response to levels comparable to healthy IgG (Figure 3D). Together, these findings suggest that CLEC7A recognizes IgG glycans, triggering calcium flux and subsequent podocyte injury in lupus nephritis.

CLEC7A leads to SYK activation in LN IgG exposed podocytes.

We further evaluated the downstream cytoplasmic signaling mediated by the immunoreceptor tyrosine-based activation motif (ITAM) motif of CLEC7A. ITAM motifs have docking sites for SYK, which, upon phosphorylation in B cells, can trigger robust calcium flux (23).

SYK is a protein kinase that facilitates signaling in hematopoietic cells by binding to ITAMs in immune receptors (24–27). This interaction activates cellular responses, such as proliferation and differentiation (27). SYK has several phosphorylation sites. Tyr323 is a negative regulatory phosphorylation site within the SH2-kinase linker region in SYK while phosphorylation of both Tyr352 and Tyr525/526, located in the activation loop of the SYK kinase domain, are also associated with increased SYK activity (28). We observed that Curdlan exposure activated SYK through phosphorylation of Tyr 525, as indicated by a significant increase in pSYK (Tyr525/526) expression and the pSYK525/SYK ratio compared to untreated podocytes (Figure 4A; t-test P<0.05, N=5). We did not detect any differences in the magnitude of phosphorylation of other SYK phosphorylation sites (Supplementary Fig 2).

Figure 4: Curdlan and LN-IgG activate SYK signaling and de-glycosylated LN-IgG and CLEC7A silencing inhibited SYK activation in human podocytes;

(A): Curdlan exposure activated SYK protein by increasing pSYK expression and pSYK525/SYK ratio in comparison to untreated podocytes, t-test P<0.05, N=5; (B): LN-IgG exposure activated SYK by increasing pSYK expression and PSYK525/SYK ratio in comparison to podocytes exposed with Healthy IgG, t-test P<0.05, N=5; (C) Deglycosylation of LN-IgG by PNGase treatment decreased activated SYK expression, t-test P<0.05, N=5; (D) Silencing CLEC7A by siRNA transfection inhibited LN IgG induced SYK activation in Podocytes, t-test P<0.05, N=3; (E) Immunoprecipitation with anti-SYK and immunoblotting with anti-CLEC7A shows binding of CLEC7A with SYK in Curdlan (50μg/ml), LN IgG, and Deglycosylated LN IgG, treated podocytes.

Immortalized human podocytes exposed to LN-IgG also expressed higher levels of activated SYK (pSYK525) compared to those exposed to healthy IgG (Figure 4B; t-test P<0.05, N=5). Next, we investigated the impact of deglycosylated LN-IgG (treated with PNGase-F) on podocytes and found reduced SYK activation in podocytes exposed to deglycosylated LN-IgG compared to those exposed to untreated LN-IgG (Figure 4C; t-test P<0.05, N=5). We also performed a similar experiment where we exposed cultured podocytes to SLE (without LN) IgG and LN-DG IgG, but we did not find significant upregulation of activated SYK or CLEC7A (Supplementary Fig 3). Additionally, silencing CLEC7A using siRNA inhibited LN-IgG-induced SYK activation in podocytes (Figure 4D; t-test P<0.05, N=3), further confirming the critical role of the CLEC7A-glycan interaction in SYK activation.

To further confirm the direct interaction between CLEC7A and SYK kinase in podocytes, we performed co-immunoprecipitation experiments. Podocytes were treated with curdlan, LN-IgG and LN-DG IgG to activate CLEC7A, and SYK kinase was pulled down. We found that SYK kinase isolated from cells exposed to curdlan or aberrantly glycosylated LN-IgG bound to CLEC7A, whereas no such interaction was observed in cells exposed to deglycosylated IgG (Figure 4E).

These results conclusively demonstrate that SYK activation is driven by the interaction of LN derived IgG glycans and CLEC7A in podocytes.

Pharmacological inhibition of SYK in podocytes prevents injury in LN.

Next, we aimed to determine whether SYK activation is responsible for podocyte injury in LN. To assess this, we utilized the SYK inhibitor, R406, to examine its impact on LN IgG-induced podocyte damage. Pre-treatment with R406 (0.5 μM) for 30 mins and 4hrs both effectively prevented podocyte injury induced by LN IgG, as demonstrated by a restored podocyte cytoskeleton evaluated by phalloidin staining (Figure 5A).

Figure 5: SYK inhibitor prevents LN IgG induced podocytes injury.

(A): Pre-treatment of podocytes with R406 (SYK inhibitor, 0.5μM) for 30 mins or 4hrs prevented LN IgG induced injury, morphological changes shown by phalloidin staining; (B) R406 pretreatment ameliorated Nephrin gene expression downregulation in LN-IgG exposed podocytes, P_trend <0.05, N=3; and (C): significantly restored Nephrin protein expression and downregulated CAMK4 expression, P<0.05, N=4; (D) Surge in calcium flux by LN IgG was ameliorated by R406 (SYK inhibitor).

Furthermore, SYK inhibition mitigated the downregulation of nephrin mRNA expression caused by LN IgG (Figure 5B, P_trend < 0.05, N = 3) and significantly restored nephrin protein expression, however, markedly reduced CaMK4 expression P<0.05, N=4 (Figure 5C). R406 pre-treatment also attenuated the surge in calcium flux triggered by LN IgG (Figure 5D).

These findings suggest that SYK signaling is upstream of CaMK4 in podocytes and that targeting SYK can effectively prevent LN IgG-induced podocyte injury and calcium signaling pathways in podocytes.

CLEC7A and SYK upregulation is a conserved molecular signature in LN across species.

To validate our findings in other cohorts we utilized the Nephroseq database. In the Berthier Lupus Cohort (18), CLEC7A and SYK were significantly upregulated (2.46 and 1.83-fold), in glomeruli of LN patients (N=32) compared to healthy living donors (N=14) (Figure 6A, D). CLEC7A was in fact in the top 2–3% of the genes overexpressed in LN. When comparing patients with proteinuria, SYK ranked among the top 1% of upregulated genes in individuals with nephrotic-range proteinuria relative to those with sub-nephrotic proteinuria (Supplementary Fig 4). STRING analysis revealed a strong predicted interaction between CLEC7A and SYK (score 0.999) (Figure 6B). In the NZW/BXSB lupus mouse model, Clec7a expression was significantly higher in proteinuric mice (1.57-fold) compared with non proteinuric mice (N=10) (Figure 6C).

Figure 6: Synaptopodin, CLEC7A and SYK expression on human and murine kidney tissues.

(A) Comparison of CLEC7A, SYK and SYNPO in Berthier Lupus GlomOver-expression in Group: Lupus Nephritis vs. Healthy Living Donor shows SYNPO expression decreases but CLEC7A and SYK expressions increase in Lupus Nephritis patients in comparison to Healthy subjects; 1. Healthy Living Donor (14) 2. Lupus Nephritis (32), Blue= least expressed, Red= most expressed; (B) STRING interaction of CLEC7A with its predicted functional partners shows highest prediction score with SYK (Score 0.999); (C) CLEC7A expression in Berthier Lupus mouse kidney (NZW/BXSB proteinuria model) shows significant over-expression (P=3.90E-7; fold change 1.54) in proteinuria mice (10 no proteinuria vs 6 proteinuria mice); (D) CLEC7A Expression in Berthier Lupus Glom cohort shows significant over-expression (P=4.77 E-10; fold change 2.457) in human subjects (32 Lupus Nephritis vs. 14 Healthy Living Donor); Human kidney biopsies from Healthy controls (HC) vs Lupus nephritis (LN): CLEC7A expression increases and SYNPO expression decreases in LN (E), SYK expression increases and SYNPO expression decreases in LN (F); Mouse kidney (WT vs LN): SYNO expression decreases in LN mice glomerulus (G-H) whereas Clec7a (G) and Syk expression (H) increases in LN mice glomerulus than WT mice. Yellow color in merged images shows colocalization of CLEC7A/ SYK (Red) with SYNPO (Green).

In our cohort, human lupus nephritis kidney biopsies showed elevated CLEC7A and SYK levels (Figure 6E–F). Similarly, MRL/lpr mice (lupus prone mice) exhibited increased Clec7a and Syk expression in glomeruli compared to controls (Figure 6G–H), supporting a conserved dysregulation pattern in podocytes during active proteinuric lupus nephritis.

Discussion

LN is a devastating complication of SLE leading to End Stage Renal Disease (ESRD) in ~20 percent of those affected. Podocyte injury is a critical early event in its pathogenesis (29). Furthermore, proteinuria in LN is attributed to podocyte injury and previous studies have shown that preventing podocyte injury by either utilizing cell targeted therapies or through cell specific genetic manipulation can prevent nephritis in lupus-prone mice (7, 30). Podocyte injury also plays a critical role in the development of various other glomerular diseases, including diabetic kidney disease, minimal change disease (MCD), focal segmental glomerular sclerosis (FSGS), and membranous nephropathy (31–34). The causes of podocyte injury in various glomerular diseases range from genetic and immune factors to infections, toxins, and hemodynamic stress (35–37). Our previous studies have demonstrated that in LN, IgG can cause podocytes injury, and specific glycosylation patterns on these LN derived IgG play a pivotal role in this process (6). However, the mechanisms by which aberrantly glycosylated LN IgG induces injury in podocytes remain unclear.

Glycosylation, a key post-translational modification of antibodies, influences their functional diversity and receptor interactions (38). Human IgG, with four subclasses and 36 possible glycoforms, can exhibit up to 144 functional states (39), impacting inflammatory responses (40). Altered glycosylation, particularly in the Fc region (N-glycosylation), has been linked to autoimmune diseases, converting antibodies from protective to autoreactive and this modification offers a potential target for therapeutic strategies in autoimmune conditions (41). Most of these data are from immune cells that express Fcγ receptors for IgG binding. For example, high-dose intravenous immunoglobulins (IVIg) with specific glycosylation patterns could be utilized to enhance anti-inflammatory effects (42) Further, IvIG can be engineered with enzymes to modify antibody glycosylation to reduce inflammation through the Fcγ affinity pathway (43). We have demonstrated that IgG glycosylation can alter pathogenic signaling in epithelial cells like podocytes and hence identifying receptors that may specifically bind aberrant glycans in these cells that lack Fcγ receptors may be key in developing therapeutic strategies for organ involvement in systemic autoimmunity or in organ specific inflammation.

CLEC7A is a C-type lectin receptor that functions as a transmembrane pattern-recognition receptor, capable of binding β-glucan carbohydrates, endogenous ligands on T cells, and glycan patterns on immunoglobulins (19, 44, 45). We and others have identified pathogenic glycan patterns that signify SLE and lupus nephritis (6, 46, 47). Notably, we have found that fucose contributes to the pathogenesis of podocyte injury in lupus nephritis, and CLEC7A is the only known mammalian receptor capable of specifically recognizing and binding to it (14, 48). In this study, we systematically demonstrate the presence of CLEC7A in human podocytes (Figure 1) and that CLEC7A can be activated by Curdlan (Figure 2) or by glycans that decorate LN-IgG (Figure 3). We further found that this interaction leads to podocyte injury through SYK activation (Figure 4). Translationally we demonstrate enhanced expression of CLEC7A and SYK in active LN compared to controls. Our findings also suggest that deglycosylation of LN-IgG or silencing of CLEC7A or the use of a SYK inhibitor may offer novel therapeutic strategies in lupus nephritis (Figure 5). The significance of these findings is highlighted by the transcriptome database analysis demonstrating overexpression of CLEC7A in LN patients as well as in lupus mouse kidney (18). Our findings highlight a conserved molecular signature in lupus nephritis, characterized by upregulation of CLEC7A and SYK and downregulation of SYNPO (Figure 6), suggesting their potential roles in disease pathogenesis across species.

Autonomous calcium signaling in podocytes is a prominent and evolutionary conserved feature of glomerular morphogenesis that is essential for the foot process formation (49). In contrast, podocyte foot process effacement is a consistent response of the podocytes to injury (50). While calcium flux appears necessary for the normal formation of foot processes, we and others have shown that increased calcium flux and signaling in podocytes can be detrimental (51).

We demonstrate that Ca⁺⁺ flux increases upon CLEC7A activation through the binding of curdlan or LN-IgG followed by SYK activation. Our findings linked SYK activation to CaMK4 which regulates cytoskeletal dynamics and cellular responses, and inhibition of SYK limits CAMK4 expression. We also observed removing glycans from LN IgG or inhibiting SYK ameliorated Ca⁺⁺ flux, suggesting the importance of the Glycan-CLEC7A-SYK axis in modulating calcium flux of podocytes which may be applicable in other calcium mediated podocytopathies.

Increased SYK signaling has been implicated in the pathogenesis of SLE (52), where it contributes to inflammation and tissue damage (53, 54). SYK inhibition suppresses skin and kidney injury in the MRL/lpr lupus-prone mouse, including reduced renal pathology and improved immune dysregulation (55). In addition, SYK inhibition suppresses ischemia-reperfusion injury in the gut (56) and the lungs (57). Lastly, SYK is increased in the kidney tissue of patients with COVID-19 (58) and a SYK inhibitor (fostatinimib) has been claimed as a drug to be repurposed to treat patients with COVID-19 and severe lung injury (59). We demonstrate here that silencing CLEC7A which recruits SYK can prevent podocyte injury in LN. Further, this data proposes the repurposing of SYK inhibitors currently approved for diseases like immune thrombocytopenic purpura for LN (60). These findings support a model in which podocytes function as active glycan-sensing cells, and the CLEC7A–SYK signaling representing a previously unrecognized pathway whereby immunoglobulin-derived glycans can modulate intrinsic cellular responses independent of classical immune complex–driven inflammation. Importantly, this pathway may operate in parallel with established pathogenic mechanisms in lupus nephritis, thereby expanding the current paradigm of glomerular injury to include direct, glycan-mediated signaling events within podocytes.

A limitation of this study is the reliance on siRNA-mediated knockdown rather than stable genetic deletion of CLEC7A. Future studies using podocyte-specific CLEC7A knockout models or CRISPR/Cas9-mediated gene editing will be important to definitively establish the causal role of CLEC7A in LN-IgG–induced podocyte injury.

In conclusion, our study identifies CLEC7A as a key receptor mediating podocyte injury in LN through the glycan-CLEC7A-SYK axis. Targeting IgG glycosylation, CLEC7A or SYK offers promising therapeutic strategies for treating lupus nephritis and other autoimmune diseases.

Data Statement:

The gene expression data of CLEC7A, SYK and SYNPO in Berthier Lupus Glom (Lupus Nephritis vs. Healthy Living Donor) and Clec7a gene expression in Berthier Lupus mouse kidney (NZW/BXSB proteinuria model), both were obtained from Nephroseq transcriptome database (https://www.nephroseq.org/resource/login.html). Additional data supporting the conclusions of this research are available from the corresponding author on reasonable request.