C3dg and MAC complement depositions in the renal histopathology of patients with lupus nephropathy

Introduction

The pathogenesis of systemic lupus erythematosus (SLE) is related to clinical and immunologic manifestations, among which lupus nephritis (LN) is the most common cause of death (1). Childhood-onset SLE refers to SLE with an onset before the age of 18 years (2). The etiology of patients with early-onset SLE often has a larger genetic component, multiple system involvement, and a more severe course. The 5- and 10-year mortality rate is lower than adult SLE (3). LN can occur in up to 50% of cases (4).

The key to the pathogenesis of SLE tissue inflammation and injury is complement activation (5,6). In the past 70 years, many researchers have described the role of the complement system in the pathogenesis of SLE. Complement protein levels (C3, C4) may be used as diagnostic markers for SLE and to monitor disease activity (7). However, the limitation of using them to measure disease level in SLE has been well documented (8). The central component in complement activation is C3. When activated, C3 is cleaved into two fragments: C3a and C3b. C3b is further cleaved into iC3b and finally to C3dg and C3c. Both C3a and C3c have a shorter half-life than C3dg. Some studies have suggested that C3dg may be a valuable diagnostic biomarker in SLE (9). Since these fragments are formed when the complement cascade is activated, the products of complement split can more accurately reflect complement activation than the level of a single intact protein (9,10). Some studies have also suggested that membrane attack complex (MAC) deposition may be a biomarker for more severe LN disease and poor response to treatment (11-13). Therefore, MAC and C3dg staining of lupus biopsies is essential to investigate whether their presence indicates a worse prognosis.

This study was conducted to observe the changes in complement split C3dg and MAC depositions in children with LN and investigated the value of using complement split C3dg and MAC depositions to predict kidney injury in children with LN. We present the following article in accordance with the STARD reporting checklist (available at https://tp.amegroups.com/article/view/10.21037/tp-22-310/rc).

Methods

Patients

This study was a retrospective case-control study of all active LN patients without treatment or serious contraindications that had undergone renal biopsy in our center. The medical records of 78 patients with renal biopsy-confirmed LN admitted to our hospital (The Children’s Hospital of Fudan University) between April 2018 and December 2020 were retrospectively reviewed. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study protocol was approved by the Ethics Committee of The Children’s Hospital of Fudan University (No. [2021]240), and individual consent for this retrospective analysis was waived.

The inclusion criteria required a diagnosis of LN as defined by clinical and laboratory manifestations meeting the American College of Rheumatology (ACR) criteria as follows: (I) persistent proteinuria greater than 0.5 gm per day or greater than 3+ by dipstick; (II) and/or cellular casts including red blood cells (RBCs), hemoglobin, granular, tubular, or mixed. A review of the ACR criteria has recommended that a spot urine protein/creatinine ratio >0.5 can be substituted for the 24-hour protein measurement, and “active urinary sediment” [>5 RBCs/high-power field (HPF), >5 white blood cells (WBCs)/HPF in the absence of infection, or cellular casts limited to RBC or WBC casts] can be substituted for cellular casts. An additional, perhaps optimal, criterion is a renal biopsy sample demonstrating immune complex-mediated glomerulonephritis compatible with LN (14).

Patients were excluded if a diagnosis of LN was ruled out during follow-up or if family members refused to sign the consent for their child.

Definition of LN

The diagnosis of LN was established based on the criteria of the American Rheumatism Association and verified by its characteristic changes, including glomerular hypertrophy, thickened capillary basement membranes, and nodular mesangial sclerosis on renal histology. The biopsies were assessed as per the International Society of Nephrology/Renal Pathology Society (ISN/RPS) classification for LN (15).

Clinical evaluation

The disease activity was assessed by the SLE disease activity index (SLEDAI) (16). Also, the following data from the study subjects were collected through the Electronic Health Information System: gender, fever, malar rash, photosensitivity, oral ulcer, alopecia, arthritis, serositis, neurological disorder, anemia, leukocytopenia, thrombocytopenia, hematuria, and leukocyturia.

Laboratory assessment

For the laboratory parameters, the study subjects’ serum and plasma were collected on the day of their renal biopsy to measure renal function and complement values.

Renal histopathology

LN was classified according to the ISN/RPS 2018 LN classification system by two experienced renal pathologists (HL and GL) who also scored the indicators independently based on the average score according to the activity index of renal tissue in lupus nephropathy (17). The intensity of immunostaining was reported by GL and blindly assessed by HL. Based on our previous research on the treatment of complement inhibitors in lupus mice, our center has carried out C3dg and MAC immunostaining in renal tissue since April 2018 (18).

Statistical analysis

The statistical analysis was performed with SPSS version 19.0 software (IBM, Armonk, NY, USA). Continuous variables are presented as means ± standard deviations. Differences between groups were determined using a one-way analysis of variance (ANOVA), and a P value less than 0.05 was considered statistically significant.

Results

General data

Of our 78 patients with LN, 15 were male, and 63 were female, with a mean age of 12.12±2.91 years at the time of renal biopsy.

Comparison of clinical manifestations

On direct immunofluorescence microscopy, C3dg and MAC were both detected in specimens from 48/78 (61.5%) patients. Of the 48 patients with C3dg deposition, 40 (83.3%) also had MAC deposition. Specimens with C3dg deposition were evaluated as grades. 1+, 2+, and 3+ after pathological staining in 16/48 (33.3%), 23/48 (47.9%), and 9/48 (18.8%), respectively. The degree of MAC deposition in the 48 patients was also evaluated in three grades: 1+, 2+, and 3+ in 19/48 (39.6%), 21/48 (43.8%), and 8/48 (16.7%), respectively.

There was a significantly greater proportion of neurological disorders in patients with MAC depositions vs. those without such deposits (22.9% vs. 3.3%, P=0.044). Similarly, a significantly greater proportion of patients with nephrotic syndrome (77.1% vs. 46.7%, P=0.006, respectively) and acute renal failure had MAC and C3dg depositions vs. those without such deposits (27.1% vs. 3.3%, P=0.018, respectively). In addition, patients with vs. without MAC and C3dg depositions on renal histopathology had significantly higher levels of serum creatinine (65.5±20.6 vs. 47.9±11.8 µmol/L, P=0.001; 63.9±21.5 vs. 50.5±12.4 µmol/L, P=0.001, respectively), urinary protein (2.9±2.2 g/24 h vs. 0.8±1.9 g/24 h, P=0.003; 3.1±2.4 g/24 h vs. 0.5±0.6 g/24 h, P<0.001, respectively), urinary RBC (67.2±75.6/HP vs. 20.9±40.1/HP, P<0.001; 70.0±74.3/HP vs. 16.5±38.1/HP, P<0.001, respectively), and urinary WBC (16.6±24.5/HP vs. 4.8±12.4/HP, P=0.001; 18.2±25.5/HP vs. 2.2±1.6/HP, P<0.001, respectively), but significantly lower proportions of serum C3 decreased (79.2% vs. 36.7%, P<0.001; 79.2% vs. 36.7%, P<0.001, respectively) and C4 decreased (68.7% vs. 33.3%, P=0.002; 66.7% vs. 36.7%, P=0.010, respectively). Moreover, circulating hemoglobin levels were significantly lower in patients with MAC depositions than those without such deposits (107.3±16.4 vs. 117.7±24.3 g/L, P=0.003) (Table 1).

In general, there were significant differences in serum creatinine, urinary protein, and the estimated glomerular filtration rate (eGFR) in the four groups of patients with differing degrees of C3dg and MAC depositions. Further analyses showed that compared with patients with neither depositions, patients with ++ C3dg or MAC staining depositions had significantly higher serum creatinine, urinary protein, and SLEDAI levels and significantly lower eGFRs and serum C3 and C4 levels (all P<0.05) (Table 2).

In a further investigation of patients with two types of deposition and patients with only one or no complement deposition, significant differences were found for serum creatinine, urinary protein, serum C3 and C4, SLEDAI, and eGFR in the four study groups (Table 3). In particular, when comparing patients with only C3d deposition and patients with two types of complement protein deposition, the latter’s urinary protein, RBC, WBC, and SLEDAI levels were significantly increased, but serum C3 and C4 levels were lower (all P<0.05).

Comparison of renal histopathology

Patients with C3dg depositions had a significantly greater proportion of type II (P=0.005), type III (P=0.029), and type IV LN (P<0.001) than patients without C3dg deposits. Patients with C3dg deposition also had significantly higher cellular crescents, subendothelial hyaline deposits, glomerular leukocyte infiltration, and activity index scores (all P<0.05). Similarly, patients with MAC deposition had a significantly greater proportion of type IV LN (P<0.001). They also had significantly higher cellular crescents, fibrinoid necrosis, glomerular leukocyte infiltration, and interstitial fibrosis (all P<0.05) (Table 1).

Overall, there were significant differences in activity index scores and cellular crescents in all four groups of patients with varying degrees of C3dg deposition. On the other hand, there were no significant differences in glomerular category or interstitial inflammation and vascular lesion scores between patients with and without MAC deposition. Those with ++ MAC-stained deposition had a greater proportion of interstitial fibrosis (P=0.002) (Table 2).

On further investigation of patients with both C3dg and MAC depositions vs. those with only one or no complement deposition, significant differences were found in the four study groups (Table 3). In particular, when comparing patients with only C3dg deposition vs. those with deposits of both complement proteins, the latter showed significantly higher levels of interstitial inflammatory cell infiltration and glomerular leukocyte infiltration but lower tubular atrophy (all P<0.05). Similarly, when comparing patients with only MAC deposition vs. those with deposits of both complement proteins, the latter showed significantly higher levels of interstitial inflammatory cell infiltration (all P<0.05).

The treatment follow-up of C3dg/MAC positive patients at 6 and 12 months

All patients received steroids as part of their induction therapy (as shown in Table 4), defined as between 0 and 6 months. A complete response (CR) was defined as a urinary protein creatinine ratio (UPCR) <500 mg/g, eGFR decrease <10% of pre-treatment level, or eGFR ≥90 mL/min per 1.73 m², and was not considered a treatment failure. Treatment aims to preserve and improve kidney function, represented by at least a 50% reduction in proteinuria 6 months after treatment and a UPCR level <500 mg/g at 12 months after treatment. Thirty-one out of 48 patients who were glomerular C3dg positive received cyclophosphamide. Similarly, 30 out of 48 patients who were glomerular MAC positive received cyclophosphamide. At 12 months, there were more non-responders to therapy in the C3dg positive group (22.9% vs. 3.3%, P=0.017) and in the MAC positive group (22.9% vs. 0%, P<0.001) than in the negative groups.

Receiver operating characteristic (ROC) curves and the predictive value of C3dg and MAC depositions

We used the treatment outcome at 12 months as the primary outcome variable and constructed ROC curves with C3dg deposition, MAC deposition, C3dg deposition with MAC simultaneously, and C3dg or MAC deposition as four independent predictors to compare the area under the ROC curve of the four prediction models. The modeling results showed that the area under the curve was 0.868 when C3dg deposition was an independent predictor, 0.939 when MAC deposition was an independent predictor, 0.958 when C3dg simultaneous deposition with MAC (in series) was an independent predictor, and 0.925 when C3dg or MAC deposition (in parallel) was an independent predictor. When C3dg simultaneous deposition with MAC (in series) was the independent predictor, the model had the highest sensitivity of 100%. When C3dg or MAC deposition (in parallel) was an independent predictor, the model had the highest specificity, at 75% (Figure 1).

Figure 1 ROC curves and predictive value of C3dg and MAC deposition. ROC, receiver operating characteristic; MAC, membrane attack complex.

Discussion

Due to the importance of complement binding fragments in LN, our center began to detect C3dg and MAC in kidney tissues from 2018. In this study, we explored the distribution of complement split C3dg and MAC depositions in the pathogenesis of LN. Our results indicate that C3dg and MAC depositions may be very important indicators of LN.

After the complement system activation, the activation of immune complexes can drive type III hypersensitivity, leading to inflammatory responses in target tissues (18-20). Tissue targeting is accomplished through the binding of the fusion protein to complement C3 fragments that contain a surface-exposed C3d domain and which are covalently deposited on tissues where complement is being activated (21). Complement activation on cell surfaces leads to the massive deposition of C3dg, the main complement opsonin. Complement receptor type 3 (CR3) fosters pathogen opsonophagocytosis by macrophages and the stimulation of adaptive immunity by complement-opsonized antigens that recognize the complement opsonin (22). In the European multinational initial cohort of 200 newly diagnosed (within two years) lupus patients, many had active disease, and only 54% had low C3 and/or C4 levels at baseline (23). In a recent study, although 30% of patients converted to SLE at follow-up, only 36% of patients historically had low C3 and/or C4 levels (24). Low complement level as a marker of disease activity in SLE is not reliable because the level is persistently low or normal, may not be related to disease activity, and is not sensitive to predicting disease onset (25,26). We found that patients with MAC deposition had a greater proportion of neurological disorders. Patients with MAC and C3dg deposition on renal histopathology had significantly higher levels of serum creatinine, urinary protein, urinary RBC, and urinary WBC. There were significant differences in serum creatinine, urinary protein, and eGFR in all four groups of patients with differing degrees of C3d and MAC deposition. Complement C3dg and MAC fragments are expected to be used as biomarkers for LN.

There were significantly more patients with C3dg and MAC depositions that had type IV LN (P<0.001). They also had significantly higher cellular crescents, fibrinoid necrosis, glomerular leukocyte infiltration, and interstitial fibrosis. These findings indicate that C3dg and MAC depositions can sensitively reflect the severity of disease in children with LN. Lupus children with more C3dg and MAC depositions require more aggressive treatment. The differences between the treatment outcomes at 6 and 12 months reminded us that although lupus children with C3dg and MAC depositions are given active hormone and immunosuppressive therapy at the initial stage of treatment, the effect is still not good. The deposition of C3dg and MAC in the kidney may indicate a poor prognosis in LN.

C3dg and MAC are also good clinical predictors of treatment outcome in LN. When C3d simultaneous deposition with MAC (in series) was the independent predictor, the model had the highest sensitivity at 100%. When C3d or MAC deposition (in parallel) was an independent predictor, the model had the highest specificity, at 75%.

Conclusions

This study suggests that C3dg and MAC depositions may be potential biomarkers for greater disease severity and tissue injury in LN. MAC and C3dg staining may be useful in routine studies of lupus biopsies to identify patients at risk of severe disease who may need more aggressive treatment.

Acknowledgments

The authors appreciate the academic support from the East China SLE Alliance.

Funding: This work was supported by the 2021 Pujiang Young Rheumatologists Cultivation Plan (No. SPROG2101).

Footnote

Reporting Checklist: The authors have completed the STARD reporting checklist. Available at https://tp.amegroups.com/article/view/10.21037/tp-22-310/rc

Data Sharing Statement: Available at https://tp.amegroups.com/article/view/10.21037/tp-22-310/dss

Peer Review File: Available at https://tp.amegroups.com/article/view/10.21037/tp-22-310/prf

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tp.amegroups.com/article/view/10.21037/tp-22-310/coif). All authors report the support from the 2021 Pujiang Young Rheumatologists Cultivation Plan (No. SPROG2101). The authors have no other conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study protocol was approved by the Ethics Committee of Children’s Hospital of Fudan University (No. [2021]240), and individual consent for this retrospective analysis was waived.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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