Glomerular C4d Staining Can Be an Indicator of Disease Activity in Lupus Nephritis

Abstract

Background: Abnormalities in complement activation and clearance of immune complexes by erythrocytes are the central pathogenic mechanisms in systemic lupus erythematosus (SLE). Serum C4d level, which is a degradation product of complement factor C4, was found to be a sensitive indicator of SLE activity. Our aim was to determine whether glomerular C4d staining could be a useful marker of disease activity in patients with lupus nephritis. Methods: This retrospective study included all consecutive patients who underwent a renal biopsy at our center between January 2005 and December 2009. A total of 29 patients with IgA nephritis were enrolled, and renal biopsy specimens of 24 patients have been evaluated. We evaluated baseline age, sex, hypertension, serum creatinine level, glomerular filtration rate (GFR), urine protein, and glomerular C4d staining. The primary endpoint of this study was the onset of end-stage renal disease (ESRD) in the course of study. Results: Fourteen (58%) patients were C4d+ and 10 (42%) patients C4d–. Urinary protein excretion was more elevated in C4d+ group (p = 0.0001). The renal biopsy showed that activity index score >12 was a higher proportion in C4d+ patients. The patients were followed up for 3.5 years. Four patients in the C4d+ group evolved to ESRD in the follow-up, but none of the patients in the C4d– group (p = 0.064). Discussion: We found a relationship between glomerular C4d staining and activity of lupus nephritis. C4d staining may be a useful marker to predict the prognosis of lupus nephritis.

• lupus nephritis
• glomerular C4d
• disease activity

INTRODUCTION

Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease with polymorphic clinical manifestations that range from moderate symptoms to life-threatening multiorgan failure. Renal involvement, which contributes to morbidity and mortality, occurs in up to 60% of patients in the course of disease.¹ Numerous studies have documented abnormalities in complement activation and clearance of immune complexes by erythrocytes as central pathogenic mechanisms in SLE.² The dominant pathway for complement activation in SLE is the classical pathway, triggered by the interaction of C1q with immune complexes. In several studies, it was shown that classical pathway complement protein levels reduced in association with active disease, especially C1, C4, and C2 levels.^3,4

Serum C4d level, which is a degradation product of complement factor C4, was found to be a sensitive indicator of SLE activity in patients whose C3 and C4 levels remain in normal range despite clinical disease activity.⁵ Recently, Espinosa et al. showed that mesangial C4d deposition could be a new prognostic factor in patients with IgA nephritis.⁶ In the present study, we aimed to determine whether glomerular C4d immunofluorescence staining could be a useful marker of disease activity in patients with lupus nephritis.

MATERIALS AND METHODS

This retrospective cohort study included all patients with lupus nephritis who underwent renal biopsy at our center from January 2005 to December 2009. All patients met four or more of the American Rheumatism Association’s revised criteria for SLE and lupus nephritis were confirmed with renal biopsy.⁷ A total of 29 patients included to the study, but five patients who had insufficient renal tissue (fewer than six glomeruli) remaining in the paraffin block were excluded. The diagnosis of lupus nephritis was based on histological assessment of renal biopsy tissue with hematoxylin and eosin, Masson’s trichrome, periodic acid-Schiff, methenamine silver and Kongo red for light microscopy, and staining with IgG, IgA, IgM, C1q, and C3 for immunofluorescence. The biopsy specimens were reevaluated and classified according to the International Society of Nephrology/Renal Pathology Society (ISN/RPS) 2003 classification.⁸ Activity and chronicity scores were calculated according to the method of Austin et al.⁹

C4d immunohistochemical staining was performed on 4-μm deparaffinized and rehydrated sections of formaldehyde-fixed renal tissue, using rabbit polyclonal anti-human C4d (polyclonal antibody E17344, Spring Bioscience, Pleasanton, CA, USA). Tubular C4d staining was scored as negative (0) or positive (1). Patients were classified as “positive” when staining in peritubular capillaries was observed. Glomerular C4d staining was evaluated semi-quantitatively, was scored 0 when absent, 1 when mild, and 2 when intense.

Twenty nine patients with lupus nephritis were evaluated for the study. The medical records were reviewed and the following information at the time of the renal biopsy was recorded: patient age, sex, presence or absence of hypertension (defined as blood pressure >140/90 mm Hg or the use of antihypertensive agents), spot urine protein/creatinine excretion and serum creatinine levels. We calculated the estimated GFR (eGFR) using the abbreviated Modification of Diet in Renal Disease (MDRD) study equation¹⁰:

Statistical Analysis

Baseline demographical, clinical, and laboratory data were recorded retrospectively. SPSS version 13.0 software (SPSS, Chicago, IL, USA) was used for statistical analysis. Results were expressed as mean ± standard deviation. The Mann–Whitney U test was used to compare the continuous variables and the chi-square test was used to compare categorical variables. A p-value of less than 0.05 was considered to be statistically significant.

RESULTS

Between January 2005 and December 2009, 29 patients were diagnosed with lupus nephritis by the renal biopsy at our institution. C4d staining was not performed on five patients who had insufficient renal tissue. Glomerular staining for C4d was observed in 14 biopsies (58.33%). In five patients, glomerular staining for Cd4 was intense, and in nine patients it was moderate. Staining in peritubular capillaries was observed in one patient.

About 79.2% of the patients were female, 50% were hypertensive, 45.8% had a GFR <60 mL/min, and 83.3% had urine protein levels >1.0 g/24 h at the time of renal biopsy diagnosis. Table 1 shows the clinical and pathological data of the patients at the time of renal biopsy. Proteinuria was more elevated in C4d+ patients (p = 0.0001).

Table 1.  Clinical and pathological data of the patients at the time of renal biopsy according to the C4d staining.

	C4d+ (N = 14)	C4d– (N = 10)	p Value
Age (year)	37.57 ± 12.53	41.80 ± 11.44	0.437*
Gender female (%)	11 (78.6%)	8 (80%)	0.668**
Hypertension (%)	7 (50%)	6 (60%)	0.473**
Urinary protein excretion (g/day)	5.43 ± 3.45	1.21 ± 0.89	0.0001*
Serum creatinine (mg/dL)	1.57 ± 1.19	1.36 ± 0.83	0.841*
eGFR (mL/min/1.73 m^2)	65.42 ± 41.02	72.0 ± 33.69	0.841*
Class 2	−	1 (10%)	0.290**
Class 3	3 (21.4%)	5 (50%)
Class 4	9 (64.3%)	3 (30%)
Class 5	1 (7.1%)	–
Class 6	1 (7.1%)	1 (10%)
Evolution to ESRD in the follow-up (%)	4 (28.6%)	–	0.064

Note: *Mann–Whitney U test, **chi-square test.

The renal biopsy showed that activity index (AI) score ≥12 was a higher proportion in C4d+ patients. In addition, the correlation between proteinuria, AI, and C4d was found to be significant (Table 2).

Table 2.  The correlation between glomerular C4d, lupus nephritis activity index, and proteinuria at the time of renal biopsy.

	Patients (n)	C4d+	R	p*
Activity index (≥12)	13	10	0.410	0.047
Proteinuria (nephrotic)	12	10	0.676	0.0001

Note: *Spearman’s rank correlation.

Table 3.  Clinical and pathological data of the patients at the time of renal biopsy according to the evolution to ESRD.

	ESRD evolution	No ESRD evolution	Total	p Value
N	4	20	24
Age (year)	35.25	40.15	39.33	0.682*
Gender female	2 (50%)	17 (85%)	19 (79.2%)	0.116**
Serum creatinine (mg/dL)	5.94	1.22	1.48	0.023*
eGFR (mL/min/1.73 m^2)	9.75	75.05	68.16	0.029*
Hypertension	1 (25%)	10 (50%)	11 (45.8%)	0.363**
Urinary protein excretion (g/day)	6.39	3.13	3.67	0.056*
C4d staining	4 (100%)	10 (50%)	14	0.064**

Note: *Mann–Whitney U test, **chi-square test.

The patients were followed up for 3.5 years. Four patients in the C4d+ group evolved to end-stage renal disease (ESRD) in the follow-up, but none of the patients in the C4d group (p = 0.064).

Among demographic and clinical factors, serum creatinine levels and GFR were univariately associated with evolution to ESRD (Table 3). Proteinuria was more elevated in patients who evolved to ESRD, but was not significant p = 0.056)

DISCUSSION

Several clinical and histological parameters were reported to be related with disease activity in patients with lupus nephritis. In the current study, we questioned whether glomerular C4d staining may be another marker of disease activity. Manzi et al. demonstrated that plasma and urine levels of C4d were superior than C3 and C4 to show the activity of disease.⁵ In another study platelet-bound C4d was found to be a biomarker for lupus activity and was associated with antiphospholipid antibodies.¹² Muso et al. reported that glomerular deposition of C3d in patients with lupus nephritis is significantly correlated with disease activity.¹³

Some previous studies indicated a strong relationship between the glomerular deposition of C4d and the presence of microthrombi in renal biopsies of patients with lupus nephritis; however, the association between C4d staining and AI was not evaluated.^14,15 Cohen et al. suggested that the presence of intense C4d staining may be associated with a poor clinical outcome.

Proteinuria and the score of AI in the renal biopsies defined by Austin et al. are the most known factors that predict poor evolution in patients with lupus nephropathy.⁹ In our study, we found a significant association between glomerular C4d staining and AI score ≥12 in patients with lupus nephritis (p = 0.047). In contrast to our findings, it was not demonstrated such a relationship between glomerular C4d staining and lupus disease activity.^16,17 We showed that glomerular C4d staining was also associated with nephrotic proteinuria and the correlation was significant (p < 0.0001). In the study of Hoon et al., they did not demonstrate such an association.¹⁶ First, these differences between the two studies may be due to ethnic origin. Second, in their study, the majority of patients had AI score <12; however, in our study, the number of patients were similar according to AI score. Lastly, the number of patients with nephrotic proteinuria was lower than the nephrotic group, but in our study the distribution of patients was equal in the two groups.

C4d staining did not differ in subgroups according to WHO classification of lupus nephritis. This is the first study in the literature that evaluates the subgroup analysis. However, the number of patients is not enough to comment accurately about this subject.

In the follow-up period of 3.5 years, four patients evolved to ESRD. When we compared the patients who evolve to ESRD according to C4d staining, all the patients were C4d+ (p = 0.064). Although the difference was not significant, we thought this can be important. If we have had more patients or more follow-up period, the difference could be significant. In addition, baseline serum creatinine levels and eGFR were univariately associated with evolution to ESRD.

There are several limitations for the present study. First, this is an observational retrospective study. Other limitations are the small number of patients and the short period of follow-up.

In conclusion, we found a relationship between glomerular C4d staining and activity of lupus nephritis. C4d staining may be a useful marker to predict the prognosis of lupus nephritis. Further studies on larger populations are required to validate our reports and to evaluate the potential prognostic utility of C4d staining in lupus nephritis.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.