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NDT Advance Access published online on October 8, 2008
Nephrology Dialysis Transplantation, doi:10.1093/ndt/gfn563
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© The Author [2008]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org


Mesangial C4d deposition: a new prognostic factor in IgA nephropathy

Mario Espinosa1, Rosa Ortega2, Jose Manuel Gómez-Carrasco1, Fernando López-Rubio2, María López-Andreu1, María Ovidia López-Oliva1 and Pedro Aljama1

1 Servicio de Nefrologia 2 Servicio de Anatomía Patológica, Hospital Universitario Reina Sofia, Cordoba, Spain

Correspondence and offprint requests to: Mario Espinosa, Servicio de Nefrología, Hospital Universitario Reina Sofía, Avda Menéndez Pidal s/n, Cordoba 14004, Spain. Tel: +34-957-010440; Fax: +34-957-010307; E-mail: espinosahe{at}supercable.es



   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Background. It has been shown that patients with IgA nephropathy can be divided into two groups on the basis of the pattern of complement activation. Activation of the lectin pathway of complement is associated with more severe renal disease. Glomerular deposition of C4d is a marker of activation of the lectin pathway of complement. The aim of our study was to determine whether C4d staining at the time of the renal biopsy could identify patients with a different long-term prognosis in IgA nephropathy.

Methods. This retrospective cohort study included all patients with IgA nephropathy who underwent renal biopsy at our centre from January 1992 to December 2006. We evaluated baseline age, sex, presence of macroscopic haematuria, hypertension, serum creatinine and glomerular filtration rate (GFR), urine protein, mesangial C4d staining, glomerulosclerosis, interstitial fibrosis and extracapillary proliferation. Kaplan–Meier survival and Cox proportional hazards analyses were performed, with end-stage renal disease (ESRD) being defined as onset of dialysis or transplantation.

Results. Nineteen patients (32.2%) were C4d positive and 40 patients (67.8%) C4d negative. Age, hypertension, absence of macroscopic haematuria, serum creatinine levels, GFR, glomerular sclerosis, interstitial fibrosis and C4d-positive staining were all univariately associated with evolution to ESRD. Renal survival at 10 years was 43.9% in C4d-positive patients versus 90.9% in C4d-negative patients (log-rank, P = 0.0005).

Conclusion. Negative mesangial C4d staining in glomeruli in patients with IgA nephropathy helps to identify patients with a good long-term prognostic for whom aggressive treatments are not justified.

Keywords: C4d; complement; IgA nephropathy; lectin



   Introduction
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 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Immunoglobulin A nephropathy (IgAN) is the most common primary glomerulonephritis in the world and is defined by the predominant deposition of IgA in the glomerular mesangium. It is characterized by a highly variable course ranging from a totally benign condition to progressive long-term renal failure; ~15–40% of patients will require renal replacement therapy within 20–25 years of presentation [1–4]. This variability in clinical course justifies the efforts to determine clinical and histological features that predict the development of renal failure in IgAN.

The initiating event in the pathogenesis of IgA nephropathy is the mesangial deposition of IgA. Recently it has been demonstrated that variable amounts of aberrantly glycosylated IgA molecules are present in the immune deposits of patients with IgAN [5]. This deposit induces the activation of mesangial cells [6–8] and local complement activation [9]. The contribution of this in situ complement synthesis and activation to progressive glomerular injury is not known [10,11]. The complement system can be activated via three pathways: the classical pathway, the alternative pathway and the lectin pathway.

Roos et al. [12] showed that patients with IgAN can be divided into two groups on the basis of the pattern of complement activation. In patients with negative glomerular staining for mannose-binding lectin (MBL), L-ficolin, MBP-associated serine protease (MASP) C4d, and C4-binding protein, the activation of the complement occurs via the alternative pathway. When these stainings are positive, activation of complement occurs via the lectin pathway of complement. Mesangial C4d deposition is a good marker to study the activation of complement in patients with IgAN.

Roos et al. also showed [12] that glomerular activation by the alternative pathway of complement (C4d negative) in IgAN is associated with a more benign renal disease at the time of the renal biopsy. However, it is not known if this could be associated with a better long-term prognosis. The aim of our study was to determine whether C4d staining could be used as a factor prognostic in patients with IgAN.



   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
This retrospective study included all consecutive patients who underwent a renal biopsy at our institution between January 1992 and December 2006. The study was approved by the Ethics Committee and the Research Board of our institution. Patient information was managed according to applicable Data Protection Regulations. All patients with a diagnosis of IgAN were included in the study. The diagnosis of IgAN was based on histological assessment of renal biopsy tissue with haematoxylin and eosin, Masson's trichrome, periodic acid-Schiff and methenamine silver for light microscopy and staining with IgG, IgA, IgM and C1q for immunofluorescence. Eighty-seven patients with nephropathy IgA 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 mmHg or the use of antihypertensive agents), 24-h urine protein excretion and serum creatinine level. We calculated the estimated GFR (eGFR) using the abbreviated Modification of Diet in Renal Disease (MDRD) Study equation [13]: eGFR (ml/min per 1.73 m2) = 186.3*(serum creatinine in mg/dl–1.154)*(age–0.203)*(0.742 if female)*(1.21 if black).

C4d staining and histological study
C4d inmunohistochemical staining was performed on 3-µm deparaffinized and rehydrated sections of formaldehyde-fixed renal tissue, using rabbit polyclonal anti-human C4d (Biomedica, Vienna, Austria) as the antibody, diluted 1:30 in PBS. In order to block non-specific staining, antigen retrieval was performed in advance of slide treatment by pressure cooking (10 min at 1 bar, 10 mM citrate buffer, pH 6.0). The detection system used was Dako EnVision HRP (Dako A/S, Copenhagen, Denmark) according to the following protocol: block endogenous peroxidase with 3% H2O2 in PBS for 10 min, wash sections in PBS, apply anti-C4d antibody for 20 min at room temperature, wash in PBS, apply DAB chromogen for 5 min, wash sections in tap water for 10 min and coverslip using an aqueous mounting medium.

C4d inmunohistochemical staining was scored as negative (0) or positive (1). Patients were classified as ‘positive’ when >75% of the glomeruli were positive for C4d. This staining was classified as ‘global’ when >50% of mesangial area was affected and ‘segmental’ when <50% of the mesangial area was positive for C4d. No staining was observed in peritubular capillaries. Tubular staining was observed irregularly in some patients. This positive glomerular staining was predominantly mesangial. Figure 1 shows a patient with global (A), segmental (B) and negative (C) staining.


Figure 1
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  		Fig. 1 Renal tissue from patients with IgAN was stained for the presence of C4d. Representative images are shown. (A) A representative glomerulus contains global staining of C4d predominantly in mesangial areas. (B) A glomerulus of a patient with segmental C4d staining in mesangial areas. (C) A glomerulus of a patient with negative C4d staining. Magnification x400. No staining was observed in peritubular capillaries. Patchy tubular staining was observed in some patients.

 
Extracapillary proliferation, global sclerosis and segmental sclerosis were calculated as percentage of the total number of glomeruli. Mesangial proliferation was scored as mild when there were less than six cells per mesangial area and moderate–severe when there were more than six cells per mesangial area. Interstitial fibrosis was evaluated semiquantitavely, was scored 0 when absent, 1 when mild (involving <30% of the interstitium), 2 when moderate (30–60% of the interstitium involved) or 3 when intense (when present in >60% of the renal interstitium). Interstitial fibrosis was evaluated as a categorical variable, mild (interstitial fibrosis grade 0–1), moderate–severe (interstitial fibrosis grade 2–3). Fifteen patients with nephroangiosclerosis and serum creatinine > 2 mg/dl were used as control to evaluate if the positive staining of C4d was associated with the glomerular sclerosis and chronic renal damage. Evaluation of renal tissue was reviewed by one of us (R.O.) who was unaware of the evolution of the patients.

Study end points
Patient case notes were reviewed by M.E. in March 2007, recording the last serum creatinine and the development of end-stage renal disease (ESRD) defined as chronic repetitive dialysis or renal transplantation. The primary end point of the study was the cumulative percentage of patients who developed ESRD in the course of the study.

Statistical analysis
The chi-squared test was used to compare qualitative values. Student's t-test was used to compare normally distributed quantitative variables. Results are expressed as mean ± standard deviation. All statistical tests are two-tailed. A P-value < 0.05 was considered to be statistically significant.

Two analyses were performed to evaluate the impact of C4d staining on the renal survival: the Kaplan–Meier analysis and the Cox proportional hazards analysis. The primary end point was ESRD (transplant or dialysis). No patient died before the time of ESRD. One patient was lost in the course of the follow-up. Univariate survival comparisons were made using the log-rank test. All univariate tests were two-sided, with an alpha level of 0.05. The Cox proportional hazards model was used to estimate the adjusted relative risk (RR) of each parameter with regard to renal survival. Variables previously found to impact on renal survival (age, hypertension, absence of macroscopic haematuria, serum creatinine levels, eGFR, glomerulosclerosis, interstitial fibrosis and C4d staining) were included in Cox's proportional hazard model. Variables were selected by backward elimination using likelihood ratio tests. Calculations were performed using SPSS statistical software version 12.0 (SPSS, Chicago, IL, USA).



   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Between January 1992 and December 2006, 87 patients were diagnosed with IgAN by the renal biopsy performed at our institution. C4d staining analysis was not performed on 21 patients who had insufficient renal tissue (fewer than two glomeruli) remaining in the paraffin block. C4d staining was thus performed on the biopsy material from 66 patients. A mean of 11 ± 7 glomeruli was identified. Seven patients (three C4d positive and four C4d negative) were excluded from the final analysis on the grounds of liver cirrhosis (1), HIV infection (1), HCV infection (3) and severe psoriasis (1) (Figure 2). In one patient there was no information in the follow-up, and he was also excluded. Patients with Henoch–Schönlein purpura (HSP) were included. Fifty-nine patients thus comprised the final study cohort.


Figure 2
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  		Fig. 2 Flow chart of the study population. Patients with IgA nephropathy who underwent renal biopsy at our centre between January 1992 and December 2006.

 
Glomerular staining for C4d was observed in a predominantly mesangial pattern in 19 biopsies (32.2%). In 9 patients, the glomerular staining for C4d was segmental (Figure 1B) and in 10 patients it was global (Figure 1A). Both were regarded as C4d positive. Glomeruli in 40 biopsies (67.8%) stained negative for C4d (Figure 1C).

Eight patients were classified as HSP and 51 patients as IgA idiopathic. Two of the eight patients (25%) with HSP were C4d positive. The frequency of positive C4d staining was similar between patients with HSP and patients with idiopathic IgAN (P = 0.7). Table 1 shows the glomerular deposition of C3, IgG, IgM and C1q according to the C4d staining. It must be stressed that no patient was positive for C1q. There were no differences between C4d-positive or C4d-negative patients.


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  		Table 1 Glomerular deposition of C3, IgG, IgM and C1q in patients with IgAN according to the C4d staining

 
It is noteworthy that 74.6% of the patients were male, 30.5% were hypertensive, 30.5% had a GFR < 50 ml/min and 66.1% had urine protein levels > 1.0 g/24 h at the time of renal biopsy diagnosis. Table 2 shows the clinical and pathological data of the patients at the time of renal biopsy. It should be emphasized that C4d positive patients were older, had macroscopic haematuria in less proportion, had hypertension in major proportion and had a more impaired renal function (mean serum creatinine 2.6 ±1.5 versus 1.3 ± 0.8 mg/dl, P = 0.004) than C4d-negative patients at the time of renal biopsy. The renal biopsy showed that C4d-positive patients had more glomerulosclerosis (mean 35 ± 50% versus 13 ± 22%, P = 0.01) and a more severe interstitial fibrosis, and that a higher proportion evolved to ESRD in the follow-up (42.1% versus 7.5%, P = 0.03). To analyse if C4d deposition was a consequence of chronic renal failure and glomerulosclerosis, this study was performed in 15 patients with nephroangiosclerosis (mean serum Cr 2.6 ± 0.8 mg/dl, glomerulosclerosis 50 ± 27%). Only in two of them (13.3%) was a focal staining of C4d observed.


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  		Table 2 Clinical and pathological data of the patients at the time of renal biopsy and evolution to ESRD in the follow-up according to the C4d staining

 
Eleven patients (18.6%) evolved to ESRD in the follow-up. Global renal survival was 79% at 5 years and 74% at 10 years. Renal survival at 10 years was 43.9% in C4d-positive patients versus 90.9% in C4d-negative patients (log-rank, P = 0.0005) (Figure 3).


Figure 3
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  		Fig. 3 Kaplan–Meier renal survival according to C4d positive (+) and negative (–) staining.

 
Among demographic and clinical factors, age, hypertension, absence of macroscopic haematuria, serum creatinine levels and GFR were all univariately associated with evolution to ESRD (Table 3). Proteinuria was more elevated in patients who evolved to ESRD but was not significant (P = 0.06). In addition, glomerular sclerosis, a more severe interstitial fibrosis, and C4d staining were univariately associated with long-term evolution to ESRD. In the Cox proportional hazards model only eGFR was found to be an independent prognostic value of evolution to ESRD (RR 0.93, IC 0.90–0.97, P < 0.001).


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

 


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Our study shows that mesangial C4d staining in glomeruli in patients with IgA nephropathy identifies patients with a different long-term prognosis. Renal survival at 10 years was 90.9% in C4d-negative patients and 43.9% in C4d-positive patients.

In the course of the past decade, predictors of renal failure in IgAN have been assessed in several clinicopathologic studies; clinical and laboratory features found to be independently associated with progressive renal disease included the degree of proteinuria [14–23], decreased renal function [15,18,19], the presence of hypertension [15,17,21,23] and the absence of macroscopic haematuria [14]. Histologic features [14,17,18,20,23] identified as independent predictors of renal failure included glomerulosclerosis, interstitial fibrosis and mesangial hypercellularity. It is important to identify other risk factors associated with a poor evolution. The different pathway of complement activation could be one of them.

There is now increasing evidence that in the pathogenesis of IgAN, an abnormal O-glycosylation of polymeric IgA1 is involved [5,25]. Deposition of this IgA in the mesangium leads to renal inflammation, potentially involving direct interactions of IgA with resident and infiltrating cells in the glomerulus, as well as complement activation. Mesangial C4d staining identifies patients with a different pathway of complement activation [12]. In patients with negative mesangial C4d staining, the activation of the complement probably occurs via the alternative pathway. In contrast, in patients with positive C4d staining, activation of complement occurs via the lectin or classical pathway of complement. The absence of C1q in the 100% of our patients with IgAN makes it very unlikely that the classical complement pathway plays any role in the pathogenesis of IgA nephropathy. This different pathway of complement activation could be associated with a different long-term prognosis.

It has recently been shown by Roos et al. [12] that glomerular activation of the lectin pathway of complement in IgAN is associated with more severe renal disease at the time of the renal biopsy. In our study, patients with positive mesangial C4d staining also had a more impaired renal function at the time of renal biopsy and more glomerulosclerosis. However, this is the first study that shows that patients with IgAN and positive C4d glomerular staining have a poor long-term renal survival in comparison with C4d-negative patients in whom the long-term prognosis is excellent. It is important to stress that only 7.5% of the C4d-negative patients evolved to ESRD in the follow-up (mean 12.7 years).

Our work has limitations; this is an observational study, the relationship between C4d staining and progression of the disease must be interpreted cautiously in terms of association rather than causality. In our study performed in paraffin using a rabbit polyclonal antibody, 32.2% of the patients were classified as C4d positive. Roos et al. [12] using the same polyclonal antibody in unfixed renal tissue observed a mesangial C4d deposition in 25% of the patients. Several studies have demonstrated acceptable sensitivity and specificity of C4d polyclonal antiserum staining of paraffin-embedded tissue as compared to immunofluorescent staining of frozen tissue [25]. The likelihood of selection bias seems low because we included all patients with renal biopsy in our hospital in the last 15 years. One could speculate that the deposit of C4d is secondary to the renal damage because these C4d-positive patients were older, had poorer renal function, more glomeruloesclerosis and interstitial fibrosis at the time of the renal biopsy. However, this mesangial C4d deposition was only observed (and focally) in 13% of patients with a histological diagnosis of nephroangiosclerosis and severe chronic renal disease (mean serum Cr 2.6 ± 0.8 mg/dl). Given these data, and early evidence [12] it is most reasonable to suppose that the C4d deposition is an indication of a distinct pathway of activation of complement rather than a consequence of chronic renal failure and glomerulosclerosis.

In conclusion, age, absence of macroscopic haematuria, hypertension, a poor renal function at the time of renal biopsy, glomerulosclerosis, interstitial fibrosis and positive mesangial C4d staining in glomeruli are associated with evolution to ESRD in patients with IgAN. Renal survival after 10 years was 90.9% in C4d-negative patients and 40.9% in C4d-positive patients. Negative mesangial C4d staining in glomeruli in patients with IgAN helps to identify patients with a good long-term prognostic for whom aggressive treatments are not justified.

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Received for publication: 19. 2.08
Accepted in revised form: 16. 9.08


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