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Saudi Journal of Kidney Diseases and Transplantation
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Year : 2021  |  Volume : 32  |  Issue : 1  |  Page : 42-48
Clinicopathological Correlation with Mesangial C4d Deposition in Primary Immunoglobulin A Nephropathy - A Descriptive study

1 Department of Pathology, St. John’s Medical College and Hospital, Bengaluru, Karnataka, India
2 Department of Nephrology, St. John’s Medical College and Hospital, Bengaluru, Karnataka, India

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Date of Web Publication16-Jun-2021


In immunoglobulin A (IgA) nephropathy, activation of lectin pathway leads to severe renal damage and more pronounced histological damage. As C4d is a marker of lectin pathway activation, the presence of mesangial C4d positivity will help in identifying those patients at risk. The study was conducted to study the prevalence of mesangial C4d positivity in patients with primary IgA nephropathy and to compare the clinical and histopathological features with C4d-positive and C4d-negative cases. It is a retrospective study conducted for four years. The inclusion criterion was IgA nephropathy with a minimum of four viable glomeruli. Biopsies with >25% of nonsclerotic glomeruli with mesangial positivity will be considered as positive for C4d. Seventy-six patients of IgA nephropathy were included of which mesangial C4d positivity was noted in 33 patients (43%). The mean age was 35 years. The male:female ratio was 2.3:1. The most common presentation was edema (56%) followed by microscopic hematuria (28%). Fifty-four patients were hypertensive. Among the clinical and laboratory parameters, absence of hematuria (P = 0.04) and presence of proteinuria (P = 0.02) showed a significant association with C4d positivity. The histological parameters in Oxford classification which had significant association with C4d positivity were segmental sclerosis (P = 0.01) and tubular atrophy (P = 0.001). Among 45 patients on follow-up with a maximum duration of 51 months, 10 developed end-stage renal disease of which four had C4d expression (0.05%) in the biopsy. Nearly half of IgA nephropathy patients have mesangial C4d positivity. Elevated creatinine with chronicity changes is more common in C4d-positive patients. Hence, C4d can be used as a marker for poor prognosis.

How to cite this article:
Vellaisamy G, Malipatel R, Renuka S, Rout P. Clinicopathological Correlation with Mesangial C4d Deposition in Primary Immunoglobulin A Nephropathy - A Descriptive study. Saudi J Kidney Dis Transpl 2021;32:42-8

How to cite this URL:
Vellaisamy G, Malipatel R, Renuka S, Rout P. Clinicopathological Correlation with Mesangial C4d Deposition in Primary Immunoglobulin A Nephropathy - A Descriptive study. Saudi J Kidney Dis Transpl [serial online] 2021 [cited 2021 Dec 4];32:42-8. Available from: https://www.sjkdt.org/text.asp?2021/32/1/42/318547

   Introduction Top

Immunoglobulin A (IgA) nephropathy is characterized by dominant or codominant deposits of IgA in the mesangium. The pathogenesis involves the isolated deposition of polymeric IgA1 or as immune complexes in the form of IgG/polymeric IgA1 or IgA/IgA immune complexes. These deposits either via interaction with mesangial cell receptors or through direct activation of complements result in activation of mesangial cells, leading to proliferation, matrix expansion, and tissue damage.[1] Complements are activated via two nonmutually exclusive mechanisms: alternative pathway and mannose-binding lectin (MBL) pathway.[2],[3],[4] The most common pathway which is activated in IgA nephropathy is the alternative pathway and is characterized by the presence of IgA deposits along with C3, C5b-9, and properdin.[5],[6] In 25%–30% of patients, IgA deposits are accompanied by C3, MBL, and C4d in the absence of C1q, indicating alternative pathway activation.

As the presence of C4d indirectly serves as a marker of lectin pathway activation, several studies have investigated its prognostic role in IgA nephropathy. Roos et al first demonstrated its association with more pronounced histological damage and heavy proteinuria.[7] In a study by Espinosa et al, C4d staining was identified as a risk factor for the development of end-stage renal disease (ESRD) both in univariate and multivariate analysis.[8],[9] The above studies evaluated C4d role in patients with various stages of renal failure. A recent study by Segarra et al evaluated C4d presence in patients with normal renal function and reported that it serves as an independent predictor of long-term renal survival.[10]

There are a few Indian studies that have evaluated C4d role in relation to clinical and histological parameters in a small number of patients with IgA nephropathy. Hence, this study was done to:

(i) Study the clinical parameters and histopathological features of IgA nephropathy

(ii) Evaluate the prevalence of mesangial C4d positivity in a larger population of primary IgA nephropathy

(iii) Compare the clinical and histopathological features with C4d-positive and -negative cases.

   Materials and methods Top

This is a retrospective study for a period of four years, from January 2014 to December 2017. The study was approved by the institutional ethics committee. This study included all patients diagnosed as IgA nephropathy based on histopathology and mesangial IgA deposits by direct immunofluorescence. The exclusion criteria were (i) secondary causes of IgA nephropathy such as liver diseases, connective tissue disorders, inflammatory bowel disease, Celiac disease, psoriasis, neoplasms, and chronic infections; (ii) Henoch–Schonlein purpuric nephritis; (iii) cases with inadequate clinical data or tissue in the paraffin blocks. Clinical presentation including hypertension [systolic blood pressure (BP) >140 mm Hg and/or diastolic BP >90 mm Hg or the use of hypertensive medications], laboratory values such as serum creatinine, proteinuria (nephrotic range defined as >3.5 g/day), microscopic hematuria (>5 RBC/HPF), pyuria (>5 WBC/ HPF), and follow-up details were obtained from clinical records and hospital information system. Estimated glomerular filtration rate (eGFR) was calculated in adults using the abbreviated Modification of Diet in Renal Disease (MDRD) study equation: eGFR (mL/min/1.73 m2) = 186.3 × (serum creatinine in mg/dL − 1.154) × age − 0.203) × (0.742 if female) × (1.21 if Black). In children, eGFR was calculated using the Schwartz formula.

Paraffin-embedded biopsies were stained with hematoxylin and eosin, periodic acid–Schiff, periodic silver methenamine, and Masson trichrome. The renal biopsies were classified according to the Oxford classification.[11],[12] Immunohistochemistry for C4d was performed using polymer technique on tissue sections of 4–5 μm thickness adhesive slides. The slides were incubated overnight at 60°C. Antigen retrieval was done by pressure cooker method in EDTA buffer. The slides were incubated with primary mouse monoclonal antibody (C4D204, Pathnsitu) at room temperature for 30 min. Subsequently, the slides were incubated with secondary antibody and immuno-reactivity was detected using diaminobenzidine as chromogen. The slides were counterstained with Harris’ hematoxylin. C4d-positive antibody-mediated rejection was used as positive control. Cases with >25% of nonsclerotic glomeruli with mesangial positivity alone or mesangial and capillary wall positivity were considered as positive for C4d.[8]

The primary end point of the study was development of ESRD in the course of the study (defined by onset of chronic dialysis or renal transplantation).

   Statistical Analysis Top

Statistical analysis was done using Statistical Package for the Social Sciences (SPSS) software version 16.0 (SPSS Inc., Chicago, IL, USA). Continuous variables were compared using Student’s t-test. Categorical data were analyzed using Chi-square test, and P <0.05 was considered as statistically significant.

   Results Top

One hundred and thirty patients of IgA nephropathy were reported within the study period. After exclusion, 76 patients were included in the study. The mean age of presentation was 35 ± 13 years. The male: female ratio was 2.3:1. The most common presentation was edema (56.5%) followed by gross hematuria (23.7%). Fifty-four patients (71%) were hypertensive. The mean serum creatinine and eGFR was 2.6 ± 2.8 mg% and 64.9 ± 60 mL/min/1.73 m2. Nephrotic range proteinuria was noted in 37 patients.

The mean number of glomeruli for light microscopy was 10 ± 5. Crescents were noted in 34% of patients, of which cellular and fibrocellular crescents (C1 and C2) were seen in 30% of cases. Mesangial hypercellularity (M1) and endocapillary hypercellularity (E1) were noted in 67.1% and 21% of patients. Moderate-to-severe tubular atrophy (T1 and T2) was noted in 48.6% of IgA nephropathy patients. Segmental sclerosis was seen in 52.6% of biopsies. Blood vessels show evidence of hyaline arteriolosclerosis and arterio-sclerosis in 35.5% of biopsies. Thrombotic microangiopathy was noted in two biopsies. One showed evidence of fibrinoid necrosis, whereas the other had thrombi with recanalization. On immunofluorescence, IgA, IgG, IgM, and C3 were noted in 100%, 7%, 10.5%, and 50%, respectively. C1q was absent in all the biopsies.

Mesangial C4d expression was noted in 33 (43%) biopsies, of which four biopsies in addition showed granular capillary wall positivity [Figure 1] and [Figure 2]. Among the demographic and clinical parameters, C4d expression showed significant association with absence of hematuria (P = 0.04) but not with age, sex, edema, and hypertension. Urine protein was significantly higher in C4d-positive patients as compared to C4d negative patients (P = 0.02). Although the C4d-positive patients had increased mean serum creatinine and decreased eGFR, it did not attain statistical significance [Table 1].
Figure 1: Immunostain for C4d showing positivity in the mesangium (×20).

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Figure 2: Immunostain for C4d showing granular positivity in the mesangium and capillary wall (×20).

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Table 1: Relationship of c4d with clinical and laboratory parameters.

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Among the parameters included in Oxford classification, the features which are associated with chronicity such as segmental sclerosis and tubular atrophy showed a significant association with C4d deposition, whereas mesangial cellularity, endocapillary hyper-cellularity, and crescents (cellular and fibro-cellular) did not [Table 2]. Immunofluorescence positivity for IgG and IgM and C3 positivity also did not show any association with C4d and had P = 0.6, 0.69, and 0.48, respectively.
Table 2: Relationship of c4d with pathological parameters

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Forty-five patients were on follow-up with a maximum duration of 51 months. Median follow-up was 26 months. Ten patients (13.1%) developed ESRD of which four had C4d expression in their biopsies. On univariate analysis, absence of gross hematuria, increased serum creatinine, low eGFR, tubular atrophy and interstitial fibrosis were significantly associated with the development of ESRD whereas C4d staining did not show any such association [Table 3].
Table 3: Relationship of clinical and pathological parameters at the time of biopsy with end-stage renal disease.

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   Discussion Top

IgA nephropathy is the most common glome-rulonephritis worldwide, with a prevalence ranging from 15% to 40% from different parts of the world.[13] The mean age of presentation was 35 ± 13 years with male predominance similar to other Indian studies.[14],[15] Approximately three-fourth of patients in this study were hypertensive which was higher than the previously reported studies.[14],[16] Crescents were noted in 34% of patients, whereas in an Indian study, 12.5% of biopsies showed crescentic transformation.[16] El Karoui et al had reported thrombotic microangiopathy in 53% of patients with poor prognosis.[17] In this study, only two patients (2.6%) show evidence of thrombotic microangiopathy. Both of them were hypertensive and presented with increased serum creatinine and nephrotic range proteinuria. One of them developed ESRD.

Similar to its various morphological manifestations, the course of IgA nephropathy is also variable from totally benign to slow progression of development of ESRD in 15%–40% of patients.[17],[18],[19] Prognostic factors of IgA nephropathy include hypertension, proteinuria, renal function at presentation, and pathological variables which include parameters of Oxford classification.[11],[12] In addition to these factors, several investigators have tried to identify the role of complement system, particularly lectin pathway, in the pathogenesis of IgA nephropathy.[4],[7],[8],[9]

In the absence of C1q deposits, C4d was identified as an indirect marker of lectin pathway and also an independent prognostic factor of IgA nephropathy.[9] C4d has no biological function and is derived from C4b. Because of its covalent bond, the half-life is longer as compared to antibodies thus serves as a marker of complement activation.[20] The prevalence of C4d positivity in different studies ranges from 20% to 80%.[7],[8],[9],[10],[21],[22],[23],[24],[25] C4d positivity was noted in 43% of biopsies in this study. Thrombotic microangiopathy was reported in two biopsies of which one had mesangial C4d expression. C4-binding protein acts as a bridge between complement system and coagulation by which thrombotic micro-angiopathy can occur in a subset of patients.[17],[26],[27] Heybeli et al reported an association of IgM deposition with C4d expression, which can also activate the lectin pathway both in vitro and in vivo. However, in this study, there was no such association.

In contrast to studies by Espinosa et al, this study did not find an association of univariate analysis C4d expression with renal survival. However, C4d expression was associated with worse renal presentation and more chronic changes in histology. Hence, C4d indirectly identifies patients with poor long-term outcome. The absence of association of C4d with renal survival does not negate the findings reported by previous studies. It could be due to a shorter period of follow-up. A prospective Indian study with long-term follow-up is necessary to confirm the same. It is not clear as to why lectin pathway is activated in a subset of patients with IgA nephropathy. The possible reasons may provide new insights in the pathogenesis and also help in identifying those patients who may benefit from aggressive treatment with follow-up at an earlier stage.

   Conclusions Top

Approximately 15% of patients with IgA nephropathy developed ESRD. C4d expression was associated with worse renal presentation and histology. Hence, C4d might be a useful marker in identifying this subset of patients where aggressive treatment with follow-up is necessary.

Conflict of interest: None declared.

   References Top

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Bene MC, Faure GC. Composition of mesangial deposits in IgA nephropathy: Complement factors. Nephron 1987;46:219.  Back to cited text no. 5
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Correspondence Address:
Gnanapriya Vellaisamy
Department of Pathology, St. John's Medical College, Bengaluru, Karnataka
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DOI: 10.4103/1319-2442.318547

PMID: 34145113

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