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| Year : 2021 | Volume
: 64
| Issue : 1 | Page : 69-77 |
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| Glomerular C4d deposition in proliferative glomerular diseases |
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Sarojini Raman1, Pallavi Mishra1, Ansuman Panigrahi2, Nikunj K Rout3, Kanakalata Dash1
1 Department of Pathology, Kalinga Institute of Medical Sciences, Bhubaneswar, Odisha, India
2 Department of Community Medicine, Kalinga Institute of Medical Sciences, Bhubaneswar, Odisha, India
3 Department of Nephrology, Kalinga Institute of Medical Sciences, KIIT University, Bhubaneswar, Odisha, India
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| Date of Submission | 18-Apr-2020 |
| Date of Decision | 30-Jun-2020 |
| Date of Acceptance | 08-Sep-2020 |
| Date of Web Publication | 8-Jan-2021 |
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 Abstract | | |
Introduction: The aim of this study was to evaluate the immunohistochemical expression of C4d in native renal biopsies of proliferative glomerular diseases, complement pathways in these diseases, and assess the relationship of C4d with histological and clinicopathological parameters, other complement proteins, and immunoglobulin markers. Methods: This cross-sectional study was conducted during the year 2018-19 involving 107 native renal biopsies with histologically diagnosed cases of proliferative glomerular diseases. C4d immunohistochemical evaluation of renal tissue sections was performed using polyclonal antihuman C4d as the primary antibody. Patients were classified as positive and negative groups based on their glomerular C4d deposition. Results: The overall prevalence of C4d positivity was 80.4% in proliferative glomerular diseases ranging between 60.0% in C3 glomerulonephritis to 92.9% in membranoproliferative glomerulonephritis. Mixed capillary and mesangial deposition were noted in all cases of proliferative glomerulonephritis. Classical pathway was dominantly involved in all glomerular diseases except C3 glomerulonephritis and IgA nephropathy. Multivariate logistic regression analysis revealed that glomerular IgG staining (aOR: 5.86, 95% CI: 1.26–27.14) and IgM staining (aOR: 3.90, 95%CI: 1.07–14.18) were significantly associated with C4d positivity. Conclusion: C4d staining along with immunoglobulin markers such as IgG and IgM and complement proteins can be useful in delineating different complement activation pathways in glomerular diseases and understanding the disease pathogenesis.
Keywords: C4d positivity, complement proteins, immunoglobulin markers, proliferative glomerular disease, renal biopsy
How to cite this article:
Raman S, Mishra P, Panigrahi A, Rout NK, Dash K. Glomerular C4d deposition in proliferative glomerular diseases. Indian J Pathol Microbiol 2021;64:69-77 |
How to cite this URL:
Raman S, Mishra P, Panigrahi A, Rout NK, Dash K. Glomerular C4d deposition in proliferative glomerular diseases. Indian J Pathol Microbiol [serial online] 2021 [cited 2023 Dec 1];64:69-77. Available from: https://www.ijpmonline.org/text.asp?2021/64/1/69/306512 |
| Introduction | |  |
Proliferative glomerulonephritis encompasses a broad group of native renal diseases including lupus nephritis (LN), membranoproliferative glomerulonephritis (MPGN), post-infectious glomerulonephritis (PIGN), IgA nephropathy (IgAN), C3 glomerulonephritis (C3GN), etc. These are characterized by varied histological features ranging from mesangial hypercellularity, endocapillary proliferation, crescents to increased mesangial matrix synthesis, thickening of glomerular capillary membranes, and presence of immune deposits.[1],[2] The pathogenesis of this proliferative group of diseases is mostly immune-mediated and complement proteins such as C3 and C1q have a significant role in it. Complement activation in glomerular diseases involves 3 pathways: classical pathway (CP), lectin pathway (LP), and alternative pathway (AP).[3] C4d generated by the activation of CP and LP has been widely used as a marker of antibody-mediated rejection, but its diagnostic application in native renal diseases is still largely unexplored.[4],[5]
Several studies have examined C4d in specific glomerular diseases,[2],[3],[6],[7],[8],[9],[10],[11] while C4d expression in glomerulonephritis as a whole has not been comprehensively studied in India. In an earlier study, Drachenberg et al. reported that strong C4d staining was present in immune-complex glomerulonephritis including LN, MPGN, and PIGN except for IgAN.[12] Another study by Sethi et al. revealed that specimens of LN, MPGN, and PIGN showed positive (2+ to 3+) C4d staining whereas, in all the specimens of IgAN and 80% of C3GN, C4d staining was completely negative.[3] Chandra et al. reviewed data on C4d in native kidney biopsies in different glomerular diseases and observed that C4d is deposited mostly in the mesangium and along the glomerular capillary wall.[1] In the present study, we aimed to evaluate the immunohistochemical expression of C4d in native renal biopsies of proliferative glomerular diseases, complement pathways in these diseases, and assess the relationship of C4d with histological and clinicopathological parameters, other complement proteins, and immunoglobulin markers.
| Methods | | |
This cross-sectional study was undertaken during the year 2018-19 involving 107 native renal biopsies with histologically diagnosed cases of proliferative glomerular diseases. Cases with inadequate renal biopsies and cases without immunofluorescence correlation were excluded from the study. According to earlier studies, the prevalence of C4d positivity in proliferative glomerular diseases varies widely from 38.5% to 83.3%.[2],[8],[11],[13] Assuming the prevalence of glomerular C4d positivity in proliferative glomerular diseases as 50% with absolute precision 10% and a confidence interval of 95%, the sample size was calculated as 96. However, 107 renal biopsies fulfilling the eligibility criteria were included in the study.
Initially light microscopic diagnosis of diseases using four stains, namely hematoxylin and eosin, periodic acid Schiff, Jones methenamine silver, and Masson's trichrome was done and findings were interpreted to make a definite diagnosis. Immunofluorescence was done with the following fluorescein isothiocyanate (FITC) labeled antibodies (from Dako Inc. Denmark): anti-IgG (FITC-F-0202), anti-IgM (FITC-F-0203), anti-IgA (FITC-F-0204), anti C3c (FITC-F-0201), anti C1q (FITC-F-0254), anti-kappa light (FITC-F-0198), anti-lambda light chain (FITC-F-0199), anti-fibrinogen and anti-albumin.
C4d immunohistochemical evaluation was performed on 3 μm deparaffinized and rehydrated sections of formaldehyde-fixed paraffin-embedded (FFPE) renal tissue sections on poly-L-lysine coated slides, using polyclonal antihuman C4d as the primary antibody. The antibodies and chemicals were obtained from Dako (Master Diagnostica, Spain). C4d primary antibody was diluted 1:50 in antibody diluent (Dako, Glostrup, Denmark). To unmask the target protein epitopes, heat-induced antigen retrieval (HIER) was performed in advance using the microwave (10 minutes at high power, 10 mM citrate buffer, pH 6.0). After cooling for 15 minutes at room temperature, endogenous peroxidase was blocked with 3% H2O2 for 5 minutes, followed by washing the sections in Tris-buffered saline (TBS). The anti-C4d antibody was applied for 30 minutes, followed by washing in TBS. Sections were incubated with a link antibody (Dako EnVision Flex™) for 30 min at room temperature followed by washing in TBS. The signal was detected with diaminobenzidine (DAB) chromogen (Dako, Glostrup, Denmark) for 15 minutes, resulting in a brown color. Following washing in tap water for a few minutes, slides were nuclear counterstained with 10% hematoxylin for 2 minutes. After dehydrating, the slides were cover slipped using the mounting medium.
C4d expression was noted in the glomeruli. Glomerular C4d deposition was defined as a glomerular capillary wall and/or mesangial staining. Intensity and pattern of glomerular C4d distribution were studied as per Gupta et al.[2] and Min-Kyung Kim et al.[14] The intensity of C4dwasgraded semi-quantitatively: no glomerular staining (-), mild glomerular staining (+), moderate glomerular staining (++), and intense glomerular staining (+++). Stain distribution was scored negative when glomeruli were with global 0 to trace C4d staining or glomeruli of any intensity with <15% segmental C4d staining. Patterns of C4d stain distribution were of 4 types: Diffuse (D) staining (=50% of the total glomeruli staining); Focal (F) staining (=50% of the total glomeruli staining); Global (G) staining (= 50% of a glomerulus with =1+ intensity); Segmental (S) staining (=50% of a glomerulus with =1+ intensity). The location of C4d stains such as glomerular capillary (C) or mesangial (M) or both (G + M) was noted. Sclerotic, scarred or collapsed glomerulus, whether global or segmental were not scored.
The medical records of all patients were reviewed to gather various demographic, laboratory, and clinical information at the time of biopsy. Relevant information regarding age, gender, clinicopathological parameters such as lipid profile, serum urea, serum creatinine, urine albumin creatinine ratio (UACR), urine pus cells, urine RBCs, urine epithelial cells, liver function test (LFT), antinuclear antibody (ANA), double-stranded DNA (dsDNA), etc., were collected using a structured schedule.
Ethical issues
Before initiation of the study, informed consent was obtained from all the study participants whose renal biopsies were considered for the study. The research protocol was approved by the Institutional Ethics Committee of the authors' institution (KIMS/KIIT/IEC/55/2017).
Statistical analysis
All the categorical variables were expressed as frequency and percentage whereas the continuous variables were presented as mean ± standard deviation. The differences in proportions between various groups were assessed by Chi-square test or Fischer's exact test. An independent t-test was used to compare the mean differences between patient groups. The variables with P value <0.15 in the univariate analyses were included in the multivariate logistic regression model to assess the relationship between c4d positivity and various independent variables after adjustment of confounders. A P value <0.05 was considered statistically significant. All the statistical analyses were performed by SPSS 21.0 statistical software.
| Results | | |
C4d staining was performed on the renal biopsy specimens of 107 patients with proliferative glomerulonephritis which included cases of LN (48, 44.9%), MPGN (14, 13.1%), PIGN (13, 12.1%), CrGN (13, 12.1%) C3GN (10, 9.3%), and IgAN (9, 8.4%). The mean age of the study population was 40.3 ± 13.8 years and 62.6% of them were females. The mean serum urea and creatinine levels were 77.2 ± 53.3 mg/dL and 3.8 ± 2.4 mg/dL, respectively, at the time of renal biopsy [Table 1]. Other demographic and clinicopathological data of the patients at the time of renal biopsy are presented in [Table 1]. Male and female patients were similar with respect to all variables except ANA and dsDNA. | Table 1: Gender wise distribution of clinical and laboratory data of patients with proliferative glomerular diseases (n=107)
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The intensity and pattern of glomerular C4d distribution in the patients are given in [Table 2]. Glomerular staining for C4d was positive in 60%, 61.5%, 66.7%, 87.5%, 92.9%, and 84.6% cases of C3GN, CrGN, IgAN, LN, MPGN, and PIGN respectively. LN and MPGN had consistently strong C4d staining intensity (2+ to 3+) in the majority of cases. Glomerular staining for C4d was dominantly diffuse in MPGN, LN, C3GN, and PIGN cases whereas mixed capillary and mesangial deposition were noted mostly in MPGN and LN cases. | Table 2: Intensity and pattern of Glomerular C4d distribution in patients with different proliferative glomerular diseases (n=107)
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[Table 3] shows the complement pathways in proliferative glomerular diseases. Almost all the glomerular diseases showed dominant classical pathway involvement except C3GN and IgAN which had dominant lectin pathway activation. Univariate analyses showing the relationship between C4d positivity and glomerular diseases, immunoglobulin markers, complement proteins, and histological features are depicted in [Table 4]. Among the proliferative glomerulonephritis irrespective of the disease entity, 72.9%, 78.5%, and 70.1% had endocapillary proliferation, mesangial hypercellularity, and crescent formation respectively. The presence of subendothelial and subepithelial immune deposits was relatively low, 39.2%, and 15.9% respectively. Glomerular IgG staining, glomerular IgM staining, and lambda positivity were found significantly associated with C4d positivity. | Table 3: Complement pathways in proliferative glomerular diseases (n=107)
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 | Table 4: Univariate analyses showing the association between glomerular C4d positivity and glomerular diseases, immune complex deposition, and histological features of activity (n=107)
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[Table 5] reveals the relationship between C4d positivity and various clinic-pathological parameters. None of the parameters showed a significant association with C4d positivity. In the multivariate logistic regression analysis, it was observed that only two variables such as glomerular IgG and glomerular IgM staining were significantly associated with C4d positivity after adjustment of other confounders [Table 6]. | Table 5: Univariate analyses showing the association between glomerular C4d positivity and clinicopathological parameters in patients with glomerular diseases (n=107)
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 | Table 6: Multivariate logistic regression analysis showing the association of factors with c4d positivity
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[Figure 1],[Figure 2],[Figure 3],[Figure 4] show glomerular C4d staining in cases of lupus nephritis, post-infectious glomerulonephritis, pauci-immune crescentic glomerulonephritis, and membranoproliferative glomerulonephritis. | Figure 1: (a): Diffuse proliferative Lupus nephritis, Class IV. (b): Membranous Lupus nephritis, Class V
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| Discussion | | |
In our study, the overall prevalence of C4d positivity was 80.4% (range: 60 – 92.9%) in proliferative glomerular diseases ranging between 60% in C3GN to 92.9% in MPGN. This observation is higher than the results reported in several other studies.[2],[15],[16],[17] Out of 13 cases of CrGN, 8 (61.5%) were pauci-immune crescentic GN (PICrGN) and 5 (4.7%) were immune complex-mediated crescentic GN (ICCrGN).
Our study showed that 92.9% of MPGN cases had diffuse C4d stain distribution which is consistent with the finding of another study that showed 92% of MPGN cases with diffuse glomerular c4d staining.[16] In the present study, the variation in the pattern of glomerular C4d deposition with the disease categories may be due to the size, quantity, location, and stability of the immune complexes. When excessive amounts of intermediate or large size complexes are formed by antibodies, they surpass the mesangial ability of clearance of these molecules and accumulate in paramesangial subendothelial region, and then ultimately in the peripheral capillary loops. These immune complexes are exposed to blood inflammatory mediators, causing the inflammation-mediated injury of the endothelium, leading to enhanced permeability of the capillary wall, resulting in passage and deposition of immune complexes in the subendothelial area and activation of the complement pathway. In contrast, small, unstable, and circulating immune complexes formed by low-affinity antibodies, may dissociate with the antigen or antibody and deposited at the outer aspect of the glomerular basement membrane as in situ immune complexes and induce complement activation.[14],[18]
In our study, the majority (81.3%) of LN cases were associated with the classical pathway activation. Earlier studies have also reported that the classical pathway activation was the predominant pathway involved in lupus nephritis.[1],[14],[19] Both lectin and alternate pathways were involved in IgAN cases in our study. Our observations are consistent with the finding of Roos et al., who reported that both alternative and lectin pathways are involved in the pathogenesis of IgA nephropathy.[20] However, according to Sethi et al.,[1] the alternate pathway and Chandra et al.,[3] the lectin pathway was predominantly activated in IgA nephropathy cases. The mechanism and clinical significance of complement activation in IgA nephropathy are unclear. Activation may occur through an alternate pathway,[21] but it has recently been proposed that the lectin pathway is involved in complement activation in IgA nephropathy.[22],[23] The observations in our study regarding classical complement pathway activation in PIGN patients were consistent with the findings of previous literature.[1],[3] According to our study, the classical pathway was predominantly activated in MPGN cases and lectin pathway was the major pathway for the pathogenesis of C3GN. These results are similar to the findings of the study done by Gupta et al.[2] whereas Sethi et al. in their study reported that lectin pathway was predominantly activated in MPGN and the alternate pathway was the major pathway in C3GN.[3] However, due to the close resemblance of C3GN and PIGN in terms of clinical, histological, and immunofluorescence features, few cases of PIGN might be misdiagnosed as C3GN. In our study, the classical pathway was mainly involved in the pathogenesis of both ICCrGN and PICrGN cases. In an earlier study, Xing et al. observed the activation of the alternate pathway and lectin pathway in CrGN.[24],[25] Villacorta et al. hypothesized that the alternate pathway might have a role in the pathogenesis of PICrGN.[26]
The multivariate regression model in our study revealed that the odds of C4d positivity increases 5.9 times in renal biopsies with positive glomerular IgG staining as compared to biopsies with negative IgG staining. In an earlier study, Faria et al. showed that glomerular C4d positivity was associated with the presence of IgG in patients of IgAN.[27] Also, in other diseases such as MPGN, LN, and PIGN C4d staining in the glomeruli was associated with the presence of IgG in the mesangium/capillary loop.[3],[4],[14] Further, we observed that biopsies with C4d positivity had a 3.9 times higher chance of being associated with positive glomerular IgM staining than negative IgM staining. Heybali et al. studied 37 IgAN patients and reported that glomerular C4d was associated with mesangial IgM deposition.[28]
The current study has few limitations, which include lack of molecular testing of the lectin pathway, and lack of confirmatory genetic testing of the alternative pathway. We used biopsies of patients with a wider age range as well as a variety of disease entities, which might affect the validity of the results of the study.
Glomerular C4d positivity can be expected in proliferative glomerular diseases where immunoglobulins and complements are in the etiopathogenetic backgrou?nd. C4d staining along with immunoglobulin markers such as IgG and IgM and complement proteins can help in delineating different complement activation pathways in glomerular diseases and assessing the severity of disease processes, besides understanding the disease pathogenesis. As newer drugs affecting complement pathways become available, identifying key complement pathways in different glomerular diseases will be crucial in the future.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
| References | | |
| 1. | Chandra P. C4d in native glomerular diseases. Am J Nephrol 2019;49:81-92.  |
| 2. | Gupta N, Wakefield DN, Clapp WL, Garin EH. Use of C4d as a diagnostic tool to classify membranoproliferative glomerulonephritis. Nefrol 2017;37:78-86. |
| 3. | Sethi S, Nasr SH, De Vriese AS, Fervenza FC. C4d as a diagnostic tool in proliferative GN. J Am Soc Nephrol 2015;26:2852-9. |
| 4. | Cohen D, Colvin RB, Daha MR, Drachenberg CB, Haas M, Nickeleit V, et al. Pros and cons for C4d as a biomarker. Kidney Int 2012;81:628-39. |
| 5. | Collins AB, Schneeberger EE, Pascual MA, Saidman SL, William WW, Tolkof-Rubin N, et al. Complement activation in acute humoral renal allograft rejection: Diagnostic significance of C4d deposits in peritubular capillaries. J Am Soc Nephrol 1999;10:2208-14. |
| 6. | Batal I, Liang K, Bastacky S, Kiss LP, McHale T, Wilson NL, et al. Prospective assessment of C4d deposits on circulating cells and renal tissues in lupus nephritis: A pilot study. Lupus 2012;21:13-26. |
| 7. | Kusunoki Y, Itami N, Tochimaru H, Takekoshi Y, Nagasawa S, Yoshiki T. Glomerular deposition of C4 cleavage fragment (C4d) and C4-binding protein in idiopathic membranous glomerulonephritis. Nephron 1989;51:17-9. |
| 8. | Bus P, Chua JS, Klessens CQF, Zandbergen M, Wolterbeek R, Van Kooten C, et al. Complement activation in patients with diabetic nephropathy. Kidney Int Reports 2018;3:302-13. |
| 9. | Ohsawa I, Ohi H, Tamano M, Endo M, Fujita T, Satomura A, et al. Cryoprecipitate of patients with cryoglobulinemic glomerulonephritis contains molecules of the lectin complement pathway. Clin Immunol 2001;101:59-66. |
| 10. | Nasri H, Rafieian-Kopaei M, Ahmadi A, Mubarak M. Association of glomerular C4d deposition with various demographic data in IgA nephropathy patients; a preliminary study. Immunopathol Persa 2016;2:e18. |
| 11. | Hilhorst M, Van Paassen P, Van Rie H, Bijnens N, Heerings-Rewinkel P, Van Breda Vriesman P, et al. Complement in ANCA-associated glomerulonephritis. Nephrol Dial Transplant 2017;32:1302-13. |
| 12. | Drachenberg CB, Papadimitriou JC, Chandra P, Haririan A, Mendley S, Weir MR, et al. Epidemiology and pathophysiology of glomerular C4d staining in native kidney biopsies. Kidney Int Reports 2019;4:1555-67. |
| 13. | Espinosa-Hernandez M, Ortega-Salas R, Lopez-Andreu M, Gomez-Carrasco JM, Perez-Saez MJ, Perez-Seoane C, et al. C4d as a diagnostic tool in membranous nephropathy. Nefrologia 2012;32:295-9. |
| 14. | Kim MK, Maeng YI, Lee SJ, Lee IH, Bae J, Kang YN, et al. Pathogenesis and significance of glomerular C4d deposition in lupus nephritis: Activation of classical and lectin pathways. Int J Clin Exp Pathol 2013;6:2157-67. |
| 15. | Daram SR, Yalamanchili P, Salinas-Madrigal L, Bastani B. Patterns of C4d staining in patients with lupus nephritis. J Appl Res 2006;6:176-81. |
| 16. | Yadav S, Singhai A, Babu S, Singh VK, Wakhlu A, Sonkar S, et al. Utility of C4D deposits in native renal diseases and relation with disease progression. Indian J Heal Sci Biomed Res 2019;12:50-5. |
| 17. | Val-Bernal FJ, Francisca GM, Val D, Rodrigo E, Arias M. C4d immunohistochemical staining is a sensitive method to confirm immunoreactant deposition in formalin-fixed paraffin-embedded tissue in membranous glomerulonephritis. Histol Histopathol 2011;26:1391-7. |
| 18. | Fogo AB. Glomerular hypertension, abnormal glomerular growth, and progression of renal diseases. Kidney Int Suppl 2000;57:15-21. |
| 19. | Bheemavathi A, Durga K, Gowrishankar S. Correlation of glomerular C4d staining with disease activity in lupus nephritis- A prospective study. Int J Sci Res Publ 2014;4:4-8. |
| 20. | Roos A, Rastaldi MP, Calvaresi N, Oortwijn BD, Schlagwein N, Van Gijlswijk-Janssen DJ, et al. Glomerular activation of the lectin pathway of complement in IgA nephropathy is associated with more severe renal disease. J Am Soc Nephrol 2006;17:1724-34. |
| 21. | Wyatt RJ, Julian BA. Activation of Complement in IgA Nephropathy. Am J Kidney Dis 1988;12:437-42. |
| 22. | Endo M, Ohi H, Ohsawa I, Fujita T, Matsushita M, Fujita T. Glomerular deposition of mannose-binding lectin (MBL) indicates a novel mechanism of complement activation in IgA nephropathy. Nephrol Dial Transplant 1998;13:1984-90. |
| 23. | Matsuda M, Shikata K, Wada J, Sugimoto H, Shikata Y, Kawasakfi T, et al. Deposition of mannan binding protein and mannan binding protein-mediated complement activation in the glomeruli of patients with IgA nephropathy. Nephron 1998;80:408-13. |
| 24. | Xing GQ, Chen M, Liu G, Heeringa P, Zhang JJ, Zheng X, et al. Complement activation is involved in renal damage in human antineutrophil cytoplasmic autoantibody associated pauci-immune vasculitis. J Clin Immunol 2009;29:282-91. |
| 25. | Xing GQ, Chen M, Liu G, Zheng X, Jie EJ, Zhao MH. Differential deposition of C4d and MBL in glomeruli of patients with ANCA-negative pauci-immune crescentic glomerulonephritis. J Clin Immunol 2010;30:144-56. |
| 26. | Villacorta J, Diaz-Crespo F, Acevedo M, Guerrero C, Campos-Martin Y, Garcia-Diaz E, et al. Glomerular C3d as a novel prognostic marker for renal vasculitis. Hum Pathol 2016;56:31-9. |
| 27. | Faria B, Henriques C, Matos AC, Daha MR, Pestana M, Seelen M. Combined C4d and CD3 immunostaining predicts immunoglobulin (Ig) A nephropathy progression. Clin Exp Immunol 2015;179:354-61. |
| 28. | Heybeli C, Unlu M, Yildiz S, Cavdar C, Sarioglu S, Camsari T. IgA nephropathy: Association of C4d with clinical and histopathological findings and possible role of IgM. Ren Fail 2015;37:1464-9. |
Correspondence Address:
Ansuman Panigrahi
Professor, Community Medicine, Kalinga Institute of Medical Sciences, KIIT University, Bhubaneswar - 751 024, Odisha
India
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/IJPM.IJPM_364_20
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6] |
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