Study of Correlation between Renal CD68 with Oxford MEST-C Scores and C3 Immunoreactivity in IgA Nephropathy

Introduction

IgA nephropathy (IgAN) is the most frequent primary glomerulonephritis globally and an important cause of ESKD. It has a variable clinical presentation, renal histology, and patient outcome.¹ These patients usually present with gross or microscopic hematuria, with or without proteinuria. Renal biopsy is required for diagnosis, and to assess the severity of the histopathologic changes.² Independent predictors of progression to kidney failure include low estimated glomerular filtration rate (eGFR), proteinuria >1 g/d or persistent proteinuria, hypertension, and glomerular or interstitial sclerosis at biopsy.^3-6 Several clinical and histologic factors (arterial hypertension, proteinuria, renal function, and Oxford classification score) determine the outcome of patients with IgAN.^7,8 The Oxford MEST C classification of IgAN was developed to improve prognostication in IgAN. The MEST criteria include M 0/1 (≤/>50% of glomeruli showing mesangial hypercellularity), E 0/1 (endocapillary hypercellularity absent/present), S 0/1 (segmental glomerulosclerosis absent/present), T 0/1/2 (tubular atrophy/interstitial fibrosis involving <=25%, 26 to 50% and >50% of the cortex) and C 0/1/2 (no crescents, involving <25% and >25% of glomeruli).^7-10 The identification of new prognostic markers could provide insights into the pathogenesis of IgAN and unveil new therapeutic avenues. Many new prognostic markers have been studied in IgAN, among which CD68 (a macrophage marker) is under debate. In this study, we investigated the prognostic value of CD68, as well as its associations with Oxford MEST scores and C3 immunoreactivity in IgAN.

Materials and Methods

This study included 35 adult patients diagnosed with IgAN between 2011 and 2020. Clinical and laboratory details, including the IgAN international risk prediction score to estimate the risk of a 50% decline in eGFR over 36 months were collected.⁵ The IgA immunostaining pattern (diffuse or segmental granular mesangial deposits), and intensity [graded semi-quantitatively as zero (nil), 1+ (weak), 2+(moderate), and 3+(strong)], were also recorded. Immunohistochemical staining was performed with monoclonal antibodies against CD68 [Figure 1].

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Figure 1:

(a) High power photomicrograph showing mesangial hypercellularity (blue arrow) (Hematoxylin & Eosin stain; 40x) (b) Glomerular CD68 positivity in mesangium (black arrow) (Immunohistochemical stain, 40x), (c) Interstitial fibrosis and tubular atrophy (red arrow) (Jones methamin silver stain, 20x), and (d) Interstitial CD68 positivity (black arrows) (Immunohistochemical stain, 40x).

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Diagnosed cases of idiopathic IgAN on renal biopsies with ≥5 glomeruli were included. Patients with any other concomitant renal diseases, diabetes mellitus, autoimmune diseases, abnormal hypergammaglobulinemia, or liver disease were excluded. Data were collected and evaluated by one pathologist and one clinician and cross-checked by the principal investigator. Prognosis was assessed by considering the correlation with MEST-C scores. The ethical committee of our institute approved the study and all patient consent was obtained.

Results

Clinico-pathological characteristics of the study cases have been presented in Table 1. We included 35 IgAN patients, including 25 (71.43%) males and 10 (28.57%) females. The median age at diagnosis was 30 (range 18-66) years. Ten (28.57%) patients had macroscopic hematuria, while 22 (62.86%) had microscopic hematuria at biopsy. Proteinuria <1 g/dL was found in 13 (37.14%) patients and >1 g/dL in 22 (62.86%) cases. Eleven (31.43%) patients had high blood pressure. The mean eGFR (mL/min/1.73m²) was 45.66 ± 39.91.

Correlation of CD68+ cells with clinical parameters

Both mean glomerular and interstitial CD68 were correlated with proteinuria (p< 0.05) [Figures 2, 3]. There was a statistically significant difference in CD68 staining between the patients with proteinuria >1 g/day versus the patients with proteinuria <1 g/day. Hypertension correlates with the mean interstitial CD68⁺ cells (p< 0.05) but not with the mean glomerular CD68. Correlation was also observed between eGFR and the mean interstitial CD68 [Table 2]. However, eGFR did not correlate with the mean Glomerular CD68. No correlation was found between mean glomerular and interstitial CD68 with age, sex, and hematuria.

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Figure 2:

Association of mean glomerular CD68⁺ cells with proteinuria.

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Figure 3:

Association of mean interstitial CD68⁺ cells with proteinuria.

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Table 2: Correlation of mean glomerular CD 68+ cells and mean interstitial CD68+ cells with age, eGFR, number of globally sclerosed & normal glomeruli, and prediction score

Variables	Age (years)	eGFR (mL/min/1.73m2)	No. of GS glom	No. of N glom	Prediction score (%)
Mean glomerular CD68+ cells
Correlation coefficient	-0.102	0.003	-0.061	-0.297	0.141
p value	0.557	0.989	0.727	0.083	0.418
Mean interstitial CD68+ cells
Correlation coefficient	0.154	-0.611	0.414	-0.583	0.781
p value	0.375	0.0001	0.014	0.0003	<0.0001

Discussion

In the present study we evaluated glomerular and interstitial macrophages in 35 IgAN biopsies by performing a standard procedure of immunohistochemistry using the widely available marker CD68. We showed that high number of interstitial CD68-positive cells are associated with hypertension and proteinuria. The association was also observed between interstitial CD68 and eGFR, risk prediction score, mesangial cellularity (M), IFTA, and number of globally sclerosed and normal glomeruli. The high number of glomerular CD68-positive cells was associated with mesangial cellularity (M), the number of globally sclerosed and normal glomeruli.

Macrophage infiltration is a general feature of glomerular injury. Macrophage infiltration into the renal cortex has been reported to be associated with progressive renal disease and can be used as an important indicator to assess the development and prognosis of kidney diseases.^11,12 M1 macrophages exhibit pro-inflammatory function by secreting inflammatory factors like IL-1, IL-6, IL-12, TNF-α, and reactive oxygen species. If the inflammation persists, it further spreads and causes tissue fibrosis by releasing several fibrogenic cytokines like TGF-β, PDGF, and also synthesizes collagen I and fibronectin. Alternatively, M2 macrophages show an anti-inflammatory role, reduce cell apoptosis, and promote tissue repair and remodelling.^13-16 Studies showed that CD68 can be used as a surface marker for M1 macrophages, and CD163 is a highly specific mannose receptor expressed in M2 macrophages.^17,18

Multivariate analyses revealed a significant relationship between macrophage infiltration and levels of proteinuria, tubulointerstitial injury, and the progression of renal fibrosis.¹⁹ Thus, an increased number of macrophages in the tubulointerstitial area may serve as a predictive factor for poor prognosis in patients with IgA. On the other hand, glomerular infiltration of macrophages has been shown to play a role in glomerular lesions by TGF-β synthesis.²⁰ TGF-β has been shown to be involved in the excessive deposition of extracellular matrix, leading to glomerulosclerosis.²¹ Macrophages mediate immune responses and tissue injury by antigen presentation, cell signalling, and mesangial matrix remodeling. CD68 has been successfully used as a macrophage marker in lupus nephritis.²² In pediatric IgAN, macrophage infiltration into the interstitium and glomeruli is associated with unfavorable activation and persistent inflammatory response.²³ Silva GE et al. suggested that the presence of CD68-positive macrophagesin IgAN, positively correlated with proteinuria, serum creatinine levels, progression of renal disease and worse outcomes.²⁴ Some studies found that when the macrophages infiltrated the tubulointerstitium in IgAN, the patients had proteinuria, lower eGFR, and IFTA.^25,26 Macrophages in the glomeruli can be regarded as the marker of mesangial and endocapillary hypercellularity. The count of macrophages per glomerulus is associated with masengial hypercellularity, crecent, and thus the final MEST-C score positively.^25,27

Our study has some limitations: it was retrospective and conducted at a single center, with a modest sample size, which may limit the generalizability and statistical power of the findings. Follow-up data and outcome measures (e.g., eGFR decline, ESKD progression) were not available, precluding us from making direct inferences about the long-term prognostic significance of CD68⁺ macrophage infiltration. The use of CD68 as a pan-macrophage marker does not allow us to distinguish between M1 (pro-inflammatory) and M2 (anti-inflammatory/repair) phenotypes, limiting our ability to interpret the functional role of macrophage infiltration in IgAN pathogenesis. Finally, the study did not include repeat or longitudinal biopsies, so we could not assess changes in macrophage burden over time or in response to treatment. Thus, future studies incorporating phenotypic markers (e.g., CD163, iNOS) and longitudinal clinical outcome are needed.

To conclude, this study highlights a potential link between interstitial macrophage burden and composite measures of disease severity, including the IgAN risk score. However, further studies on a larger scale with follow-up biopsies are required to elucidate the definitive functional and prognostic roles of macrophages in IgAN.