Morphological changes in kidneys and clinical presentation in patients with primary and secondary membranous nephropathy

Introduction/Aim: Membranous nephropathy (MN) typically presents as nephrotic syndrome and may be idiopathic (primary MN, pMN) or occur secondary to other diseases (secondary MN, sMN). Given the differing therapeutic approaches, this study aimed to compare clinical and pathohistological parameters between patients with pMN and sMN.

Methods: Through a retrospective analysis of kidney biopsy samples diagnosed at the Institute of Pathology, Faculty of Medicine, Belgrade, between 2011 and 2023, 99 patients with membranous nephropathy were identified. These patients were divided into two groups: patients with pMN (n = 44) and patients with sMN (n = 55). Clinical and pathohistological parameters (by optical and immunofluorescence microscopy) were compared among study participants.

Results: While males more frequently suffered from MN, females more frequently had sMN (p = 0.036). Haematuria was more common in subjects with sMN (p = 0.009). Proteinuria values were significantly higher among patients with pMN (p = 0.018), related to a significantly greater decrease in serum total protein values in them (p = 0.011). Endocapillary proliferation was significantly more frequent among those with sMN (p = 0.032). Marked positivity of several immunofluorescent markers was significantly more common in subjects with sMN: IgA, IgM, C1q, κ-chains, and λ-chains (p < 0.05).

Conclusion: Females are more commonly affected by sMN. Characteristics of sMN include presence of haematuria, endocapillary hypercellularity and immunofluorescence positivity for markers not seen in pMN (IgA, IgM, C1q), while characteristics of pMN include significantly higher levels of proteinuria, accompanied by significantly reduced serum protein values.

INTRODUCTION

Membranous glomerulonephritis (MGN) is a chronic kidney disease characterized by subepithelial deposition of immunoglobulins and complement components within the glomeruli, most commonly presenting clinically as nephrotic syndrome [1]. The disease most commonly occurs between 50 and 60 years of age and is approximately twice as prevalent in men, while it is rarely observed in children [2],[3]. Based on etiology, it is classified into primary and secondary MGN [4]. Primary MGN is characterized by an autoimmune response against podocyte antigens in the absence of an identifiable secondary cause; antigens identified to date include PLA₂R and THSD7A [5],[6]. Secondary MGN may be associated with autoimmune diseases (systemic lupus erythematosus, Sjögren’s syndrome, autoimmune thyroiditis), infections (hepatitis B and C, HIV, syphilis, malaria), drugs and toxins (non-steroidal anti-inflammatory drugs, penicillamine, gold salts, mercury), as well as malignant diseases (lung, prostate, colon, and uterine cancers, and hematological malignancies), in which the NELL-1 antigen has been identified [7],[8].

The gold standard for diagnosing MGN is the histopathological evaluation of kidney biopsy samples. In patients presenting with nephrotic syndrome who are serum-positive for PLA₂R antibodies and have preserved renal excretory function, a kidney biopsy is not required [9]. On light microscopy, a diffuse and uniform thickening of the glomerular basement membrane is observed, with the matrix gradually forming “spike”- like projections between the deposited immune complexes [10]. A hallmark of immunofluorescence microscopy is the presence of fine, granular deposits of IgG and the complement component C3 along the glomerular basement membrane [10].

Treatment of MGN depends on its underlying etiology. Patients with primary MGN receive immunosuppressive therapy, including corticosteroids, cyclophosphamide, and calcineurin inhibitors, as well as rituximab, a monoclonal antibody targeting the CD20 antigen on B lymphocytes [11],[12]. In patients with secondary MGN, treatment is focused on the underlying condition. Supportive therapy, including diuretics, ACE inhibitors, angiotensin receptor blockers, and statins, is used in all patients to manage blood pressure, edema, proteinuria, and lipid levels [11].

Considering the significance of MGN etiology for therapeutic strategy, this study aimed to compare clinical parameters, such as gender and age distribution, proteinuria levels, presence of hematuria, serum total protein and albumin concentrations, markers of renal excretory function, and the most common comorbidities, with pathohistological features, including glomerular, tubulointerstitial, and vascular changes on light microscopy, as well as the intensity of immunoglobulin, light chain, and complement component deposits on immunofluorescence microscopy, between patients with primary and secondary MGN.

METHODS

The retrospective study included 99 kidney biopsy samples diagnosed as MGN between 2011 and 2023 at the Institute of Pathology “Dr. Đorđe Joannović,” Faculty of Medicine, Belgrade. Of these 99 samples, 64 were from patients treated at the Clinic for Nephrology, University Clinical Center of Serbia, 27 from the Clinical Hospital Center “Zvezdara”, 4 from the Clinical Hospital Center “Zemun”, 2 from the University Children’s Clinic; and 2 from the Institute for Mother and Child Health Protection of Serbia “Dr. Vukan Čupić”. Patients were divided into two groups based on disease etiology: those with primary MGN (n = 44) and those with secondary MGN (n = 55).

Clinical and laboratory data recorded at the time of biopsy were collected through review of patients’ medical records. Proteinuria values < 3,5 g/24 h were classified as subnephrotic, while values of ≥ 3,5 g/24 h were classified as nephrotic [11]. Normal serum total protein levels range from 62 to 82 g/L, and albumin from 35 to 52 g/L [13]. Glomerular filtration rate was estimated using the CKD-EPI 2021 equation, and the stage of chronic kidney disease was determined based on the estimated glomerular filtration rate values [11]. Pathohistological data were obtained from the reports of previously analyzed kidney biopsy samples.

Statistical analysis was performed using IBM SPSS Statistics 22 (SPSS Inc., Chicago, Illinois, USA). Data normality was assessed using the coefficient of variation, skewness, and kurtosis. For numerical variables with a normal distribution, differences between patients with primary and secondary MGN were evaluated using the Student’s t-test for two independent samples. For data with a non-normal distribution or ordinal scale, the Mann–Whitney U test was applied. Differences in the frequencies of nominal variables between the two patient groups were assessed using the χ² test for r×k tables and Fisher’s exact test. Hypotheses were tested at a significance level of 0.05.

RESULTS

Clinical parameters

A detailed overview of the clinical parameters in patients with primary and secondary MGN is presented in Table 1. Among the 99 patients, 63.6% (n = 63) were male and 36.4% (n = 36) were female. The secondary form of the disease was observed in 69.4% (n = 25) of female patients compared to 47.6% (n = 30) of male patients, a difference that was statistically significant (p = 0.036). The mean age of patients with primary MGN was 52.8 ± 14.7 years, while that of patients with secondary MGN was 51.7 ± 15.9 years (p = 0.704).

Table 1. Clinical parameters of subjects with membranous nephropathy

Most patients exhibited nephrotic-range proteinuria at the time of biopsy. Proteinuria was subnephrotic in 9.1% (n = 9) of cases, the majority of whom (n = 7) had secondary MGN. The median 24-hour urine protein level in patients with primary MGN (median 8.8 g/24 h; range 2.9-55.0 g/24 h) was significantly higher than in those with secondary MGN (median 6.9 g/24 h; range 1.9-45.1 g/24 h; p = 0.018). Hematuria, mostly microscopic, was observed in 34.3% (n = 34) of patients, with 73.5% (n = 25) of these cases occurring in patients with secondary MGN (p = 0.009).

The mean total protein level in patients with primary MGN was 44.8 ± 7.9 g/L, representing a significantly greater reduction (p = 0.011) compared to patients with secondary MGN (50.1 ± 7.7 g/L). Although serum albumin levels were also reduced, no significant difference was observed between the two groups (Table 1).

Indicators of excretory renal function, including serum urea and creatinine levels, estimated glomerular filtration rate, and chronic kidney disease stage, did not differ significantly between patients with primary and secondary MGN, although higher stages of chronic kidney disease (G3, G4, and G5) were more frequently observed in patients with secondary MGN (Table 1).

The most frequent comorbidities in the study population were hypertension and diabetes mellitus. Among patients with hypertension, the distribution was nearly even, with 47.1% (n = 32) having primary MGN and 52.9% (n = 36) having secondary MGN (p = 0.438). Diabetes mellitus was present in only 8.1% (n = 8) of patients, yet 75.0% (n = 6) of these cases were associated with secondary MGN (p = 0.248).

Histopathological parameters

On light microscopy, all patients exhibited uniform thickening of the glomerular basement membrane, a hallmark of MGN regardless of etiology (Figure 1). Indicators of chronic glomerular damage, such as the proportion of globally and segmentally sclerotic glomeruli, did not differ significantly between primary and secondary MGN (Table 2). Crescentic glomerular formations were more common in patients with secondary MGN, although this difference was not statistically significant (p = 0.091). Endocapillary proliferation was noted in 9.1% (n = 9) of the examined tissue samples, of which 88.9% (n = 8) were from patients with secondary MGN (p = 0.032) (Table 2, Figure 1D). Karyorrhexis was observed in only two patients with secondary MGN (p = 0.500).

Figure 1. Changes in glomeruli on light microscopy in primary (A) Periodic acidSchiff (PAS), x200, (B) Masson’s trichrome (MTS), x200, (C) Jones methenamine silver (JMS), x400; and secondary membranous nephropathy, with evident endocapillary hypercellularity (D) PAS, x200, (E) MTS, x200, (F) JMS, x200

Table 2. Pathohistological parameters of patients with membranous nephropathy by light microscopy

Unlike chronic vascular lesions (including medial hypertrophy, intimal fibroelastosis, and vascular hyalinization), indicators of chronic tubulointerstitial damage, such as interstitial fibrosis and tubular atrophy, differed significantly between the groups, occurring more frequently in patients with secondary MGN (Table 2).

On immunofluorescence microscopy, in addition to IgG and the complement component C3, whose fine granular deposits along the glomerular basement membrane represent a characteristic finding in MGN (Figure 2), pronounced positivity for other markers (IgA, IgM, C1q, κ chains, and λ chains) was significantly more frequently observed in secondary MGN (Table 3).

Table 3. Pathohistological parameters of patients with membranous nephropathy by immunofluorescence

Figure 2. Immunofluorescence microscopy findings: fine granular deposits of (A) IgG and (B) C3 along the glomerular basement membrane, x400

DISCUSSION

In this retrospective study, clinical and pathohistological parameters were compared between patients with primary and secondary MGN. Male patients accounted for 63.6% of the study population, which is consistent with published data indicating a male-to-female ratio of approximately 1.5-2:1 in the incidence of MGN [14, 15]. Moreover, as many as 69.4% of female patients had a secondary form of the disease. This gender-based distribution may be explained by the substantial contribution of autoimmune diseases to the etiology of secondary MGN, as women are considerably more likely to develop autoimmune disorders, which can underlie the secondary form of MGN [7],[16]. The autoimmune disease most frequently associated with this pattern is systemic lupus erythematosus, in which renal involvement may manifest as membranous glomerulonephritis, corresponding to class V lupus nephritis [17]. Systemic lupus erythematosus occurs 2 to 15 times more frequently in females [18],[19].

Membranous glomerulonephritis most commonly presents with nephrotic syndrome, characterized by marked proteinuria, hypoalbuminemia, peripheral edema or anasarca, hyperlipidemia, and lipiduria [20]. Approximately one third of patients with primary MGN achieve spontaneous remission after several months or years. [21]. Proteinuria levels are an important factor in disease progression – values < 4 g/24 h are associated with a low risk, whereas values > 8 g/24 h are associated with a high risk of progression [22]. Our results demonstrated significantly higher 24-hour urinary protein levels in patients with primary MGN. Consequently, owing to greater urinary protein loss, patients with the primary form of the disease exhibited significantly reduced serum total protein levels compared with physiological values.

Literature data indicate that the presence of microscopic hematuria, arterial hypertension, and impaired renal function increases the likelihood of a secondary etiology of MGN [23],[24]. The results of our study showed that microscopic hematuria was significantly more common in patients with secondary MGN (73.5%) compared to those with primary MGN. Additionally, patients with secondary MGN had lower estimated glomerular filtration rates and a higher frequency of advanced chronic kidney disease stages (G3, G4, and G5) than those with primary MGN, although these differences were not statistically significant. In contrast, the prevalence of arterial hypertension did not differ between the groups.

In addition to serving as the gold standard for diagnosing MGN, histopathological evaluation of kidney biopsy samples can also suggest a possible etiology, whether primary or secondary. Specifically, the presence of mesangial and endocapillary hypercellularity on light microscopy is more indicative of a secondary etiology [23]. The results of our study support this conclusion–of the nine tissue samples exhibiting endocapillary proliferation, 88.9% were from patients with secondary MGN. Conversely, positivity for PLA2R and THSD7A antigens indicates a primary etiology [25],[26]. On the other hand, positivity for EXT1 and EXT2 antigens is linked to autoimmune diseases as the underlying cause of secondary MGN, primarily systemic lupus erythematosus, whereas NELL-1 positivity is associated with malignancy in approximately one-third of cases [27],[28].

The “full house” pattern on immunofluorescence microscopy, characterized by positivity for all five markers, i.e., IgG, C3, as well as IgA, IgM, and C1q, is primarily indicative of secondary MGN in the context of systemic lupus erythematosus [29]. Deposits of IgG and the complement component C3 along the glomerular basement membrane are a characteristic feature of membranous glomerulonephritis, regardless of disease etiology; however, marked positivity for these markers was more frequently observed in secondary MGN. The distribution of IgA, IgM, and C1q positivity differed significantly according to MGN etiology, with moderate to strong positivity for these markers being significantly more common in secondary MGN. Differences in κ-chain and λ-chain positivity could not be adequately interpreted, as these markers were not routinely assessed at the Institute of Pathology, Faculty of Medicine in Belgrade, until 2018.

CONCLUSION

Females are more likely to develop secondary MGN. Features of secondary MGN include hematuria, endocapillary hypercellularity, and immunofluorescence positivity for markers not typically seen in primary MGN (IgA, IgM, C1q). In contrast, primary MGN is characterized by significantly higher levels of proteinuria, accompanied by markedly reduced serum protein concentrations.

ABBREVIATION LIST

MGN – membranous glomerulonephritis
pMGN – primary membranous glomerulonephritis
sMGN – secondary membranous glomerulonephritis