Characterization of patients with IgA nephropathy with and without associated minimal change disease

Introduction Immunoglobulin A nephropathy (IgAN) presents various clinical manifestations and pathological phenotypes. Approximately 5% of patients with IgAN present with early onset nephrotic syndrome, mild mesangial lesions, and diffuse foot process effacement of podocytes, which resemble minimal change disease (MCD). These patients are defined as MCD-IgAN. Whether MCD-IgAN is a special type of IgAN or simply MCD accompanied by IgA deposition remains controversial. Methods A total of 51 patients diagnosed with MCD-IgAN at Beijing Anzhen Hospital from January 2010 to September 2022 were recruited. The clinical and pathological characteristics of IgA-MCD were analyzed. Patients with IgAN but without MCD (non-MCD-IgAN) and healthy participants were enrolled as controls. Galactose-deficient immunoglobulin A1 (Gd-IgA1) and complement C3 were detected both in the circulation and in renal tissues. Results We found that the levels of serum Gd-IgA1 were lower in participants with MCD-IgAN than in those with non-MCD-IgAN, but higher than in healthy participants. Gd-IgA1 was rarely deposited in the glomeruli of participants with MCD-IgAN, with a positive rate of only 13.7% (7/51); in contrast, the positive rate in participants with non-MCD-IgAN was 82.4% (42/51). Among renal Gd-IgA1-positive patients, Gd-IgA1 and immunoglobulin A (IgA) colocalized along the glomerular mesangial and capillary areas. Interestingly, we found that the circulating levels of complement C3 were significantly higher in participants with MCD-IgAN than in participants with non-MCD-IgAN. In addition, the intensity of C3c in glomeruli in participants with MCD-IgAN was significantly weaker than in participants with non-MCD-IgAN. Conclusions Our study suggests that, in MCD-IgAN, most of the IgA that is deposited on glomeruli is not the same pathogenic Gd-IgA1 as found in general IgAN. Complement activation both in the circulation and in the renal locality was much weaker in MCD-IgAN than in non-MCD-IgAN. Our study suggests that IgAN with MCD might be MCD with coincidental IgA deposition.


Introduction
Immunoglobulin A nephropathy (IgAN) is the most frequent form of primary glomerulonephritis worldwide (1). Up to 20%-40% of patients will have reached end-stage kidney disease approximately 20 years after diagnosis (2). The clinical presentation and histologic features of IgAN are highly variable (3). In addition to the classic presentation (3), such as recurrent episodes of gross hematuria after mucosal infections, some patients with IgAN present with onset nephrotic syndrome, mild mesangial lesions under light microscopy, and diffuse foot process effacement of podocytes under electron microscopy, which resemble minimal change disease (MCD) (4,5). These patients are defined as MCD-IgAN. Some studies have reported these patients to have a special clinical subtype of IgAN (5,6), whereas others have considered this to be MCD accompanied by IgA deposition (7,8).
Today, the pathophysiology of IgAN is considered to be a multi-hit mechanism (9). The elevated serum levels of aberrantly O-glycosylated polymeric immunoglobulin A1 (IgA1), i.e., galactose-deficient IgA1 (Gd-IgA1), as the pivotal component at the beginning of IgAN pathogenesis (10,11), are associated with a poor prognosis in IgAN (11). Staining for Gd-IgA1 was relatively specific in the glomeruli of patients with IgAN (12). Gd-IgA1 combined with the glycan-specific immunoglobulin G (IgG) or immunoglobulin A (IgA) autoantibodies, (13) and formed of Gd-IgA1-IgG/IgA immune complexes, deposited in the glomeruli and then induced the activation of the complement system and an inflammation reaction (14). The components of complement activation are usually detected in both the circulation and renal biopsy tissues of IgAN (15). Complement component 3 (C3) accompanies IgA deposited in the renal tissue of approximately 90% patients with IgAN (16). In recent years, increasing evidence has confirmed the important role of abnormal complement activation in the pathogenesis of IgAN (15), whereas Gd-IgA1 and abnormal activation of the complement system are rarely observed in MCD. It is unclear whether or not the IgA deposited in MCD-IgAN is the same pathogenic Gd-IgA1 as in general IgAN, and whether or not the degree of complement system activation in MCD-IgAN is the same as in general IgAN.
In this study, we explored the role of Gd-IgA1 and complement C3 in IgAN with MCD (MCD-IgAN) and IgAN without MCD (non-MCD-IgAN). We aimed to determine whether unusual cases of MCD-IgAN are a special clinical subtype of IgAN or MCD accompanied by coincidental IgA deposition.

Ethics statement
This research was conducted according to the principles of the Declaration of Helsinki. The ethics committees of Beijing Anzhen Hospital approved the study and all enrolled individuals provided informed consent to participate in this investigation.

Study participants
This was a retrospective study of patients with IgAN in Beijing Anzhen Hospital. A total of 926 patients were diagnosed with primary IgAN from January 2010 to September 2022. Among them were 51 patients who had unremarkable glomeruli under light microscopy (only one patient had mild mesangial hypercellularity; see Table 1) and diffuse (over 80%) foot process effacement under electron microscopy ( Figure 1). Because these findings, other than the mesangial IgA deposition, indicate MCD, this group was defined as the MCD-IgAN group. In addition, 51 patients with IgAN without MCD (i.e., the non-MCD-IgAN group), from June 2019 to February 2020, were consecutively enrolled as the disease control.

Clinical and histologic characteristics
The clinical characteristics of recruited patients at the time of renal biopsy were collected from the medical records. These included age, gender, history of high blood pressure [i.e., a systolic blood pressure (SBP) of 140 mmHg or more, a diastolic blood pressure (DSP) of 90 mmHg or more, or taking antihypertensive medication to prevent hypertension], prodromic infection, gross hematuria, microscopic hematuria, 24-hour urine protein excretion, serum creatinine levels, and plasma C3 and C4 levels. Microscopic hematuria was defined as more than three red blood cells per high-power field under light microscopic examination of sediment after centrifugation. The estimated glomerular filtration rate (GFR) was calculated using the modified GFR estimating equation (17). Histologically, the Oxford classification was used to evaluate the pathological lesions for those with more than eight glomeruli in biopsy specimens (18).

Detection of glomerular Gd-IgA1 by immunofluorescence in renal biopsies
The paraffin-embedded biopsy renal tissues were cut into slices of 4 mm thickness, deparaffinized, and incubated with pepsase for 35 minutes at 37°C. After being washed with phosphate-buffered saline (PBS), sections were incubated in 3% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) blocking solution for 30 minutes at 25°C. Anti-human Gd-IgA1 antibody, produced by clone KM55 (Immuno-Biological Laboratories, Fujioka, Japan), with a dilution of 1:200 in PBS were incubated overnight at 4°C. After being washed, Cy ™ 3-conjugated AffiniPure donkey anti-rat antibodies (diluted 1:200 in PBS; Jackson ImmunoResearch Laboratories, Philadelphia, PA, USA) were added for 1 hour at 37°C. After being washed with PBS three times for 5 minutes each, the sections were air dried in the dark and mounted with mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI).
Primary antibodies were replaced by PBS as blank controls. = Glomerular immunofluorescence was scored using fluorescence microscopy (Nikon 80i, Japan). The staining intensity of anonymized renal biopsies as negative: 0 (absent), positive: 1+, 2+, 3+ and 4+ were grades by two observers (Wy G and Lj S), who were blinded to clinical data. We excluded sections that contained fewer than two glomeruli.
Detection of serum Gd-IgA1 by enzymelinked immunosorbent assay Serum samples from 37 patients with MCD-IgAN and 48 patients with non-MCD-IgAN were collected on the morning of biopsy, divided into aliquots, and stored at -80°C until the measurement of circulation Gd-IgA1. Serum was also collected from 54 age-, gender-, and geographically matched healthy adults selected as healthy controls. The levels of serum Gd-IgA1 were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) test kit with KM55 (Immuno-Biological Laboratories, Fujioka, Japan), in accordance with the manufacturer's protocol.

Statistical analysis
Quantitative variables were summarized as the mean ± standard deviation (for normally distributed data), or median and interquartile range (IQR) (for non-normally distributed data). Categorical variables were expressed as the number and percentage. The Kolmogorov-Smirnov test was used to analyze the normality of the distribution of variables. For continuous variables, if the data were normally distributed, an independent-samples t-test was used; if not, a Mann-Whitney U-test or Kruskal-Wallis H-test was performed. For categorical variables, a chisquared test was used. The statistical software Statistical Product and Service Solutions (SPSS; IBM SPSS Statistics), version 23.0 (IBM Corporation, Armonk, NY, USA), was used for the analysis. Statistical significance was set at a p-value of less than 0.05.

Discussion
Patients with IgAN display various clinical manifestations and pathological phenotypes. Approximately 5%-10% of patients with IgAN exhibit onset nephrotic syndrome (5,20), with histologic features of mild mesangial lesions, by light microscopy, and diffuse foot process effacement of podocytes, by electron microscopy, representative of MCD, which is defined as MCD-IgAN. The specific pathogenesis of MCD-IgAN is still not entirely clear (3,9). In this study, we found not only that the clinical and histologic features of MCD-IgAN were different from those of classic IgAN, but also that the underlying mechanisms may differ from IgAN.
We found that the clinical manifestation of MCD-IgAN greatly differed from that of IgAN, as previous reported (21). IgAN usually presented with recurrent episodes of gross hematuria after mucosal infections, and with subnephrotic proteinuria. Conversely, MCD-IgAN usually presented with nephrotic proteinuria and hypoproteinemia, and seldom hematuria, which more closely resembles MCD (21)(22)(23)(24)(25). Previous studies also revealed that there are no significant differences in clinical manifestation and prognosis between MCD-IgAN and MCD (22). In terms of therapy, patients with MCD-IgAN presented better renal outcomes than patients with non-MCD-IgAN (21). Patients with MCD-IgAN usually achieve a comparable clinical outcome to those with MCD, requiring corticosteroid and immunosuppressive medication in addition to the treatments used for MCD (23). However, some studies have reported that patients with MCD-IgAN respond well and safely to corticosteroid therapy (24,25), MCD-IgAN experience a higher recurrence rate than non-MCD-IgAN. (25) IgAN and MCD may also coexist in children, and most of these patients who presented with nephrotic syndrome responded well to corticosteroids and had a satisfying prognosis (26). The pathophysiology of IgAN is considered to be a multi-hit mechanism (9). Gd-IgA1 is the key component in the pathogenesis of IgAN (10). In this study, we found that, compared with non-MCD-IgAN patients, MCD-IgAN patients presented with lower levels of serum Gd-IgA1, which was consistent with previous research (6). However, the levels of Gd-IgA1 were much higher in patients with MCD-IgAN than in healthy control participants. Furthermore, unlike previous studies (6,22), we found that Gd-IgA1 was rarely deposited in the glomerulus of patients with MCD-IgAN. Gd-IgA1 was deposited in the glomerulus in only 13.7% of patients with MCD-IgAN, compared with 82.4% of patients with non-MCD-IgAN. Our study suggested that most IgA deposited in this variant form of IgAN was normal IgA but not Gd-IgA1. A previous study showed that, compared with poly-IgA1 (pIgA1) complexes from non-MCD-IgAN, pIgA1 complexes from MCD-IgAN could induce weaker effects on mesangial inflammatory cytokine production (6). These findings may suggest that the IgA deposited in patients with MCD-IgAN was not as pathophysiological as that deposited in patients in IgAN. The presence of incidental IgA deposition in donor kidneys (from individuals with no known underlying kidney disease) appears to be quite high in Asian countries, particularly in China and Japan (27,28). Unfortunately, we do not have information about the incidence of glomerular IgA depositions in donor kidney biopsies in our study hospital.
Interestingly, we found that the activation of the complement system was more severe in non-MCD-IgAN patients than in MCD-IgAN patients. Compared with patients with MCD-IgAN, plasma levels of C3 and C4 were significantly lower in patients with non-MCD-IgAN. Moreover, the intensity of C3c deposition in MCD-IgAN patients was weaker than in non-MCD-IgAN patients. Increasing evidence has implied that alternative pathway-and lectin pathway-induced complement activation has an important role in the pathogenesis of IgAN (15,29,30). In three different pathways. These three pathways converge at the C3 level and lead to the formation of C3 convertase, inducing the cleavage of C3, and then inducing the activation of the common terminal pathway and triggering the formation of C5b-9 (15). In patients with IgAN, the activation of the complement system occurs both in the systemic circulation and in the renal locality. The intensity of renal local C3c deposition and the level of plasma C3 could represent the degree of complement activation. Overactivation of systemic complement results in C3 consumption and, thus, low plasma C3 levels, and renal local complement activation leads to greater C3c deposition in glomeruli (31). A previous study showed that lower levels of C3 and stronger intensity of C3 deposition in glomeruli led to poor renal outcomes in patients with IgAN (32). In this study, we found that the activation of the complement system, both in the circulation and in the renal locality, was much weaker in patients with MCD-IgAN than in those with non-MCD-IgAN.
In conclusion, our study suggests that most IgA that was deposited in MCD-IgAN was not as pathogenic as the Gd-IgA1deposited in IgAN. Complement activation in both the systemic circulation and the renal locality was much weaker in patients with MCD-IgAN than in those with non-MCD-IgAN. Our study suggests that IgAN with MCD might be MCD with coincidental IgA deposition. We hope that all these findings might aid a better understanding of and provide a theoretical basis for intervention in MCD-IgAN in the future.

Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics statement
The studies involving human participants were reviewed and approved by the ethics committees of Beijing Anzhen Hospital. The patients/participants provided their written informed consent to participate in this study.

Author contributions
W-yG and HC made substantial contributions to the study concept and design. W-yG drafted the manuscript. HC critically revised the manuscript, supervised the entire study, and gave final approval to the article. W-yG and H-rD performed the immunofluorescence in renal biopsies. W-yG performed the ELISAs. The renal biopsies were reviewed and the staining intensity from anonymized sections were graded by W-yG and L-jS independently. W-yG and NY conducted statistical analyses. X-yX, G-qW, Z-rZ, W-rC, and YL collected the primary data and treated all the patients. All authors contributed to the article and approved the submitted version.