, Introduction
Henoch-Schönlein purpura (HSP) is a type of systemic vasculitis mediated by immune complexes, also known as immunoglobulin A vasculitis (IgAV). This disease can affect the skin, joints, digestive tract, and kidneys. When the kidneys are involved, the disease is also called IgAV nephritis (formerly known as HSP nephritis) [1] and kidney involvement is a crucial factor in determining the long-term prognoses of HSP patients. In fact, it accounts for 2.5% to 25% of all cases of end-stage kidney disease (ESKD) in children [2]. The typical kidney histopathological immunofluorescence features involve diffuse granular deposition of IgA and C3 in the mesangium, occasionally accompanied by deposits of other proteins such as IgG, IgM, and fibrin. Despite this, the clinical significance of IgM deposition in children with IgAV nephritis is still uncertain. Since IgM deposits can exacerbate glomerular injury by activating complement [3,4], we speculate that IgM deposition might play a significant role in the development of the disease in children with IgAV nephritis. However, to date, no reports have investigated IgM deposition in the glomeruli of IgAV nephritis patients.
Therefore, in the current study, we examined whether IgM deposition in children with IgAV nephritis is an indicator that not only reflects the severity and pathological damage of the disease but also predicts the disease prognosis. This new finding would enable improved management and kidney outcomes for children with IgAV nephritis.
Methods
Patients
The patients included in this study met the diagnostic criteria for IgAV nephritis as the European League Against Rheumatism/Paediatric Rheumatology International Trials Organization/Paediatric Rheumatology European Society (EULAR/PRINTO/PRES) [5]. These guidelines were released by the Pediatric Society of the Chinese Medical Association. Medical records and follow-up data were obtained from both inpatients and outpatients through the hospitalization and outpatient follow-up systems. Participants were selected according to the following criteria: 1) age ≤16 years; 2) follow-up duration ≥6 months; 3) presence of >10 glomeruli observed under light microscopy; 4) exclusion of secondary nephritis, such as hepatitis B virus-associated nephritis, lupus nephritis, antineutrophil cytoplasmic antibody-associated vasculitis, and other secondary nephritis; 5) exclusion of children with congenital and hereditary kidney diseases. Patients were classified into positive and negative groups based on the presence or absence of positively stained IgM in the immunofluorescence of kidney glomeruli.
This study was approved by the Ethics Committee of Jinling Hospital (No. 2022JLHGKJDWLS-098).
Clinical data
The study collected basic clinical and laboratory data of children who underwent kidney biopsies. The data included age of onset, sex, symptoms experienced during the initial onset such as the presence of joint swelling, hematuria (with or without macroscopic hematuria), abdominal pain (with or without gastrointestinal bleeding), purpura (whether it is necrotic), 24-hour urinary protein quantification, estimated glomerular filtration rate (eGFR), immunoglobulin (IgA, IgM, IgG), kidney tubular function, and biochemical markers. Hypertension was defined as systolic blood pressure and/or diastolic blood pressure ≥the 95th percentile of the blood pressure of children of the same age, sex, and height for three or more different time measurements during hospitalization and follow-up [6]. The eGFR was calculated using the updated Schwartz formula; if the age was over 16 years old, the CKD-EPI (Chronic Kidney Disease Epidemiology Collaboration) equation was used [7].
Kidney histopathology
Indications for renal biopsy were persistent proteinuria and/or elevated serum creatinine levels. At first, we examined the kidney tissues of all selected children using a light microscope, immunofluorescence, and electron microscope. Immunofluorescence staining for IgG, IgA, IgM, C1q, C3, and fibrin was conducted directly on frozen sections. The amount of immune complexes was evaluated using a fluorescence microscope and graded semiquantitatively as trace, 1(+), 2(+), and 3(+) in increasing order. Negative results for both low-power and high-power microscopy were reported as “–”; negative results for low-power microscopy and possible results for high-power microscopy were shown as “+/–”. For samples where possible results for low-power microscopy and clear results for high-power microscopy were observed, the results were shown as “+”; and for cases of clear results for low-power microscopy and obvious results for high-power microscopy, the results were presented as “++”. In situations where clear results were obtained for both low-power and high-power microscopy, the outcomes were displayed as “+++”, and for cases where dazzling results for low-power microscopy and visible fluorescent results for high-power microscopy were seen, results were shown as “++++”. The immunofluorescence intensity was recorded by semiquantitative method, – or +/–, +, ++, +++ represent the fluorescence intensity from negative to the strongest, recorded as 0, 1, 2, 3. Patients were grouped according to degree of IgM deposition (grade A, – or +/–; grade B, +; grade C, ++; grade D, +++/++++). The grading system used for kidney pathology classification was based on Oxford-MESTC scoring criteria; M, mesangial hypercellularity; E, endocapillary proliferation; S, glomerular sclerosis; T, tubolointerstitial fibrosis; C, crescents [8]. Additional pathological features were evaluated, including fibrinoid necrosis of vascular loops, glomeruli adhesion, and infiltration of inflammatory cells in kidney interstitium. The pathology slides were independently reviewed by two pathologists, and any disagreements were discussed before the final diagnosis.
Treatment and follow-up
All pediatric patients were treated according to the EULAR/PRINTO/PRES [5]. Basic therapy includes oral angiotensin-converting enzyme inhibitors (ACEI) or angiotensin receptor blockers (ARB), as well as prednisone, starting at 1.5 to 2 mg/kg per day and gradually reducing the dosage after 4 weeks of daily use, or using equivalent doses of methylprednisolone for at least 6 months. In addition to using the steroids, immunosuppressive agents such as tacrolimus and mycophenolate mofetil, as well as high-dose steroid/cyclophosphamide pulse therapy, were also used in some patients for at least 6 months. The follow-up deadline was June 30, 2023, and all patients were followed up at least once every 3 months after being diagnosed with IgAV nephritis by kidney biopsy. The follow-up included routine blood tests, liver and kidney function tests, 24-hour urine protein quantification, eGFR and kidney tubular function indicators (urinary retinol-binding protein [URBP], urinary N-acetyl-β-D-glucosaminidase [NAG]). Acute kidney injury (AKI) diagnosis meets the 2012 diagnostic criteria of KDIGO (Kidney Disease: Improving Global Outcomes) [9]. The endpoint events were defined as doubling of serum creatinine and/or a reduction in eGFR of at least 50%, and/or the occurrence of ESKD (eGFR <15 mL/min/1.73 m2 or the requirement for kidney replacement therapy).
Statistical analysis
All statistical analyses were conducted using R version 4.3.0 software (R Foundation for Statistical Computing). Normally-distributed quantitative data were presented as mean ± standard deviation (x ± s), while nonnormally-distributed quantitative data were expressed as median (interquartile range). Continuous variables were evaluated using analysis of variance or the Kruskal-Wallis H test. Qualitative data were expressed as frequency (n) or proportion (%), with between-group comparisons conducted using either chi-square or Fisher exact tests. Correlation analysis used Spearman rank correlation analysis. Kidney survival time for three groups was assessed using the Kaplan-Meier method. Relevant factors were screened through univariable analysis, with variables showing statistical significance included in the Cox proportional hazard model. Additionally, a multivariable Cox proportional hazard regression model was used to analyze the prognostic impact of the degree of IgM deposits in children with IgAV nephritis. Proportional hazard assumptions were assessed visually by inspection of Schoenfeld plots, log-log plots, and Schoenfield residual tests. It was found that all the proportional hazard assumptions were satisfied. The GraphPad Prism 8.0 software was used to generate the receiver operating characteristic (ROC) curve and predict the diagnostic value of IgM deposition in IgAV nephritis. The area under the ROC curve (AUC) ranges from 0.5 to 1, 0.5 to 0.7 is a low diagnostic value, 0.7 to 0.9 is a medium diagnostic value, and greater than 0.9 is a high diagnostic value. A p-value of less than 0.05 was deemed statistically significant.
Results
Patients’ demographic and clinical characteristics
A total of 359 children with biopsy-proven IgAV nephritis were enrolled in this study. Among these, 228 cases were male (63.5%), and the mean age of onset was 7.61 ± 0.59 years. During the follow-up period, endpoint events were observed in 39 cases (10.9%), while 155 cases had IgM deposition in the kidney glomeruli (Table 1).
Serum IgM, serum creatinine, blood urea nitrogen, URBP, NAG, and urine protein quantification were found to be higher in the group with elevated levels of IgM deposition. These patients also had low C3 levels, gastrointestinal bleeding, and AKI, and patients who reached primary endpoint at a higher proportion. Furthermore, their serum albumin and eGFR levels were lower. It is worth noting that patients with IgM deposits received more aggressive treatment, including early pulse therapy with cyclophosphamide and methylprednisolone (all p < 0.05) (Table 1).
Kidney histopathology data
Patients with a higher level of IgM deposits presented with higher proportions of balloon adhesions and S1 lesions and higher IgA, IgG, and C3 deposition intensity (all p < 0.05) (Table 2).
Correlation analysis of IgM deposition intensity and IgA, IgG, C3 deposition intensity in children with IgAV nephritis
Spearman rank correlation analysis of the correlation between the deposition intensity of IgA, IgG, and C3 and the deposition intensity of IgM showed that the deposition intensity of IgA, IgG, and C3 in children with IgAV nephritis was positively correlated with the deposition intensity of IgM (r = 0.410, p < 0.05; r = 0.478, p < 0.05; r = 0.372, p < 0.05).
IgM deposition and kidney outcomes
During the follow-up period from kidney biopsy, the median follow-up time was 45 months (34–56 months). A total of 39 patients (10.9%) entered the endpoint event. Notably, children with higher glomerular IgM deposition had a higher incidence of endpoint events (Table 1). Kaplan-Meier analysis showed that cumulative renal survival was significantly lower in children with higher glomerular IgM deposition (log-rank test chi-square = 55.341, p < 0.001), as shown in Fig. 1.
To investigate further the correlation between IgM deposition and the prognosis of IgAV nephritis, we included glomerular IgM deposition and other variables in univariable and multivariable Cox regression analysis and concluded that gastrointestinal bleeding (hazard ratio [HR], 27.67; 95% confidence interval [CI], 6.36–120.44; p = 0.001), serum uric acid (HR, 6.53; 95% CI, 2.09–20.41; p = 0.003), eGFR (HR, 0.90; 95% CI, 0.84–0.96; p = 0.002), T0/T1 (HR, 3.83; 95% CI, 1.47–9.94; p = 0.006), the degree of glomerular IgM deposits (grade C + D: HR, 2.78; 95% CI, 1.15–3.24; p = 0.02; grade B: HR, 2.69; 95% CI, 1.96–3.99; p = 0.03), C1/C2 (HR, 1.20; 95% CI, 1.10–1.31; p = 0.001), and S1 (HR, 3.59; 95% CI, 1.51–8.53; p = 0.004) were associated with endpoint events. The significant factors with p-values less than 0.05 from the univariable analysis were integrated into the multivariable Cox regression model. Analysis findings demonstrate that the degree of glomerular IgM deposits (grade C + D: HR, 2.04; 95% CI, 1.67–3.93; p = 0.04; grade B: HR, 2.59; 95% CI, 1.08–4.23; p = 0.03) and S1 (HR, 1.76; 95% CI, 0.42–2.98; p = 0.03) are the sole independent risk factors associated with negative renal prognosis (Table 3).
The ROC curve analysis results showed that the AUC of IgM deposition predicting poor kidney prognosis was 0.603 (95% CI, 0.51–0.70), with a sensitivity of 0.615 and a specificity of 0.591, as shown in Fig. 2.
Discussion
In this study, we assessed the relationship of the IgM deposition with the clinical characteristics and kidney survival of 359 children with biopsy-proven IgAV nephritis. The positive rate of IgM was 44.9%, which was similar to previous reports [10]. However, this rate was slightly higher than that reported in foreign literature, which may be related to racial differences [3]. In addition, the positive rate of IgM detection may be affected by different indications for kidney biopsy in different medical institutions. Levels of urinary protein quantification, serum creatinine, blood urea nitrogen, URBP, urinary NAG enzyme, and serum IgM were elevated in children with IgM deposition compared to those without. This was accompanied by increased rates of gastrointestinal bleeding, AKI, and occurrence of endpoint events. Moreover, this study incorporated the latest Oxford classification criteria for IgAV nephritis, which included fibrinoid necrosis, balloon adhesions, and interstitial inflammation in the evaluation system, so as to comprehensively evaluate the pathological condition of children and obtain more comprehensive pathological information. The results showed that the proportion of balloon adhesions and S1 lesions was higher in the IgM-positive group than in the IgM-negative group. Therefore, it can be considered that the clinical symptoms and kidney pathology of children with IgM deposition are more severe, which can be used to evaluate the degree of disease severity. The results of Cox regression analysis indicated that the deposition of IgM in the glomerulus was an independent risk factor for poor prognosis in children with IgAV nephritis. We recommend closely monitoring pediatric patients with IgM deposition and providing active treatment.
IgAV nephritis is distinguished by prevalent IgA deposition in the mesangial region, typically partnered with C3 deposition and occasionally by the deposition of IgM or IgG [11]. Presently, no evidence indicates that IgM deposition affects the prognosis of young patients with IgAV nephritis, however, previous studies have discovered IgM deposition in other glomerular ailments. For example, a study by Wang et al. [4] uncovered IgM deposition in the glomeruli of lupus nephritis contributing to complement activation and kidney injury. Deposition of IgM in focal segmental glomerulosclerosis is linked to more severe glomerular damage, proteinuria, and poor patient outcomes [12], signifying that IgM deposition can cause clinical manifestations and pathological kidney damage. While there is presently no research on the association between IgM deposition and IgAV nephritis in children, IgA nephropathy is the most relevant disease for this study because IgAV nephritis and IgA nephropathy possess very similar pathological alterations [13]. The study by Tan et al. [3] revealed that mesangial IgM deposition correlates with pathological manifestations, clinical severity, and kidney outcome, and is an independent risk factor for poor prognosis in patients with IgA nephropathy. Furthermore, Heybeli et al. [14] have demonstrated that mesangial IgM deposition is closely associated with kidney prognosis, and patients in this group have higher S1 scores. The study found that the proportion of S1 was significantly higher in the IgM-positive group than in the IgM-negative group, and IgM deposition was an independent risk factor for poor prognosis in children with IgAV nephritis, suggesting that IgM deposition may be responsible for the occurrence of S1. However, this study failed to establish a conclusive relationship between poor kidney prognosis and M1, E1, fibrinoid necrosis, balloon adhesions, interstitial inflammation cell infiltration, and C1/C2. These indicators typically arise during active pathological changes and are accompanied by severe clinical symptoms such as gastrointestinal bleeding and massive proteinuria. The alleviation of these symptoms may be linked to timely and effective pharmaceutical intervention in the IgM deposition group, thereby aiding the restoration of these indicators [15].
The role of IgM deposition in the glomerulus has not yet been fully elucidated. IgM is the earliest antibody synthesized and secreted during the immune response, and the immune complexes formed with specific antigens are large and insoluble macromolecules that can easily accumulate in the mesangial region of the glomerulus. This is consistent with what we observed under immunofluorescence and electron microscopy. In addition, deposition of C3 in the mesangial region of the glomerulus in some patients often accompanies the deposition of immune complexes and may activate complement [16], thereby causing kidney injury. Previous studies have also shown that skin and mesangial deposits in IgAV nephritis contain complement components C3 and C5b–C9, and rarely C1q deposits [17], so it is not strongly associated with the classical pathway of the complement. As we observed in this study, complement deposition in the mesangium of all children was mainly C3, and the proportion of C1q deposition was relatively small. Therefore, the alternative and lectin pathways are the primary pathogenic factors in IgAV nephritis. Furthermore, damage caused by such complement pathways may also affect the function of complement regulatory proteins, leading to enhanced alternative pathway activation, which in turn increases susceptibility to kidney dysfunction [18,19]. On the other hand, when the body is exposed to antigens, initial production of IgM antibodies takes place, which is then transfigured into IgG with the assistance of T cells. Children with IgAV nephritis cannot convert a significant amount of IgM to IgG due to alterations in T cell function. As a result, this causes the IgM to remain in the IgM stage [20], leading to an unusual rise in IgM levels in the serum, which aligns with the findings of our experimental study. Matsumoto et al. [21] discovered the presence of an antibody called anti-Gd-IgA1 IgM in the blood of IgA nephropathy patients, which forms a complex with complement C3. These findings may explain the simultaneous deposition of IgA and IgM in the renal glomeruli of IgA nephropathy patients. Nihei et al. [22] found the accumulation of a substance called apoptosis inhibitor of macrophage (AIM) in the renal glomeruli of gddY mice and IgA nephropathy patients. However, when recombinant AIM was administered to AIM-deficient gddY mice, it was found that not only IgA deposition occurred, but also IgG, IgM, and C3 deposition, leading to proteinuria [22]. These study results suggest that the formation of immune complexes may involve the function of AIM in IgA nephropathy. AIM functions by binding IgA in the renal glomeruli with other immunoglobulins and C3, but the specific mechanism is unclear. Therefore, we hypothesized that binding of glomerular IgM to injury-associated epitopes activates the complement system through the alternative or lectin pathway leading to the formation of the membrane attack complex (C5b–C9), thereby inducing long-term and more severe glomerulonephritis. Consistent with this hypothesis, we observed a correlation between the intensity of glomerular IgM deposition and the intensity of IgG, C3, and IgA deposition, as well as severe clinical manifestations. Additionally, we identified a higher proportion of lower serum C3 levels in the group with higher IgM deposition intensity compared with the IgM-negative group, presumably related to the higher intensity of complement C3 deposition in the higher IgM deposition group. Previous studies have also revealed that IgM deposition is associated with balloon adhesions and glomerulosclerosis, which is analogous to our findings that the IgM-positive group had a higher proportion of balloon adhesions and glomerulosclerosis in comparison to the IgM-negative group. This problem can be elucidated by the increased capture of IgM molecules in the sclerosis area or the physiological effect of IgM on healing damaged tissues, although its specific pathogenic mechanism needs to be further deciphered through molecular experiments [14].
The impact of IgM deposition on the prognosis of patients with kidney disease remains controversial. Nieuwhof et al. [23] discovered that IgM deposition in glomerular mesangial areas is associated with both hypertension and disease progression. Subsequent studies have reported that IgM deposition in the mesangial area of the kidney is an indicator of disease progression. Some researchers suggest reducing IgM antibody production to mitigate disease worsening [24]. Ju et al. [25] analyzed 283 children with MCD and identified IgM deposition as an independent risk factor for disease remission and prognosis. Conversely, other studies hold contradictory opinions, asserting that IgM deposition in kidney disease lacks predictive value for disease progression. Moriyama et al. [26] found that IgM deposition was related to glomerulosclerosis and tubular adhesion but not an independent risk factor for kidney function progression. Sun et al. [27] observed that IgM deposition impacts kidney survival in IgA nephropathy but does not independently influence kidney function progression. This study’s findings demonstrate that IgM deposition is an independent risk factor for poor prognosis in children with IgAV nephritis. Additionally, the results of the ROC curve analysis suggest that IgM deposition possesses predictive value for adverse kidney prognosis. However, this study’s findings indicate that T is not a risk factor for poor kidney outcomes, potentially related to the lower incidence of T in children with IgAV nephritis. Only 3.3% of patients in this study exhibited tubular atrophy/interstitial fibrosis, and no T2 patients were observed. In contrast, the proportion of T1/T2 lesions in adults with IgAV nephritis ranged from 11% to 54.1% [28]. The Oxford classification’s methodology primarily relies on data from adult patients with IgA nephropathy. Despite similarities with IgAV nephritis, distinctions in clinical manifestations, disease course, and kidney injury mechanisms exist. No statistical significance was observed for eGFR, hypertension, or urinary protein quantification in our study. This could be attributed to most participating children receiving ACEI/ARB treatment and combined steroid-immunosuppressive drugs, which can help control blood pressure, proteinuria, and improve kidney function.
This study had several limitations. Being a single-center, retrospective study, the conclusions drawn are restricted. It is important to acknowledge that the sample size of events is limited, and the average duration of follow-up is relatively brief. Furthermore, variations in the treatment process among patients and the necessity for multiple changes in strategy due to poor efficacy may introduce bias in result analysis. Additionally, as the first report elucidating the clinical significance of IgM deposits in pediatric patients with IgAV nephritis, these findings may not extend to Caucasian or African-American patients or adult patients with IgAV nephritis. Moreover, future multicenter studies with larger sample sizes are required to substantiate these findings.
There are differences in the clinicopathological features of IgAV nephritis with different degrees of mesangial IgM deposition, and kidney glomerular IgM deposition and S1 are independent risk factors for poor prognoses of IgAV nephritis in children. Moreover, future research should focus on understanding the specific mechanisms of IgM deposition and complement activation in the pathogenesis of IgAV nephritis, which could potentially serve as therapeutic targets for children with IgA nephropathy, ultimately significantly improving their prognoses.
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