Glomerular Galactose‐Deficient IgA1 Deposition in 192 Autopsy Cases in Japan

ABSTRACT

Aim

Glomerular IgA deposition is incidentally observed in asymptomatic individuals; however, it remains unclear whether these cases represent a preclinical stage of IgA nephropathy (IgAN). We aimed to address this question by staining kidney samples from autopsy cases with the anti‐galactose‐deficient IgA1 (Gd‐IgA1: the key effector molecule in the pathogenesis of IgAN) antibody, KM55.

Methods

Kidney samples from 192 autopsy cases in Japan were stained with anti‐IgA antibody and KM55. The deposition of complement C3, IgG and IgM in glomeruli, as well as the histological findings and clinical data were analysed in cases positive for glomerular IgA.

Results

Glomerular IgA deposition was detected in 13 cases (6.8%), all of which were stained with KM55. Of 10 cases with available urinary data prior to admission, six did not have abnormal urinary findings. 7 of 13 were positive for glomerular Gd‐IgA1 alone without obvious histological changes. Glomerular IgG and IgM were detected in 1 of 13 and 5 of 13 cases, respectively. 2 of 13 were positive for glomerular C3, with one showing mesangial proliferation.

Conclusion

Our results indicate that asymptomatic cases with glomerular IgA deposition may frequently include subclinical IgAN, characterised by glomerular deposition of Gd‐IgA1 (KM55‐positive) without prominent histopathological changes or obvious clinical symptoms.

Glomerular Galactose‐Deficient IgA1 Deposition in 192 Autopsy Cases in Japan.

Summary at a Glance.

• Glomerular IgA and galactose‐deficient IgA1(Gd‐IgA1) deposition was investigated in 192 autopsy cases in Japan.

• Glomerular IgA deposition was noted in 13 cases (6.8%), all of which were stained with the anti‐Gd‐IgA1 antibody.

• Our results suggest that asymptomatic cases with glomerular IgA deposition may frequently include subclinical IgAN.

1. Introduction

IgA nephropathy (IgAN) is the most common form of primary glomerulonephritis worldwide, characterised by the deposition of IgA, often with IgG and/or IgM, and complement C3 in the glomerular mesangial region [1]. As the pathogenesis of IgAN remains unclear and no curative treatment has been established, ~30% to 50% of cases reach kidney failure within 10 to 20 years from diagnosis [2, 3].

Glomerular IgA in patients with IgAN lacks galactose in the O‐glycosylated sites in its hinge region (referred as galactose‐deficient IgA1: Gd‐IgA1) [4, 5]. Gd‐IgA1 has been reported to be increased in the sera of IgAN patients [6, 7], and considered to be the key effector molecule in the pathogenesis of IgAN [1]. Previously, we developed a monoclonal antibody against Gd‐IgA1, KM55 [8], and demonstrated that glomerular Gd‐IgA1 was specifically observed in patients with IgAN. Therefore, KM55 is used worldwide to diagnose IgAN [9, 10, 11, 12].

Glomerular IgA deposition without abnormal urinary findings is observed in the general population. Waldherr et al. investigated the frequency of glomerular IgA deposition in the 250 autopsy cases without a history of kidney disease and reported that IgA deposition was observed in 12 of 250 cases (5%) [13]. Suzuki et al. analysed the frequency of glomerular IgA deposition in renal transplant donors and reported that 82 of 520 donors (16%) had IgA deposition in the glomerulus [5]. Thus, asymptomatic cases with glomerular IgA deposition are relatively common. However, it remains unclear whether such cases represent a preclinical stage of IgAN, characterised by glomerular deposition of Gd‐IgA1 (KM55‐positive) without prominent histopathological changes or obvious clinical symptoms (subclinical IgAN). To address this question, herein, we stained kidney samples from 192 autopsy cases using KM55.

2. Methods

2.1. Study Design and Patients

In this retrospective cohort study conducted from January 1, 2016 to March 31, 2023, cases of 387 autopsies performed at Juntendo Hospital were examined. Autopsies were performed with the consent of the patient's family and at the decision of individual clinicians. The exclusion criteria were patients (i) < 18 years old, (ii) without kidney samples, (iii) with missing clinical and laboratory data (no examinations within 1 year prior to the death‐related admission), (iv) with an eGFR of < 15 mL/min/1.73 m² or on renal replacement therapy prior to death, and (v) use of immunosuppressant therapy other than prednisolone within 3 months prior to death. Clinical and laboratory data were obtained at the time of death‐related admission or at the last outpatient visit preceding admission. This study was approved by the Ethics Review Board of the Juntendo University Faculty of Medicine (E23‐0302) under the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

2.2. Clinical Measurements

For each case, the following data were obtained: demographic characteristics, medical history (hypertension and diabetes), and medications [immunosuppressive drugs excluding steroids, renin–angiotensin system (RAS) inhibitors, anticancer drug and molecularly targeted therapy]. eGFR was calculated using the following formula: eGFR (mL/min per 1.73 m²) = 194 × creatinine−1.094 × age−0.287 (×0.739 for women). Hematuria and proteinuria were assessed by dipstick urine tests. Body mass index was calculated as body weight (kg) divided by height in metres squared (m²).

2.3. Immunofluorescence (IF) Microscopy and Histological Analyses

In this study, frozen sections were not available in all specimens; therefore, formalin‐fixed and paraffin‐embedded sections were used. IF staining of Gd‐IgA1 and IgA in the glomeruli was performed as described [14]. Kidney samples embedded in paraffin were cut at 4‐μm sections. For glomerular IgG, IgM, and C3 staining, enzymatic antigen retrieval for IgG and IgM or heat‐induced antigen retrieval for C3 were performed after deparaffinising using a series of xylene/ethanol and rehydration steps. The samples were then rinsed with distilled water and blocked with Protein Block (Dako Japan, Tokyo, Japan) at room temperature for 30 min and stained with FITC‐conjugated rabbit anti‐IgG antibody (F0185, Dako Japan, Tokyo, Japan) diluted 1:200 or FITC‐conjugated rabbit anti‐IgM antibody (F0058, Dako Japan, Tokyo, Japan) diluted 1:50 for 1 h. For C3 staining, the samples were stained with rabbit anti‐C3 antibody (ab97462, Abcam, Cambridge, UK) diluted 1:100 as primary antibody for 1 h and with FITC‐conjugated goat anti‐rabbit IgG antibody (Life Technologies, CA) diluted 1:1000 as second antibody for 30 min. After washing with PBS/TBST, the slides were sealed using Fluoromount (Diagnostic BioSystems, CA, USA). All samples were examined using an IF microscope (BZ‐X710; KEYENCE). Glomerular Gd‐IgA1, IgA, IgG, IgM and C3 levels were determined as positive or negative by two independent nephrologists. Positive results were defined as when more than half of the glomeruli observed were stained. For histological evaluation, sections were stained with periodic acid‐Schiff stain.

2.4. Statistical Analyses

Continuous variables are presented as medians and interquartile ranges or means and standard deviations, as appropriate. Nonparametric continuous variables were compared using the Mann–Whitney U and Kruskal–Wallis tests. Categorical variables are presented as percentages and compared using Pearson's chi‐squared and Fisher's exact tests. All statistical analyses were performed using Stata/MP version 17.0 (StataCorp, College Station, TX, USA). Statistical significance was set at p < 0.05.

3. Results

3.1. Incidence of Glomerular IgA and Gd‐IgA1 Deposition in Autopsy Cases

Of the 387 autopsy cases eligible for study inclusion, 195 met the exclusion criteria. Kidney samples from the remaining 192 cases were stained with anti‐IgA antibody and KM55 (Figure 1). Table 1 shows the characteristics of the included cases. Glomerular IgA deposition was detected in 13 cases (6.8%), all of which were stained with KM55 (Figures 2 and S1). There were no cases positive for KM‐55 but negative for IgA.

FIGURE 1.

Flowchart of the study design. Overall, 387 autopsy specimens performed at Juntendo Hospital were examined. The exclusion criteria were cases (i) < 18 years old, (ii) without kidney samples, (iii) with missing clinical and laboratory data, (iv) with an eGFR of < 15 mL/min/1.73 m² or on renal replacement therapy prior to death, and (v) use of immunosuppressant therapy other than prednisolone within 3 months prior to death. In total, 192 cases that were not excluded underwent further analysis.

TABLE 1.

Clinical characteristics of the 192 autopsy cases.

	n = 192
Age, year (range)	67.1 (24–93)
Male sex (%, [n])	60.4 [116]
BMI (kg/m2)	20.5 ± 4.2
Time from death to autopsy (min)	711.0 [214.8–1020.3]
Medication at baseline
RAS‐I (%, [n])	13.0 [25]
Corticosteroid (%, [n])	30.7 [59]
Anticancer drug or molecularly targeted therapy (%, [n])	26.6 [51]
Past history
Hypertension (%, [n])	26.6 [51]
Diabetes (%, [n])	21.6 [42]
Renal function
BUN (mg/dL)	18.0 [13.0–26.0]
s‐Cr (mg/dL)	0.74 [0.59–0.99]
eGFR (mL/min/1.73 m2)	72.1 [53.9–92.0]
CKD G1 (%, [n])	26.0 [50]
CKD G2 (%, [n])	42.7 [82]
CKD G3a (%, [n])	13.5 [26]
CKD G3b (%, [n])	12 [23]
CKD G4 (%, [n])	5.7 [11]

Note: Values for continuous variables are presented as medians (interquartile ranges).

Abbreviations: BUN, blood urea nitrogen; eGFR, estimated glomerular filtration rate; RAS‐I, renin–angiotensin system inhibitors; s‐Cr, serum creatinine.

FIGURE 2.

IF study showing glomerular IgA and Gd‐IgA1 deposition. Kidney samples from the 192 cases were stained with anti‐IgA antibody (green) and KM55 (red). The merged images are shown in yellow. Shown are representative images of the case with (lower panel, Case 12; Patient ID is the same as those listed in Table 2) or without (upper panel) glomerular IgA and Gd‐IgA1 deposition.

3.2. Clinical Characteristics of Autopsy Cases With Glomerular Gd‐IgA1 Deposition

Clinical characteristics of 13 glomerular IgA‐positive and 179 glomerular IgA‐negative cases were analysed (Tables 2 and S1). No obvious differences were found in age, sex, medical history, presence of diabetes and hypertension, and renal function on admission. Although cancer was the most common underlying disease in 13 glomerular IgA‐positive cases, there was no significant difference in cancer incidence between glomerular IgA‐positive and negative patients (p = 0.761). Glomerular IgA deposition is frequently observed in patients with liver disease [15]; however, no case had a history of liver disease, except Case 8 who had a history of primary biliary cirrhosis in this study. Of the 10 cases with available urinary data prior to admission, 6 had no abnormal urinary findings. None of the 10 cases had received immunosuppressive drugs, including steroids, at the time the urine test was performed.

TABLE 2.

Clinicopathological findings in the 13 cases with latent glomerular Gd‐IgA1 deposition.

Case	Age	Sex	Clinical diagnosis	PH		s‐Cr (mg/dL)	Urinalysis		IF
				H	D		UP	OB	IgA	Gd‐IgA1	C3	IgG	IgM
1	49	F	Pancreatic cancer	—	—	0.30	—	—	+	+	—	—	—
2	78	M	Myelofibrosis	—	—	0.61	2+	3+	+	+	+	—	+
3	46	M	Acute myelogenous leukaemia	—	—	2.31	N/A	N/A	+	+	—	—	—
4	77	F	Ovarian cancer	—	—	0.56	1+	1+	+	+	—	—	+
5	85	F	Thalamic haemorrhage	+	+	1.21	N/A	N/A	+	+	—	—	—
6	71	M	Stomach cancer	—	+	0.74	—	—	+	+	—	—	+
7	69	M	Multiple myeloma	—	—	1.25	—	±	+	+	—	—	—
8	44	F	Primary biliary cholangitis	—	—	0.40	—	—	+	+	—	—	—
9	78	F	Hodgkin lymphoma	—	—	0.57	—	2+	+	+	+	—	—
10	88	M	Diffuse large B‐cell lymphoma	—	—	1.41	N/A	N/A	+	+	—	—	—
11	66	M	Hodgkin lymphoma	—	+	0.74	1+	—	+	+	—	—	+
12	49	M	Lung cancer	—	—	1.10	—	—	+	+	—	—	—
13	84	M	Gallbladder cancer	+	—	2.20	—	—	+	+	—	+	+

Abbreviations: D, diabetes mellitus; H, hypertension; IF, glomerular immunofluorescence staining; N/A, not available; OB, occult blood; PH, past history; s‐Cr, serum creatinine; UP, urinary protein.

3.3. Histopathological Analyses of Cases With Glomerular Gd‐IgA1 Deposition

We then performed histopathological analysis of the kidney samples from the 13 cases with glomerular Gd‐IgA1 deposition (Table 2). IF staining of the kidney samples revealed that 2 of 13 were positive for C3, 1 of 13 for IgG, and 5 of 13 for IgM (Figure S2). 7 of 13 were positive for Gd‐IgA1 alone. Pathologically, only Case 9 showed mesangial proliferation (Figure 3).

FIGURE 3.

Light microscopic findings of kidney samples from cases with latent glomerular Gd‐IgA1 deposition. Light microscopic findings of the kidney sample with (right panel, Case 9) or without mesangial proliferation (left panel, Case 1). Yellow arrows indicate mesangial proliferation. Patient ID is the same as those listed in Table 2. Periodic acid‐Schiff staining was performed.

4. Discussion

In this study, we stained kidney samples from 192 autopsy cases in Japan with anti‐IgA antibody and KM55. Glomerular IgA deposition was observed in 13 of 192 cases (6.8%), and all of which were stained with KM55. Of the 10 cases for which urinalysis data were available prior to admission, 6 did not have abnormal urinary findings. Considering that Gd‐IgA1 is the key effector molecule in the pathogenesis of IgAN [16] and is specifically detected in glomeruli from patients with IgAN [14], our results indicate that asymptomatic cases with glomerular IgA deposition may frequently include subclinical IgAN.

Recently, Kuroyanagi et al. evaluated 723 transplant kidney biopsy samples obtained 1 h after transplantation [17] and reported that all cases with glomerular IgA deposition were positive for KM55. Since this report evaluated transplanted kidneys from relatives of patients with IgAN, whether asymptomatic cases with glomerular IgA deposition have IgAN should be evaluated in a population wherein genetic factors have been excluded. Thus, our study compensates for the limitations of Kuroyanagi's report.

Gd‐IgA1 has been thought to form ICs in the circulation to be deposited in glomeruli [16]. However, in this study, 7 of 13 cases were positive for Gd‐IgA1 alone. This suggests that in cases with glomerular IgA deposition, Gd‐IgA1 may be deposited in the glomeruli even without forming ICs. We reported that glomerular IgA deposition preceded IC formation in the circulation in a murine model of IgAN, gddY mice [18]. Apoptosis inhibitor of macrophages (AIM) is a secreted scavenger protein produced by tissue macrophages [19]. We found that AIM colocalized with IgA, IgG and IgM in the glomeruli in both patients with IgAN and gddY mice. In AIM‐deficient gddY mice, deposition of C3, IgG and IgM did not occur, and renal injury improved; however, IgA deposition in the glomeruli persisted. When recombinant AIM was administered to AIM‐deficient gddY mice, C3, IgM and IgG were co‐deposited, and nephritis was reproduced [20]. These results suggest that, at least in gddY mice, serum IgA may be initially deposited in the glomeruli followed by AIM‐mediated ICs formation in situ, resulting in renal injury. Following these findings, we discovered IgA‐type autoantibodies specific to mesangial cells with the properties of Gd‐IgA1 in the serum of patients with IgAN [21, 22, 23]. These data suggest the possibility that the disease onset of IgAN could be Gd‐IgA1 deposition in the glomerulus, although IC formation is necessary for subsequent glomerular injury.

This study has several limitations. First, this study is a single‐centre study in which only 13 Japanese autopsy cases with glomerular IgA deposition were identified among a total of 193 cases examined. Verification in other regions/countries with a larger number of cases is needed. Second, this study is a retrospective/cross‐sectional study using autopsy cases, which introduces a selection bias in that many cases were relatively old and had a history of cancer. IgAN typically presents at a relatively young age, most often during the second and third decades of life [1], thus it is possible that the prevalence of subclinical IgAN is higher among younger individuals than in older populations. Although we could not find the studies which have shown an increased prevalence of incidental glomerular IgA in patients with cancer, such IgA deposition has been reported more frequently in individuals with pulmonary [24] or liver diseases [15]. Further investigation of glomerular KM55 staining in patients with these comorbidities may help elucidate the true prevalence of subclinical IgAN in the general population. Finally, whether in situ or circulating IC formation is more important in the pathogenesis of IgAN, or whether both are important, should be examined in detail in the future. As previous studies have shown that ICs containing Gd‐IgA1 are deposited in the kidney and induce inflammation when administered to mice [25], the present study does not rule out that ICs containing Gd‐IgA1 formed in the circulation are involved in the pathogenesis of IgAN.

In conclusion, our data suggest the existence of subclinical IgAN cases within the general population. As Waldherr previously stated [13], the diagnosed cases of IgAN may represent only the tip of the iceberg. Further investigations are needed to identify which subclinical IgAN cases progress to clinically overt IgAN and to elucidate the mechanisms underlying this progression.

Author Contributions

Research idea and study design: Y.N., R.A. and A.K. Data acquisition: Y.N., R.A., A.K., H.S. and I.S. Data analysis/interpretation: Y.N., R.A., K.M. and A.K. Statistical analysis: Y.N., R.A. and K.M. Writing – original draft: Y.N. Writing – review and editing: M.K., T.Y. and Y.S. Supervision: M.K., T.Y. and Y.S. Each author contributed important intellectual content during manuscript drafting or revision and accepted accountability for the overall work by ensuring that questions pertaining to the accuracy or integrity of any portion of the work were appropriately investigated and resolved.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: IF study showing glomerular IgA and Gd‐IgA1 deposition in autopsy cases. Thirteen autopsy cases positive for IgA and Gd‐IgA1 and one negative for both (negative case) are shown. Images from cases with IgAN are shown (IgAN).

Figure S2: IF study showing glomerular C3, IgG, and IgM deposition in cases positive for glomerular Gd‐IgA1. Kidney samples of 13 cases positive for IgA and Gd‐IgA1were stained with anti‐C3, anti‐IgG, or anti‐IgM antibody.

Table S1: Clinical characteristics of the 13 cases positive for Gd‐IgA1 and the 179 cases negative for Gd‐IgA1. Values for continuous variables are presented as medians [interquartile ranges]. BMI, body mass index; BUN, blood urea nitrogen; s‐Cr, serum creatinine; eGFR, estimated glomerular filtration rate.

Nihei Y., Aoki R., Matsuzaki K., et al., “Glomerular Galactose‐Deficient IgA1 Deposition in 192 Autopsy Cases in Japan,” Nephrology 30, no. 9 (2025): e70128, 10.1111/nep.70128.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.