A cross-sectional analysis of clinicopathologic similarities and differences between Henoch-Schönlein purpura nephritis and IgA nephropathy

Motonori Sugiyama; Yukihiro Wada; Nobuhiro Kanazawa; Shohei Tachibana; Taihei Suzuki; Kei Matsumoto; Masayuki Iyoda; Hirokazu Honda; Takanori Shibata

doi:10.1371/journal.pone.0232194

. 2020 Apr 23;15(4):e0232194. doi: 10.1371/journal.pone.0232194

A cross-sectional analysis of clinicopathologic similarities and differences between Henoch-Schönlein purpura nephritis and IgA nephropathy

Motonori Sugiyama ^1,^#, Yukihiro Wada ^1,^*,^#, Nobuhiro Kanazawa ¹, Shohei Tachibana ¹, Taihei Suzuki ¹, Kei Matsumoto ¹, Masayuki Iyoda ¹, Hirokazu Honda ¹, Takanori Shibata ¹

Editor: Fabio Sallustio²

PMCID: PMC7179927 PMID: 32324811

Abstract

Introduction

Recent studies noted that Henoch-Schönlein purpura nephritis (HSPN) and IgA nephropathy (IgAN) share the feature of galactose-deficient IgA1 (Gd-IgA1)-oriented pathogenesis, although there are distinct clinical differences. We aimed to clarify the clinicopathologic differences between these 2 diseases.

Methods

We cross-sectionally analyzed adult patients with HSPN (n = 24) or IgAN (n = 56) who underwent renal biopsy (RB) between 2008 and 2018 at Showa University Hospital. Serum Gd-IgA1 (s-Gd-IgA1) levels at the time of RB were compared among study groups using enzyme-linked immunosorbent assay (ELISA) with anti-human Gd-IgA1-specific monoclonal antibody (KM55). We also immunohistochemically stained paraffin-embedded sections for glomerular Gd-IgA1 (g-Gd-IgA1)-deposition using KM55. Serum inflammatory cytokines were measured using ELISA.

Results

Glomerular endothelial injury with subendothelial IgA deposition was significant in patients with HSPN. Serum IL-8, MCP-1, TNF-α, and IL-6 levels were significantly higher in patients with HSPN than IgAN. Levels of s-Gd-IgA1 were comparable among patients with HSPN and IgAN, and a similar degree of g-Gd-IgA1-deposition was detected in both diseases. Furthermore, g-Gd-IgA1-deposition was evident in patients with histopathologically advanced HSPN or IgAN. In HSPN, significant positive correlations between s-Gd-IgA1 levels and crescent formation or IL-6 elevation were confirmed, and g-Gd-IgA1 intensity showed a significant positive correlation with MCP-1 and a tendency to positively correlate with IL-8. Meanwhile, patients with IgAN showed no correlation between inflammatory cytokines and both-Gd-IgA1. Moreover, most g-Gd-IgA1-positive areas were not double stained with CD31 in HSPN.

Conclusions

Although assessing both-Gd-IgA1 alone was insufficient to distinguish between HSPN and IgAN, patients with HSPN showed considerable glomerular capillaritis with subendothelial IgA deposition and significant elevation of serum inflammatory cytokines. Furthermore, such glomerular subendothelial IgA deposition might not contain Gd-IgA1, and factors associated with Gd-IgA1 were inconsistent among these 2 diseases. Thus, developmental mechanisms for IgAN might not apply to HSPN completely, and these 2 diseases still have different aspects.

Introduction

Henoch-Schönlein purpura (HSP), recently also called immunoglobulin A vasculitis (IgAV), is a systemic vasculitis characterized by the deposition of IgA immune complexes (IC) in small vessels of the skin and other organs such as the gastrointestinal tract, joints, and kidney [1, 2]. Renal involvement in IgAV, also referred to as Henoch-Schonlein purpura nephritis (HSPN), is the most common and severe complication and a major factor affecting the long-term outcome of patients with HSP [3]. The annual incidence of HSP in children is estimated to be 3–26.7 cases per 100,000 for children and infants, whereas that in adults is only 0.8–1.8 per 100,000 for adults [4–6]. However, HSPN occurs more frequently in adults than children [3, 7, 8]. In addition, the clinical presentation and renal outcome can be more severe in adults than children [9]. Furthermore, the risk for progression to chronic renal insufficiency is much higher in adults at approximately 30% [3, 9, 10], and approximately 10% of adults with HSPN reach end-stage kidney disease (ESKD) within 15 years [3]. Therefore, adult-onset HSPN is characterized by formidable glomerulonephritis (GN) as well as IgA nephropathy (IgAN).

IgAN is the most prevalent type of GN worldwide [11]. Progressive glomerular and interstitial sclerosis in severe IgAN leads to ESKD in 30–40% of patients within 20 years after diagnosis [12, 13]. Histopathologically, IgAN is characterized by mesangial cell proliferation with IgA-IC deposition in the glomerular mesangium, which is indistinguishable from pathologic findings of HSPN [2, 12, 14]. Regarding the pathogenesis of IgAN, several studies that investigated aberrant IgA1 O-glycosylation indicated that galactose-deficient IgA1 (Gd-IgA1) plays a pivotal role in the progression of IgAN [15–23]. According to these studies, patients with IgAN have aberrant IgA1 molecules with a Gal deficiency of O-linked glycans in the hinge region, which indicates that Gd-IgA1 consists of terminal N-acetyl-galactosamine (GalNAc) or sialylated GalNAc [20–23]. These studies identified excess Gd-IgA1 in both serum and glomerular immune deposits in patients with IgAN [20–23]. Furthermore, the recently proposed multi-hit theory of IgAN states that overproduced Gd-IgA1 and autoantibodies against Gd-IgA1 subsequently form circulating IC, resulting in glomerular mesangial deposits followed by accelerated nephritis [20, 24]. Thus, Gd-IgA1 is vital to the pathogenesis of IgAN.

Intriguingly, HSPN and IgAN have been described consecutively in the same patient and in identical twins [14]. Moreover, recent studies noted that these 2 diseases share the feature of Gd-IgA1-oriented pathogenesis [25, 26]. In our recent report, serum Gd-IgA1 (s-Gd-IgA1) levels, quantified by a novel lectin-independent enzyme-linked immunosorbent assay (ELISA) using an anti-Gd-IgA1 monoclonal antibody (KM55) [27], were significantly elevated in patients with HSPN or IgAN compared to other kidney diseases [26]. Similar results were obtained in other recent studies [27, 28]. Additionally, glomerular-Gd-IgA1 (g-Gd-IgA1)-deposition, assessed by immunofluorescence (IF) or immunohistochemistry (IHC) with KM55, was specific to patients with HSPN and IgAN [25, 26]. Taken together, HSPN and IgAN have similar pathologic and biological abnormalities and are closely related.

However, there are also distinct clinical differences between the 2 diseases. HSPN can be regarded as a renal symptom of systemic vasculitis. Patients with HSPN present palpable purpura and gastrointestinal bleeding or polyarthritis other than GN [2]. Meanwhile, IgAN is restricted to the kidneys. Furthermore, symptoms in the acute phase of HSP are sometimes self-limiting and resolve without intensive treatment. HSPN tends to have a good prognosis compared to IgAN [10, 13]. Therefore, it is interesting to explore whether IgAN could be recognized as renal-limited HSP and if pathogenic mechanisms proposed for IgAN may also apply to HSPN. In this study, we aimed to clarify the clinicopathologic dissimilarities between HSPN and IgAN to address these clinical questions.

Patients and methods

Study design and participants

We performed a cross-sectional study of 80 adult (≥18 years) patients with HSPN (n = 24) or IgAN (n = 56) who underwent a renal biopsy (RB) between April 2008 and December 2018 at Showa University Hospital. Data for some patients with HSPN or IgAN were previously described in our historical cohort study [26]. Furthermore, 6 patients with lupus nephritis (LN), 10 with ANCA-associated vasculitis (AAV), and 6 with minimal change disease (MCD) were enrolled as positive or negative controls for ELISA measurements.

HSPN was diagnosed according to a modification of the European League Against Rheumatism/the Pediatric Rheumatology International Trials Organization/the Pediatric Rheumatology European Society (EULAR/PRINTO/PRES) classification criteria [29]: purpura or petechiae with lower limb predominance and the presence of urinary abnormalities, renal insufficiency, and predominant mesangial IgA deposits on RB. All but 2 patients underwent a skin biopsy and were confirmed to have leukocytoclastic vasculitis with predominant IgA deposits. Purpura had developed before nephritis or at the same time as nephritis in all patients with HSPN.

All patients provided written informed consent regarding preservation of blood samples, urine samples, and kidney tissues. In addition, they provided written informed consent to make all data obtained from RB available. Opt-out methods were used to obtain informed consent regarding the measurement of s-Gd-IgA1 values and evaluation of g-Gd-IgA1 deposition. All enrolled patients agreed to participate in this study. The Ethics Committee at Showa University Hospital approved the study protocol (No. 2831), and the study proceeded in accordance with the ethical standards of the Declaration of Helsinki.

Clinical and pathologic parameters

Clinical characteristics, including information on duration from onset of abnormal urinalysis findings to the time of RB (duration from onset), age, sex, body mass index (BMI), history of hypertension (HT), mean arterial pressure (MAP), absence of complications such as gastrointestinal bleeding and polyarthritis, absence of hematuria, degree of urinary protein, urinary N-acetyl-beta-D-glucosaminidase (NAG) index, serum creatinine (sCr), estimated glomerular filtration rate (eGFR), serum IgA and C3, and therapeutic regimens were obtained from patient records. A blood pressure ≥135/85 mmHg was defined as HT. We calculated MAP according to previous report [30]. Hematuria was scored from 0 to 3+ as described previously [31]. We calculated eGFR using the modified Modification of Diet in Renal Disease (MDRD) equation for Japanese persons [32]. Therapeutic regimens at the point of RB, including information about treatment with renin-angiotensin system inhibitors (RASI) and steroids, were assessed. In addition, implementation of steroid pulse therapy combined with tonsillectomy (TSP) after RB was also analyzed. Indications for and the regimen for TSP in IgAN are detailed elsewhere [24, 33, 34].

Histologic sections were independently reviewed by 2 renal pathologists who were blinded to the clinical data of patients. The International Study of Kidney Disease in Children (ISKDC) classification based on the degree of mesangial proliferation and the rate of crescent formation was used to analyze the histologic severity of HSPN [35]. The histologic severity of IgAN was determined according to the histologic grading criteria of the Japanese Society of Nephrology (JSN) [36]. The Oxford classification was also used to evaluate or categorize histologic findings [37].

Immunofluorescence staining

Two nephrologists independently analyzed mesangial IgA, IgG, IgM, and fibrin deposits using IF staining according to our protocol [38] and graded the IF intensity of mesangial IgA as described elsewhere [31].

ELISA for s-Gd-IgA1

Levels of s-Gd-IgA1 were measured using sandwich ELISA kits with KM55 (#27600, Immuno-Biological Laboratories, Fujioka, Japan) [27]. Serum samples were diluted with EIA buffer (1:800), and levels were measured as recommended by the manufacturer.

ELISA for inflammatory cytokines

Levels of serum interleukin (IL)-8, monocyte chemoattractant protein-1 (MCP-1), IL-6, and tumor necrosis factor-α (TNF-α) were measured using specific sandwich ELISA kits from R&D Systems (IL-8, #D8000C; MCP-1, #DCP00; IL-6, #D6050, TNF-α, #DTA00D; Abingdon, U.K.). Serum samples, except for MCP-1, were measured without dilution, and levels were measured as recommended by the manufacturer. Samples for measuring MCP-1 levels were diluted with RD6Q buffer (x 2).

Immunohistochemistry for IgA

IgA deposition in the glomerulus was identified by IHC staining with an IgA-detection kit (BioGenex, Hague, Netherlands) as previously described [39, 40]. Briefly, the paraffin sections of tissues were dewaxed and washed in phosphate buffered saline (PBS). H₂O₂ (0.3%) in methanol was added to slides for 30 min to quench endogenous peroxidase. Sections were washed in PBS and incubated for 60 min at room temperature with rabbit anti-human IgA polyclonal antibody (#AR045-5R, #PU045-UP, 1:500 antibody dilution). Sections were washed 3 times in PBS, then incubated with EnVisionTM+Dual Link System -HRP (Dako, Glostrup, Denmark) for 60 min at room temperature. Next, the sections were developed using diaminobenzidine (DAB) (Dako) as the substrate to produce a brown stain, and sections were counterstained with hematoxylin.

The intensity of IgA in glomerular areas was assessed as 0, none; 1, mild; 2, moderate; or 3, severe. Two nephrologists independently scored IgA intensity in all glomeruli in each section under × 400 magnification, and the mean value per glomerulus of each section was determined. Also, the intensity of IgA in glomerular mesangial or endothelial areas was similarly assessed.

Immunohistochemistry for Gd-IgA1

Gd-IgA1 deposition in glomeruli was examined by IHC staining as described elsewhere [26, 39, 40]. Briefly, dewaxed paraffin sections were heated with Histofine (Nichirei, Tokyo, Japan) in an autoclave at 121°C for 30 min for antigen retrieval. After endogenous peroxidase was quenched with 0.3% H₂O₂ in methanol, nonspecific binding was blocked with protein blocking solution, and sections were incubated overnight at 4°C with rat monoclonal anti-human Gd-IgA1 antibody (KM55) (#10777, Immuno-Biological Laboratories) diluted to 1:100, followed by EnVisionTM+Dual Link System-HRP (Dako) for 60 min at room temperature. Color was then developed using DAB (Dako).

The intensity of Gd-IgA1 in glomerular areas was assessed as 0, none; 1, mild; 2, moderate; or 3, severe. Two nephrologists independently scored g-Gd-IgA1 intensity in all glomeruli in each section under × 400 magnification, and the mean value per glomerulus of each section was determined. Also, the intensity of Gd-IgA1 in glomerular mesangial or endothelial areas was similarly assessed.

Double immunostaining for CD31 with Gd-IgA1

Two-color immunostaining was used to detect colocalization of CD31, an endothelial cell surface marker [41], with Gd-IgA1 according to our protocol [42]. Briefly, as described above, paraffin sections were heated with Histofine in an autoclave at 121°C for antigen retrieval. Thereafter, sections were stained with KM-55 (1:100 dilution) and rabbit anti–human CD31 Ab (Abcam, Cambridge, United Kingdom, 1:200 dilution), followed by EnVisionTM+Dual Link System-HRP (Dako) and EnVisionTM G/2 System/AP (Dako). Color was then developed using DAB (Dako) or permanent red (Dako).

Statistical analysis

Data are expressed as means ± SD or SEM or ratios (%). Results were analyzed using Prism software (GraphPad Software Inc., La Jolla, CA, USA). Non-parametric variables were compared using either Mann-Whitney U tests or Kruskal-Wallis tests. Categorical variables were compared using Fisher exact tests. Correlations between parameters were assessed using Spearman correlation coefficients. P values of < 0.05 were considered to be statistically significant in all the analyses.

Results

Clinical characteristics

Table 1 summarizes the outcomes of comparisons of clinical characteristics at the time of RB and the therapeutic regimens between 24 patients with HSPN (male, 13; mean age [± SD], 44.4 ± 19.3 years) and 56 patients with IgAN (male, 26; 37.4 ± 13.9 years). The clinical characteristics did not significantly differ between groups except for duration from onset and degree of hematuria. Compared to patients with IgAN, patients with HSPN underwent RB significantly earlier after onset of urinary abnormality. In terms of renal function, the mean (± SD) for sCr level was lower, and eGFR level tended to be higher in patients with HSPN compared to IgAN, although differences were not significant. Regarding therapeutic regimens, significantly more patients with HSPN received steroid therapy (ST) than patients with IgAN. In HSPN, 9 of 24 patients had already received ST against purpura rather than GN at the point of RB. In addition, significantly more patients with IgAN were treated with RASI and TSP than patients with HSPN.

Table 1. Clinical characteristics of patients with HSPN and IgAN.

	HSPN	IgAN	P value
Characteristics	(n = 24)	(n = 56)
Age (years)	44.4 ± 19.3	37.1 ± 13.9	0.174
Male gender, No. (%)	13 (54.2%)	26 (46.4.%)	0.525
Duration from onset (months)	8.8 ± 11.8	47.6 ± 65.1	<0.001
BMI (kg/m²)	23.0 ± 5.2	22.4 ± 4.3	0.741
History of HT^a, No. (%)	3 (12.5%)	14 (25.0%)	0.210
MAP^b (mmHg)	88.7 ± 13.3	89.4 ± 11.8	0.603
Proteinuria (g/day)	1.3 ± 2.1	1.2 ± 1.4	0.305
Hematuria^c, No. (%) (±)	2 (8.3%)	2 (3.6%)	0.370
(1+)	11 (45.8%)	7 (12.5%)	0.001
(2+)	5 (20.8%)	11 (19.6%)	0.903
(3+)	6 (25.0%)	36 (64.3%)	0.001
Urinary NAG index (U/gCr)	10.7 ± 9.9	11.1 ± 10.0	0.531
sCr (mg/dL)	0.7 ± 0.2	1.0 ± 0.6	0.053
eGFR (mL/min/1.73 mm²)	81.6 ± 19.0	74.4 ± 31.2	0.282
Alb (g/dL)	3.8 ± 0.6	3.9 ± 0.6	0.373
Serum IgA (mg/dL)	326.0 ± 109.7	330.9 ± 108.8	0.829
IgA/C3	3.0 ± 1.3	3.3 ± 1.3	0.364
Treatment, No. (%)
Use of RASI at RB	1 (4.2%)	15 (26.8%)	0.020
Steroid therapy at RB	9 (37.5%)	1 (1.8%)	<0.001
Underwent TSP after RB	1 (4.2%)	13 (23.2%)	0.039

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Value are means ± SD or (percent). Mann-Whitney U test or Fisher’s test were used for statistical analysis.

Abbreviations: HSPN, Henoch-Schönlein purpura nephritis; IgAN, Immunoglobulin A nephropathy; BMI, body mass index; HT, hypertension, MAP, mean arterial pressure; sCr, serum creatinine; eGFR, estimated glomerular filtration rate; Alb, albumin; RB, renal biopsy; RASI, renin-angiotensin system inhibitor; TSP, steroid pulse therapy combined with tonsillectomy.

^aBlood pressure ≥135/85 mmHg was defined as hypertension.

^bMAP was calculated according to previous reports [30].

^cHematuria was scored from 0 to 3+ as described previously [31].

Histologic findings

Table 2 summarizes the comparisons of histologic findings between 24 patients with HSPN and 56 patients with IgAN. The proportions of grade I, II, IIIa, IIIb, and IV in ISKDC classification for HSPN were 0%, 16.7%, 83.3%, 0%, and 0%, respectively. The proportions of histological-grade (H-grade) I, II, III, and IV using the JSN classification for IgAN were 32.1%, 33.9%, 33.9%, and 0%, respectively. Compared to IgAN, the mean rate (± SD) of global sclerosis or crescent formation was significantly lower in patients with HSPN. When applying the histologic severity based on Oxford classification to HSPN, endothelial injury was significant in patients with HSPN, although mesangial proliferation, crescent formation, and tubulointerstitial injury were significantly worse in patients with IgAN. With regard to glomerular IC deposition on IF or electron microscope assessment, patients with HSPN showed a significantly higher rate of fibrin deposition and electron dense deposition in the subendothelial area compared to patients with IgAN, although there was no significant difference in the mesangial IgA or IgG depositions between the 2 diseases.

Table 2. Comparison of histologic findings between patients with HSPN and IgAN.

	HSPN	IgAN	P value
Characteristics	(n = 24)	(n = 56)
ISKDC classification^a, No (%)
I	0 (0%)
II	4 (16.7%)
IIIa	20 (83.3%)
IIIb	0 (0%)
IV	0 (0%)
Histological grade according to JSN^b, No (%)
I		18 (32.1%)
II		19 (33.9%)
III		19 (33.9%)
IV		0 (0%)
Global sclerosis rate^c (%)	7.3 ± 10.3	19.4 ± 17.2	0.002
Crescent rate^c (%)	5.4 ± 8.0	16.2 ± 18.2	0.006
Global sclerosis + crescent rate^c (%)	14.4 ± 21.5	35.8 ± 20.9	<0.001
Oxford classification^d, No (%)
M1	12 (50.0%)	45 (80.3%)	0.006
E1	18 (75.0%)	13 (23.2%)	<0.001
S1	10 (41.6%)	16 (28.6%)	0.195
T1-2	0 (0%)	15 (26.8%)	0.003
C1-2	12 (50%)	41 (73.2%)	0.044
Glomerular deposition on IF staining
IgA: weak^e	2 (8.3%)	10 (17.8%)	0.331
IgA: moderate^e	10 (41.7%)	29 (51.8%)	0.407
IgA: strong^e	12 (50.0%)	17 (30.4%)	0.094
IgG deposition	9 (37.5%)	15 (26.8%)	0.489
Fibrin deposition,	21 (87.5%)	6 (10.7%)	<0.001
Electron dense deposit, No (%)
Mesangial area	23 (95.8%)	55 (98.2%)	0.087
Subendothelial area	11 (45.8%)	3 (5.4%)	<0.001

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Value are means ± SD or (percent). Mann-Whitney U test or Fisher’s test are used for statistical analysis.

Abbreviations: HSPN; Henoch-Schönlein purpura nephritis, IgAN; Immunoglobulin A nephropathy; ISKDC, International Study of Kidney Disease in Children; JSN, Japanese Society of Nephrology, IF; immunofluorescence.

^aHistologic classification in HSPN was graded based on the ISKDC classification [35].

^bHistological grade in IgAN was classified according to the criteria of the JSN [36].

^cRates of global sclerosis, crescents, and both types of glomerular lesions (%) were calculated by dividing total number of each type of lesion by total number of glomeruli. Crescents comprise cellular, fibrocellular, and fibrous types.

^dHistological severity was graded according to Oxford classification [37].

^eIntensity of IgA deposition was described earlier [31].

Levels of s-Gd-IgA1 and intensity of g-Gd-IgA1 deposition

Fig 1A shows that mean (± SD) s-Gd-IgA1 levels were significantly elevated in patients with HSPN compared to patients with MCD (HSPN vs MCD: 13.1 ± 9.7 vs. 5.2 ± 1.8 μg/mL, p = 0.005). In addition, s-Gd-IgA1 values were significantly elevated in patients with IgAN compared to patients with MCD (16.1 ± 10.2 vs. 5.2 ± 1.8 μg/mL, p = 0.001), but did not significantly differ between patients with IgAN or HSPN (Fig 1A). Even after correction for sCr levels, s-Gd-IgA1 levels were also significantly higher in patients with HSPN or IgAN than in patients with MCD (Fig 1B). Glomerular Gd-IgA1 deposition was apparently specific to HSPN and IgAN (Fig 1C) at significantly higher intensity, and mean (± SD) values for g-Gd-IgA1 staining were significantly higher in these patients than in patients with MCD (HSPN vs. MCD: 1.0 ± 0.9 vs. 0.1 ± 0.1, p < 0.001) (IgAN vs. MCD: 0.9 ± 0.7 vs. 0.1 ± 0.1, p < 0.001) (Fig 1C).

Among patients with HSPN categorized using the ISKDC classification based on the degree of mesangial proliferation and the presence of crescents, s-Gd-IgA1 levels tended to be higher in those with grade IIIa disease than grade II disease, although differences were not significant (10.1 ± 4.7 vs. 13.7 ± 10.4 μg/mL, p = 0.255) (Fig 1D). Staining for g-Gd-IgA1 was significantly more intense among patients with grade IIIa than II disease (0.2 ± 0.1 vs. 1.2 ± 0.9, p = 0.022) (Fig 1E). Similarly, among patients with IgAN categorized as JSN H-grade based on the ratio of global sclerosis, segmental sclerosis, and crescents, s-Gd-IgA1 levels were significantly higher in those with grade II or III disease than grade I disease (17.2 ± 8.3 or 20.1 ± 12.9 vs. 10.7 ± 6.1 μg/mL, p = 0.008 and p = 0.003, respectively) (Fig 1F). Staining for g-Gd-IgA1 was significantly more intense among patients with H-grades III than I according to the JSN classification (1.0 ± 0.6 vs. 0.6 ± 0.6, p = 0.037) (Fig 1G). Additionally, g-Gd-IgA1 intensity scores positively correlated with s-Gd-IgA1 values in patients with IgAN (r = 0.082, p = 0.032) (S1B Fig), whereas no correlation was detected in patients with HSPN (S1A Fig).

Furthermore, we focused on the influence of ST on both types of Gd-IgA1 in patients with HSPN. S2 Fig shows levels of s-Gd-IgA1 and intensity of g-Gd-IgA1 deposition among HSPN patients who received ST [HSPN-ST (+), n = 9] and HSPN patients who did not receive ST [HSPN-ST (-), n = 15] at the point of RB. As shown in S2A Fig, s-Gd-IgA1 levels tended to be higher in HSPN-ST (+) compared to HSPN-ST (-) although the differences were not statistically significant. Values of g-Gd-IgA1 positivity were comparable between two groups (S2B Fig). In addition, similar to the results in S1A Fig, no correlation between s-Gd-IgA1 levels and g-Gd-IgA1 intensity was detected with either HSPN-ST (+) (S2C Fig) or HSPN-ST (-) (S2D Fig).

Levels of serum inflammatory cytokines

Fig 2 shows the levels of serum inflammatory cytokines including IL-8, MCP-1, TNF-α, and IL-6 among patients with HSPN, IgAN, AAV, LN, and MCD. Levels of serum IL-8 in patients with HSPN or IgAN were significantly higher than those in patients with LN or MCD. Mean (± SEM) IL-8 levels in patients with HSPN were significantly higher than levels in patients with IgAN (62.8 ± 24.9 vs. 21.2 ± 4.4 pg/mL, p = 0.033) (Fig 2A), and the elevation of IL-8 in patients with HSPN was comparable with patients with AAV (Fig 2A). In addition, levels of serum TNF-α in patients with HSPN were significantly higher than levels in patients with MCD (Fig 2C). Patients with HSPN showed significant elevations of TNF-α (mean ± SEM) compared to patients with IgAN (11.6 ± 1.5 vs. 5.8 ± 0.5 pg/mL, p<0.001) (Fig 2C).

Levels of MCP-1 and IL-6 in patients with HSPN or IgAN were not elevated and were significantly lower compared to patients with AAV or LN. However, serum concentrations (mean ± SEM) of those 2 cytokines were higher in patients with HSPN than in patients with IgAN (MCP-1: 313.5 ± 21.0 vs. 262.4 ± 11.4 pg/mL, p = 0.022) (Fig 2B), (IL-6: 4.6 ± 2.2 vs. 1.5 ± 0.3 pg/mL, p = 0.045) (Fig 2D).

To assess the influence of ST at the point of RB on serum inflammatory cytokines in HSPN, we compared levels of inflammatory cytokines among HSPN-ST (+) and HSPN-ST (-). As shown in S3A Fig, serum concentrations (mean ± SEM) of IL-8 were significantly higher in HSPN-ST (-) compared to HSPN-ST (+) (86.5 ± 38.1 vs. 23.4 ± 13.9 pg/mL, p = 0.034). Serum levels for MCP-1, TNF-α, and IL-6 were not significantly different between the two groups (S3B, S3C and S3D Fig).

Association of Gd-IgA1 with laboratory parameters or pathologic findings

Table 3 summarizes associations between both types of Gd-IgA1 and laboratory parameters or pathologic findings in 24 patients with HSPN and 56 patients with IgAN. Serum IgA and IgA/C3 ratios positively correlated with s-Gd-IgA1 in patients with IgAN but not in patients with HSPN (Table 3).

Table 3. Correlation between both types of Gd-IgA1 and clinicopathologic parameters in patients with HSPN or IgAN.

	HSPN (n = 24)				IgAN (n = 56)
	s-Gd-IgA1 (μg/mL)		g-Gd-IgA1 intensity		s-Gd-IgA1 (μg/mL)		g-Gd-IgA1 intensity
Variable	R value	P value	R value	P value	R value	P value	R value	P value
Serum IgA (mg/dL)	0.014	0.580	0.019	0.517	0.429	<0.001	0.049	0.102
IgA/C3	<0.001	0.884	0.004	0.766	0.276	<0.001	0.060	0.068
Proteinuria (g/day)	0.061	0.254	0.124	0.099	0.003	0.666	0.002	0.766
Serum Cr (mg/dL)	0.013	0.595	0.249	0.013	0.056	0.080	0.006	0.559
eGFR (mL/min/1.73 mm²)	0.012	0.616	0.014	0.583	-0.092	0.023	0.002	0.757
NAG (U/gCr)	0.002	0.856	0.047	0.319	0.008	0.523	0.014	0.399
Global sclerosis rate^a (%)	<0.001	0.944	0.011	0.621	0.207	<0.001	0.026	0.233
Crescent rate^a (%)	0.454	<0.001	0.003	0.811	0.008	0.522	<0.001	0.831
Global sclerosis + Crescent rate^a (%)	0.632	<0.001	<0.001	0.929	0.088	0.026	0.029	0.206

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Data were statistically analyzed using Spearman correlation tests.

Abbreviations: HSPN, Henoch-Schönlein purpura nephritis; IgAN, Immunoglobulin A nephtopathy; Cr, creatinine; eGFR, estimated glomerular filtration rate.

^aRates of global sclerosis, crescents, and both types of glomerular lesions (%) were calculated by dividing the total number of each type of lesion by the total number of glomeruli. Crescents comprise cellular, fibrocellular, and fibrous types.

In terms of renal function, g-Gd-IgA1 significantly correlated with sCr values in patients with HSPN (Table 3). S-Gd-IgA1 negatively correlated with eGFR values in patients with IgAN (Table 3). Furthermore, s-Gd-IgA1 significantly correlated with global sclerosis plus crescent rates in patients with HSPN or IgAN. Neither type of Gd-IgA1 correlated with proteinuria or urinary NAG index in either disease. Of note, s-Gd-IgA1 in patients with HSPN significantly correlated with crescent rates, whereas s-Gd-IgA1 in patients with IgAN significantly correlated with global sclerosis rates (Table 3).

Based on the Oxford classification, s-Gd-IgA1 levels were significantly higher in patients with IgAN with segmental sclerosis and tubular atrophy/interstitial fibrosis (S4B Fig), and g-Gd-IgA1 intensity in patients with IgAN was significantly higher in patients with segmental sclerosis, tubular lesions, and crescent formation (S4D Fig). On the other hand, based on the Oxford classification of patients with HSPN, neither type of Gd-IgA1 showed any significant difference among classifications (S4A and S4C Fig), although s-Gd-IgA1 showed a tendency to be higher in patients with crescent formation (S4A Fig).

Association of Gd-IgA1 with inflammatory cytokines

Table 4 shows associations between serum inflammatory cytokines and both types of Gd-IgA1 in patients with HSPN and IgAN. Although patients with IgAN showed no correlation of inflammatory cytokines with Gd-IgA1, patients with HSPN showed a significant positive correlation between s-Gd-IgA1 and IL-6 (Table 4). In addition, g-Gd-IgA1 intensity in patients with HSPN was positively correlated with IL-8 and MCP-1 elevations (Table 4).

Table 4. Correlation between both types of Gd-IgA1 and inflammatory cytokines in patients with HSPN and IgAN.

	HSPN (n = 24)				IgAN (n = 56)
	s-Gd-IgA1 (μg/mL)		g-Gd-IgA1 intensity		s-Gd-IgA1 (μg/mL)		g-Gd-IgA1 intensity
Variable	R value	P value	R value	P value	R value	P value	R value	P value
IL-8 (pg/mL)	0.079	0.184	0.165	0.052	0.049	0.100	0.002	0.727
MCP-1 (pg/mL)	0.003	0.771	0.197	0.030	0.012	0.423	0.056	0.079
TNF-α (pg/mL)	0.049	0.297	0.001	0.902	0.037	0.157	0.001	0.901
IL-6 (pg/mL)	0.487	<0.001	0.068	0.218	0.001	0.506	0.024	0.250

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Data were statistically analyzed using Spearman correlation tests.

Abbreviations: HSPN, Henoch-Schönlein purpura nephritis; IgAN, Immunoglobulin A nephtopathy.

We also evaluated those associations with HSPN-ST (+) or HSPN-ST (-). As shown in S1 Table, serum IL-6 levels showed a significant positive correlation with s-Gd-IgA1 levels in HSPN-ST (+) and a significant positive correlation with g-Gd-IgA1 intensity in HSPN-ST (-). On the other hand, other serum cytokines, including IL-8, MCP-1, and TNF-α, did not significantly correlate with either type of Gd-IgA1 in either group (S1 Table), However those 3 cytokines showed a tendency to positively correlate with g-Gd-IgA1 intensity in HSPN-ST (-) (S1 Table).

Association of inflammatory cytokines with HSPN progression

To clarify the association between inflammatory cytokines and HSPN severity, we compared levels of inflammatory cytokines determined by ELISA among patients with HSPN. S5 Fig shows levels of IL-8, MCP-1, TNF-α, and IL-6 between patients with HSPN without any systemic symptoms other than nephritis and patients with HSPN with arthritis or abdominal symptoms (HSPN-AA). As shown in S5A–S5D Fig, serum levels for IL-8, MCP-1, and IL-6 tended to be higher in patients with HSPN-AA compared to patients with HSPN only although none of the differences showed statistical significance.

We also compared serum inflammatory cytokines among groups based on the Oxford classification (Fig 3A–3H). The presence of mesangial hypercellularity, segmental sclerosis, and interstitial fibrosis/tubular atrophy lesions did not all lead to significant differences in levels of serum inflammatory cytokines among patients with HSPN (S6A–S6L Fig). However, mean (± SEM) IL-8 levels were higher in patients with HSPN with endocapillary lesions (75.6 ± 32.5 vs. 24.5 ± 14.9 pg/mL, p = 0.067) (Fig 3A). Also, mean (± SEM) MCP-1 levels were significantly elevated in patients with HSPN with crescent formation compared to the patients without crescent formation (352.6 ± 38.1 vs. 275.8 ± 16.7 pg/mL, p = 0.039) (Fig 3F).

Distinction between glomerular IgA and Gd-IgA1 deposition in HSPN vs. IgAN

We focused on the distribution of glomerular IgA and Gd-IgA1 deposition between patients with HSPN and IgAN. Consecutive paraffin-embedded sections of RB specimens, obtained form 24 patients with HSPN and 56 patients with IgAN, were stained with anti-human IgA polyclonal antibody and KM-55. Fig 4A–4D shows representative photos of IHC staining for IgA and Gd-IgA1 in the glomerulus. As shown in Fig 4A and 4B, intensity of mesangial IgA deposition in patients with HSPN was comparable with that of patients with IgAN, whereas the mean (± SEM) intensity of endothelial IgA deposition was significantly higher in patients with HSPN (1.1 ± 0.1 vs. 0.8 ± 0.1, p < 0.001) (Fig 4E).

Although the intensity of g-Gd-IgA1 deposition was generally lower than that of IgA deposition in patients with HSPN or IgAN (Fig 4C and 4D), both diseases showed a similar degree of Gd-IgA1 deposition in the mesangial area (Fig 4F). Regarding endothelial Gd-IgA1 deposition, both diseases showed weak staining compared to the Gd-IgA1 positivity in the mesangial area, although patients with HSPN showed a statistically higher degree of endothelial Gd-IgA1 deposition (Fig 4F).

Furthermore, 2-color immunohistochemistry with CD31 stained red and Gd-IgA1 stained brown was performed to confirm that g-Gd-IgA1 deposition is dominant in the mesangial area compared with the endothelial area. As shown in Fig 5A and 5B, positivity of Gd-IgA1 was significant in the glomerular mesangial region in both diseases. Most Gd-IgA1-positive mesangial areas were not double stained with CD31 in HSPN and IgAN samples, although partial double-stained areas were occasionally seen in HSPN samples (Fig 5C and 5D).

Fig 5 — Paraffin-embedded kidney sections were stained using 2-color immunohistochemistry with CD31 stained red and Gd-IgA1 stained brown. Representative photos of double staining for CD31 with Gd-IgA1 in patients with HSPN (A and C) or IgAN (B and D). The boxed area with broken lines in the upper panels (A and B) is enlarged in the lower panels (C and D). Original magnification: ×40.

Discussion

In the present study, we compared detailed clinicopathologic information, including s-Gd-IgA1 levels, g-Gd-IgA1deposition, and levels of several serum inflammatory cytokines between patients with HSPN or IgAN, which led to clarify similarities and differences between these 2 immuno-pathologically indistinguishable diseases. Therefore, our results might provide suggestive information to physicians.

Based on our findings, patients with HSPN underwent RB at a relatively early phase after the onset of GN and a greater number of patients received ST. In contrast, patients with IgAN tended to received supportive therapy such as RASI before diagnosis and undergo RB at relative late phase after the onset of GN. Consequently, histological grade in patients with HSPN was mild to moderate. Meanwhile RB findings in some patients with IgAN were advanced and showed chronic lesions, which reflected the difference in renal dysfunction and pathological severity between patients with HSPN and IgAN. Therefore, the timing of the RB and pretreatment with ST may affect the results in the current study. Hilhorst et al. also pointed out that differences in clinical presentation between these 2 diseases were due to the interval between disease onset and the time of RB [43]. However, intriguingly, glomerular endothelial injury with fibrin deposition was apparently predominant in patients with HSPN even though more of these patients received ST before RB. Similarly, recent reports also indicated that endothelial injury and fibrin deposition were distinctive characteristics in patients with HSPN rather than IgAN [14, 44], and such findings are generally considered to represent acute lesions in patients with HSPN [2, 3, 14]. Furthermore, previous research indicated that fibrin itself directly induced inflammation in endothelial cells via its C-terminal end [45]. Wang et al. demonstrated that patients with HSPN with severe fibrin deposition exhibited more severe glomerular damage with active endothelial injury and crescent formation [44]. Taken together, HSPN tends to present as acute glomerular inflammatory lesions with endothelial injury, whereas IgAN tends to develop relative slowly but is associated with progressive mesangial lesions.

With respect to a difference in Gd-IgA1 between the 2 diseases, our findings were consistent with previous reports [25, 26, 28]. Assessing Gd-IgA1 alone was not enough to distinguish between these 2 diseases. However, our study showed several intriguing findings. First, similarly to IgAN, patients with HSPN in histopathologically advanced stage showed high s-Gd-IgA1 levels and evident g-Gd-IgA1 deposition. Second, no correlation between s-Gd-IgA1 and g-Gd-IgA1 was detected in patients with HSPN in spite of the presence of a significant correlation of both forms of Gd-IgA1 in IgAN. Third, in patients with HSPN, significant positive correlations between s-Gd-IgA1 and crescent formation or IL-6 were confirmed, and g-Gd-IgA1 intensity was positively correlated with MCP-1 and IL-8 elevations. In contrast, patients with IgAN showed no correlation of inflammatory cytokines with either type of Gd-IgA1, and both Gd-IgA1 were notably associated with glomerular sclerosis or tubulointerstitial injury. Moreover, serum IgA and IgA/C3 ratios positively correlated with s-Gd-IgA1 in patients with IgAN but not in HSPN. In terms of the influence of ST on both types of Gd-IgA1 in patients with HSPN, no remarkable findings were detected. This lack of findings implies that any potential bias in our results due to ST for Gd-IgA1 may not be significant. Collectively, both types of Gd-IgA1 in patients with HSPN might serve as biomarkers that reflect disease activity, as shown in the patients with IgAN in our recent report [26]. However, mediators or other factors related to Gd-IgA1 appear to be different between patients with HSPN or IgAN. According to our results, Gd-IgA1 in patients with HSPN might be associated with inflammatory cytokines or acute lesions, whereas that in patients with IgAN might be associated with serum IgA levels or chronic lesions. Additional data are needed to provide reliable evidence of the value of Gd-IgA1 as a biomarker that reflects activity in patients with HSPN.

Another remarkable finding of the present study is that serum inflammatory cytokines were significantly higher in patients with HSPN than in patients with IgAN even though more HSPN patients received ST at the time of RB. In other words, elevation of serum inflammatory cytokines was evident in patients with HSPN, regardless of the anti-inflammatory effects of ST. Thus far, levels for serum inflammatory cytokines were mainly compared among patients with HSP with skin lesions alone and patients with HSP with systemic symptoms [2, 17, 46]. However, to our knowledge, comparisons of serum inflammatory cytokines between patients with HSPN or IgAN are limited. In the present study, serum inflammatory cytokines were higher in patients with HSPN with systemic symptoms, which is consistent with previous reports [2, 17, 46]. Furthermore, elevated serum inflammatory cytokines, especially IL-8 and MCP-1, tended to be associated with intensity of g-Gd-IgA1 deposition and degree of active lesions such as endothelial injury or crescent formation in patients with HSPN. These results led us to presume that elevated levels of serum inflammatory cytokines in HSPN may be related to the formation of Gd-IgA1 and the progression of nephritis. Indeed, a recent review by Heineke et al. mentioned that IL-8 released by vascular endothelial cells promotes an inflammatory response with neutrophil migration and eventually causes systemic small vessel damage [2], which partially supports our findings. However, the contribution of inflammatory cytokines to Gd-IgA1 production in HSPN remains unclear. Basic research to elucidate whether an acute inflammatory response in patients with HSPN affects glycosylation of IgA1 is needed. On the other hand, contrary to our expectations, serum inflammatory cytokines were not elevated in patients with IgAN, and those cytokines levels in IgAN were not associated with both types of Gd-IgA1 or other clinical parameters. However, Suzuki et al. showed that IL-6 in IgA1-producing cell lines derived from the blood of IgAN patients was involved in Gd-IgA1 production [47]. A recent review emphasized the significance of T-cell–derived inflammatory cytokines on the progression of IgAN [48]. Thereby, further studies should evaluate if serum levels of inflammatory cytokines are associated with both types of Gd-IgA1 or disease activity in patients with IgAN.

Next, we discuss localization of the Gd-IgA1 deposition in glomerulus. According to our results, distribution of the g-Gd-IgA1-deposition was dominate in mesangial area in both HSPN and IgAN, which was reasonable when considering a dedicated receptor like mesangial transferrin receptor (CD71) for Gd-IgA1 [17, 46]. However, the patients with HSPN also exhibited considerable glomerular subendothelial IgA deposition, implying that most of the subendothelial IgA-IC in HSPN may be consisted of components other than Gd-IgA1 unlike the mesangial IgA-IC in IgAN. To our knowledge, there have not been similar analysis or discussion, but previous basic researches indicated supportive results to us. First of all, the molecular size of circulating IgA1-IC was reported to be different between patients with HSPN and IgAN [14, 43]. Then, recent review mentioned as follow: IgA1 antibodies in IgAV are generated against autoantigens on endothelial cells, such as infection-related microorganisms or β₂GPI, and such anti-endothelial IgA1 antibodies (AECA) ultimately induce vessel damage via infiltration of neutrophils [2, 49]. Thus, our hypothesis might be partially plausible, and significant glomerular endothelial IgA in patients with HSPN may also represent AECA, which may cause endothelial injury with fibrin deposition. However, the meaning of subendothelial Gd-IgA1 deposition as partially seen in our results is unclear, and further analysis focusing on AECA in IgAV or glomerular endothelial Gd-IgA1 deposition in patients with HSPN or IgAN is needed.

Our study had several limitations. First, data from this study were acquired from a single center. Second, the number of enrolled patients was too small to establish strong evidence. Third, the present study mainly targeted patients with less severe HSPN. In fact, none of the patients with HSPN presented with ISKDC grade IV or V. Finally, the influence of ST on our results, especially data for both-Gd-IgA1 and inflammatory cytokines, was not completely revealed in patients with HSPN.

In conclusion, assessing both types of Gd-IgA1 alone was insufficient to distinguish between HSPN and IgAN, but patients with HSPN showed considerable glomerular endothelial injury with subendothelial IgA deposition and significant elevation of serum inflammatory cytokines. Furthermore, such glomerular subendothelial IgA might not necessarily be Gd-IgA1 itself, and factors associated with Gd-IgA1 were inconsistent among these 2 diseases. Taken together, HSPN exhibits not only Gd-IgA1-related glomerulonephritis, similar to IgAN, but also considerable glomerular inflammatory capillaritis distinct from IgAN, and developmental mechanisms for IgAN might not apply to HSPN completely. We suggest that these 2 immuno-pathologically indistinguishable diseases still have different aspects that might hinder to be categorized as variants of one disease.

Supporting information

S1 Fig. Correlations between g-Gd-IgA1 intensity and s-Gd-IgA1 level.

Scatter plots of correlations between g-Gd-IgA1 positivity and s-Gd-IgA1 levels in patients with HSPN (A) and IgAN (B). Data were statistically analyzed using Spearman correlations.

(PDF)

Click here for additional data file.^{(94.2KB, pdf)}

S2 Fig. Both types of Gd-IgA1 among HSPN patients with or without steroid therapy at the time of renal biopsy.

Comparisons of s-Gd-IgA1 levels (A) and g-Gd-IgA1 positivity (B) among MCD patients, HSPN patients who received steroid therapy [HSPN-ST (+)], HSPN patients who did not receive steroid therapy [HSPN-ST (-)], and IgAN patients. Horizontal solid lines represent means. Data were statistically analyzed using Kruskal-Wallis tests and Mann-Whitney U tests. *P<0.05, **P<0.01, and ***P<0.001. Scatter plots of correlations between g-Gd-IgA1 positivity and s-Gd-IgA1 levels in HSPN-ST (+) (C) and HSPN-ST (-) (D). Data were statistically analyzed using Spearman correlations.

(PDF)

Click here for additional data file.^{(115.2KB, pdf)}

S3 Fig. Serum inflammatory cytokines determined by ELISA among HSPN patients with or without steroid therapy at the time of renal biopsy.

Comparison of serum IL-8 (A), MCP-1 (B), TNF-α (C), and IL-6 (D) levels among MCD patients, HSPN patients who received steroid therapy [HSPN-ST (+)], HSPN patients who did not receive steroid therapy [HSPN-ST (-)], and IgAN patients. Values are presented as means ± SEM. Data were statistically analyzed using Kruskal-Wallis tests and Mann-Whitney U tests. *P<0.05, **P<0.01, and ***P<0.001.

(PDF)

Click here for additional data file.^{(104.4KB, pdf)}

S4 Fig. Comparisons of both types of Gd-IgA1 among groups based on the Oxford classification of patients with HSPN or IgAN.

Patients with HSPN (A and C) or IgAN (B and D) were assigned to groups according to mesangial hypercellularity, endocapillary hypercellularity, segmental glomerulosclerosis, and tubular atrophy/interstitial fibrosis. Values are presented as means ± SEM. Data were statistically analyzed using Mann-Whitney U tests. *P<0.05 and **P<0.01.

(PDF)

Click here for additional data file.^{(142.1KB, pdf)}

S5 Fig. Serum inflammatory cytokines determined by ELISA among HSPN patients with or without any systemic symptoms other than nephritis.

Comparison of serum IL-8 (A), MCP-1 (B), TNF-α (C), and IL-6 (D) levels between patients with HSPN without any systemic symptoms other than nephritis and patients with HSPN with arthritis or abdominal symptoms (HSPN-AA). Values are presented as means ± SEM. Data were statistically analyzed using Mann-Whitney U tests.

(PDF)

Click here for additional data file.^{(169.6KB, pdf)}

S6 Fig. Comparisons of serum inflammatory cytokines among the HSPN patients with or without mesangial hypercellularity, segmental glomerulosclerosis, and tubular atrophy/interstitial based on the Oxford classification.

Comparison of serum IL-8 (A, E and I), MCP-1 (B, F and J), TNF-α (C, G and K), and IL-6 (D, H and L) in patients with HSPN according to the presence of mesangial hypercellularity, segmental glomerulosclerosis, and tubular atrophy/interstitial fibrosis based on the Oxford classification. Values are presented as means ± SEM. Data were statistically analyzed using Mann-Whitney U tests.

(PDF)

Click here for additional data file.^{(176.9KB, pdf)}

S1 Table. Correlation between both types of Gd-IgA1 and inflammatory cytokines in HSPN patients with or without steroid therapy at the time of renal biopsy.

(RTF)

Click here for additional data file.^{(79.1KB, rtf)}

S1 Dataset. Original data.

(XLSX)

Click here for additional data file.^{(2.2MB, xlsx)}

Acknowledgments

We greatly appreciate the excellent technical assistance provided by Ms. Tomoko Suzuki.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

The authors received no funding for this work.

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PLoS One. doi: 10.1371/journal.pone.0232194.r001

Decision Letter 0

Fabio Sallustio

10 Feb 2020

PONE-D-20-00737

A cross-sectional analysis of clinicopathologic similarities and differences between Henoch-Schönlein purpura nephritis and IgA nephropathy

PLOS ONE

Dear Dr Wada,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

In particular, as highlighted from Reviewer 1, the influence of corticosteroid therapy on the showed data should be addressed and discussed.

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Academic Editor

PLOS ONE

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Reviewers' comments:

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Comments to the Author

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Reviewer #1: Partly

Reviewer #2: Yes

**********

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Reviewer #1: N/A

Reviewer #2: Yes

**********

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Reviewer #1: No

Reviewer #2: Yes

**********

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Sugiyama et al have carried out a comparative analysis of the clinicopathologic findings between 24 adult patients with Henock-Schonlein Purpura nephritis (HSPN) and 56 individuals affected by IgA Nephropathy (IgAN).

Serum gal-deficient-IgA1 (s-Gd-IgA1) were measured using KM55 ELISA kit and glomerular deposits of Gd-IgA1 (g-Gd-IgA1) were detected using KM55 antiserum. Moreover, the authors measured some inflammatory cytokines (IL-6, IL-8, TNFα and MCP-1) in the serum samples of all patients.

Results demonstrated similar amounts of Gd-IgA1 deposits in the glomeruli (g) of patients with HSPN and those with IgAN. But g-Gd-IgA1 deposits were more evident in the advanced phase of both diseases. Next, the authors found significant correlations between s-Gd-IgA1 and IL-6 in the serum samples and between g-Gd-IgA1 and IL-8 only in the patients with HSPN. In conclusion, the authors evidenced more inflammation in HSPN (glomerular nephritis) than IgAN patients. They concluded that the meccanisms of development of both diseases are not completely identical.

Results of this paper demonstrate that Gd-IgA1 deposits are present in both diseases but patients with HSPN have considerable glomerular endothelial injury caused by IgA1 deposits and significant increased levels of some inflammatory cytokines.

The paper has some bias that should be evidenced in the discussion

1. The measurement of inflammatory cytokines was carried out in serum samples of HSPN patients under corticosteroid therapy. This means that the serum levels of some cytokines were normal because patients received therapy that can modify the cytokine values.

2. The presence of a few significant correlations between s-Gd-IgA1 and clinicopathologic findings in HSPN may be explained by corticosteroid therapy

3. Correlations between significant of IL-8 and g-Gd-IgA1 are not significant (p=0.052)

4. Fig. 3 shows no statistical difference of serum IL-8 between E0 and E1 (p<0.07)

5. s-Gd-IgA1 and g-Gd-IgA1 may be considered biomarkers of HSPN in active phase only in presence of capillaritis (positivity of CD31).

6. p25 line2 fluctuation of g-Gd-IgA1 deposits and active lesion have not been demonstrated because this is not a longitudinal study.

Minor point

P9 line3 delete and

Reviewer #2: In this paper, authors aimed to clarify the clinic-pathologic differences between Henoch-Schönlein purpura nephritis and IgA nephropathy. They analyzed data from 24 HSPN and 56 IgAN in a period going from 2008 to 2018.

They measured serum levels of IL-8, MCP-1, TNF-α, and IL-6, levels of s-Gd-IgA1 and g-Gd-IgA1-deposition. They tested clinical characteristics and histological parameters, according to ISKDC) classification for HSNP and Oxford classification for IgAN.

Although the absence of relevant findings, the study design was well assessed and the statistical analysis were well performed, giving strength to the result. For these reasons, the data are worthy of publication.

Minor Points:

- In the tables 1, 2, 3 and 4 statistically significant P value should be highlighted by the use of bold

- Page 9 line 4: delete “and”

- I suggest to show the p values in the legend and not in the figures, for a better representation of the data

- Most important concern: in figure 4, I suggest using arrows to help readers to discriminate between mesangial and endothelial area.

**********

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2020 Apr 23;15(4):e0232194. doi: 10.1371/journal.pone.0232194.r002

Author response to Decision Letter 0

25 Mar 2020

March 25, 2020

Dr. Fabio Sallustio

Academic Editor

PLOS ONE

RE: PONE-D-20-00737

A cross-sectional analysis of clinicopathologic similarities and differences between Henoch-Schönlein purpura nephritis and IgA nephropathy

Dear Dr. Sallustio:

We appreciate the meticulous review of our study and are pleased to learn that our submission is of interest. We appreciate the opportunity to submit a revised manuscript. All changes, including major revisions for the problems listed below and minor modifications for English grammar, are shown in red in the revised manuscript using track changes. Furthermore, the data-set (excel sheet) including raw data and summary of the analysis in the present study was submitted as supporting information to ensure that all data underlying the findings in our manuscript are fully available.

We have addressed the concerns raised as follows:

Journal requirements

1. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

No changes are required regarding our financial disclosure.

2. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future.

Because the experimental protocols are clearly detailed in the materials and methods section, we did not prepare a separate specific laboratory protocol.

3. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.

We carefully read PLOS ONE's style requirements and prepared the revised manuscript and file names based on the instructions.

Reviewer 1

Major comments

Sugiyama et al have carried out a comparative analysis of the clinicopathologic findings between 24 adult patients with Henock-Schonlein Purpura nephritis (HSPN) and 56 individuals affected by IgA Nephropathy (IgAN). Serum gal-deficient-IgA1 (s-Gd-IgA1) were measured using KM55 ELISA kit and glomerular deposits of Gd-IgA1 (g-Gd-IgA1) were detected using KM55 antiserum. Moreover, the authors measured some inflammatory cytokines (IL-6, IL-8, TNFα and MCP-1) in the serum samples of all patients. Results demonstrated similar amounts of Gd-IgA1 deposits in the glomeruli (g) of patients with HSPN and those with IgAN. But g-Gd-IgA1 deposits were more evident in the advanced phase of both diseases. Next, the authors found significant correlations between s-Gd-IgA1 and IL-6 in the serum samples and between g-Gd-IgA1 and IL-8 only in the patients with HSPN. In conclusion, the authors evidenced more inflammation in HSPN (glomerular nephritis) than IgAN patients. They concluded that the meccanisms of development of both diseases are not completely identical. Results of this paper demonstrate that Gd-IgA1 deposits are present in both diseases but patients with HSPN have considerable glomerular endothelial injury caused by IgA1 deposits and significant increased levels of some inflammatory cytokines.

The paper has some bias that should be evidenced in the discussion

Thank you very much for this important comment. To address this concern, we compared levels of inflammatory cytokines among HSPN patients who received steroid therapy (ST) [HSPN-ST (+), n=9] and HSPN patients who did not receive ST [HSPN-ST (-), n=15] at the point of renal biopsy (RB). As shown in the newly prepared S3 Fig A, serum concentrations (mean ± SEM) of IL-8 were significantly higher in HSPN-ST (-) compared to HSPN-ST (+) (86.5 ± 38.1 vs. 23.4 ± 13.9 pg/mL, respectively; p = 0.034). Serum levels of MCP-1, TNF-α, and IL-6 were not significantly different between groups (S3 Fig B, C, and D) (Page 17, Line 14-19).

At first, we hypothesized that all inflammatory cytokines measured would be significantly higher in HSPN-ST (-) compared to HSPN-ST (+) because of the anti-inflammatory effect of ST. However, except for IL-8, our results did not support this hypothesis. This may be attributable to the small number of patients, diversity of the dose of ST, or difference of administration period of ST in each case.

We added a new S3 Fig as a supporting file in response to your question and mentioned the involvement of ST in the discussion (Page 26, Line 7-9). Furthermore, we described the influence of ST as a limitation of the study (Page 28, Line 9-10).

2. The presence of a few significant correlations between s-Gd-IgA1 and clinicopathologic findings in HSPN may be explained by corticosteroid therapy

As mentioned, we need to address the influence of ST for HSPN on the results of the present study. In particular, we focus on the influence of ST on the findings for both types of Gd-IgA1 in HSPN. In the present study, 9 of 24 patients with HSPN had already received ST against purpura rather than GN at the point of RB (Page 11, Line 23-Page 12, Line 1). No patients with HSPN had completed ST against purpura before undergoing RB.

The newly prepared S2 Fig shows the levels of s-Gd-IgA1 and the intensity of g-Gd-IgA1 deposition between [HSPN-ST (+), n=9] and [HSPN-ST (-), n=15]. As shown in S2 Fig A, s-Gd-IgA1 levels tended to be higher in HSPN-ST (+) compared to HSPN-ST (-), although the differences were not statistically significant. Values of g-Gd-IgA1 positivity were comparable between the groups (S2 Fig B). In addition, similar to the results in S1 Fig A, no correlation between s-Gd-IgA1 levels and g-Gd-IgA1 intensity was detected with either HSPN-ST (+) (S2 Fig C) or HSPN-ST (-) (S2 Fig D). (Page 15, Line 24-Page 16, Line 8).

In addition, we evaluated the associations between serum inflammatory cytokines and both types of Gd-IgA1 in HSPN-ST (+) and HSPN-ST (-). As shown in the newly prepared S1 Table, serum IL-6 levels showed a significant positive correlation with s-Gd-IgA1 levels in HSPN-ST (+) and a significant positive correlation with g-Gd-IgA1 intensity in HSPN-ST (-). On the other hand, other serum cytokines, including IL-8, MCP-1, and TNF-α, did not correlate with either type of Gd-IgA1 in either group (S1 Table). However, those 3 cytokines showed a tendency to positively correlate with g-Gd-IgA1 intensity in HSPN-ST (-) (Page 20 Line 1-7).

Taken together, additional analysis indicated no remarkable differences between HSPN-ST (-) and HSPN-ST (+). Notable findings were not obtained from HSPN patients in the present study even after we divided these patients according to the presence or absence of ST at the point of RB. Therefore, we consider that the potential bias effect due to ST may not be significant (Page 25, Line 18-21). We described the influence of ST as a limitation of the present study (Page 28, Line 9-10).

3. Correlations between significant of IL-8 and g-Gd-IgA1 are not significant (p=0.052)

Thank you for pointing this out. We did not use bold to highlight the P value in the revised Table 4. Furthermore, we did not use the term “significant” in the explanation regarding correlations between IL-8 levels and g-Gd-IgA1 deposition (Page 3, Line 6-7) (Page 19, Line 17).

4. Fig. 3 shows no statistical difference of serum IL-8 between E0 and E1 (p<0.07)

As you pointed out, Fig 3A did not show significant differences in serum IL-8 levels between E0 and E1 in patients with HSPN (P=0.0667). Thus, we modified Fig 3A and put the P value directly on the figure. In addition, we did not use the term “significant” in the explanation of the results shown in Fig 3A (Page 21, Line 10-11).

5. s-Gd-IgA1 and g-Gd-IgA1 may be considered biomarkers of HSPN in active phase only in presence of capillaritis (positivity of CD31).

As you mentioned, both types of Gd-IgA1 appear to have the potential to be biomarkers that reflect the active phase of HSPN consisting of glomerular capillaritis, crescent formation, and so on. However, it was difficult to strongly suggest this potential from our results because of the limitation of sample size as well as the study design (retrospective, cross-sectional study in a single facility). Furthermore, the absence of capillaritis or crescent depends on the timing of RB and the presence of pretreatment at the time of RB, which could be important confounding factors for reliable evidence. Thus, we described future prospects and tasks needed to establish strong evidence of the value of Gd-IgA1 as a biomarker reflecting active lesions in patients with HSPN (Page 26, Line 3-4)

6. p25 line2 fluctuation of g-Gd-IgA1 deposits and active lesion have not been demonstrated because this is not a longitudinal study.

As you pointed out, this is a retrospective cross-sectional study. Thus, it is hard for this study to explain the fluctuation (time-course) of g-Gd-IgA1 deposits and active lesions in HSPN. We modified the discussion to reflect this point (Page 26, Line 16-17).

Minor point:

P9 line3 delete and

Thank you so much for your suggestion. We deleted “and” in line 3 of Page 9 (Page 9, Line 6).

Reviewer 2

In this paper, authors aimed to clarify the clinic-pathologic differences between Henoch-Schönlein purpura nephritis and IgA nephropathy. They analyzed data from 24 HSPN and 56 IgAN in a period going from 2008 to 2018. They measured serum levels of IL-8, MCP-1, TNF-α, and IL-6, levels of s-Gd-IgA1 and g-Gd-IgA1-deposition. They tested clinical characteristics and histological parameters, according to ISKDC) classification for HSNP and Oxford classification for IgAN. Although the absence of relevant findings, the study design was well assessed and the statistical analysis were well performed, giving strength to the result. For these reasons, the data are worthy of publication.

Minor Points:

1. In the tables 1, 2, 3 and 4 statistically significant P value should be highlighted by the use of bold

As you suggested, we highlighted significant P values using bold text in Tables 1, 2, 3, 4, and S1 Table.

2. Page 9 line 4: delete “and”

We deleted “and” in line 6 of Page 9.

3. I suggest to show the p values in the legend and not in the figures, for a better representation of the data

We edited the explanation of P value in all figures regarding comparisons among study groups (Fig 1, Fig 2, Fig 3, Fig 4, S2 Fig, S3 Fig, and S4 Fig) and included the P values in the figure legends. Descriptions of P values were left in figures showing correlations (S1 Fig and S2 Fig) with sample numbers (n) and R values to help readers easily confirm the findings.

4. Most important concern: in figure 4, I suggest using arrows to help readers to discriminate between mesangial and endothelial area.

In the revised Fig 4, representative positive findings in the endothelial area are shown by white arrows, and those in mesangial area are shown by white asterisks.

We feel that we have addressed all of the concerns of the referees and that the manuscript has been significantly improved. We hope that you find the revised version of our manuscript suitable for publication in PLOS ONE.

We look forward to receiving your response.

Sincerely,

Yukihiro Wada

Division of Nephrology, Department of Medicine

Showa University School of Medicine

Tokyo, Japan

Address: 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8666, Japan

Tel: +81-3-3784-8533

E-mail address: yukihiro@med.showa-u.ac.jp

Attachment

Submitted filename: Response to Reviewer.docx

Click here for additional data file.^{(33.4KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0232194.r003

Decision Letter 1

Fabio Sallustio

3 Apr 2020

PONE-D-20-00737R1

A cross-sectional analysis of clinicopathologic similarities and differences between Henoch-Schönlein purpura nephritis and IgA nephropathy

PLOS ONE

Dear Dr Wada,

==============================

ACADEMIC EDITOR:

Just correct some typos

==============================

We would appreciate receiving your revised manuscript by May 18 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Fabio Sallustio

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: Pag 26 line 16 delete were

Pag 26 line 18 these results let us to presume

After these corrections the revised paper will be suitable for publication.

Reviewer #2: Recommendation: Accept With No Changes

The authors have completely addressed all the issues raised by reviewers

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

PLoS One. 2020 Apr 23;15(4):e0232194. doi: 10.1371/journal.pone.0232194.r004

Author response to Decision Letter 1

5 Apr 2020

April 5, 2020

Dr. Fabio Sallustio

Academic Editor

PLOS ONE

RE: PONE-D-20-00737R1

A cross-sectional analysis of clinicopathologic similarities and differences between Henoch-Schönlein purpura nephritis and IgA nephropathy

Dear Dr. Sallustio:

We really appreciate the meticulous review and are pleased to learn that our submission is of interest. We appreciate the opportunity to submit a revised manuscript. All changes, including minor modifications, are shown in red in the revised manuscript using track changes.

We have addressed the concerns raised as follows:

Journal requirements

1. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

No changes are required regarding our financial disclosure.

Because the experimental protocols are clearly detailed in the materials and methods section, we did not prepare a separate specific laboratory protocol.

Reviewer 1

After these corrections the revised paper will be suitable for publication.

1. Page 26 line 16 delete were

Thank you so much for your suggestion. We deleted “were” in line 16 of Page 26.

2. Pag 26 line 18 these results let us to presume

As you pointed out, that sentence appeared to be inappropriate. We modified as follow; These results led us to presume that elevated levels of serum inflammatory cytokines in HSPN may be related to the formation of Gd-IgA1 and the progression of nephritis.

Reviewer 2

Recommendation: Accept With No Changes

The authors have completely addressed all the issues raised by reviewers

We really appreciate your careful review of our manuscript. We completed to modify some inappropriate parts of the manuscript.

We look forward to receiving your response.

Sincerely,

Yukihiro Wada

Division of Nephrology, Department of Medicine

Showa University School of Medicine

Tokyo, Japan

Address: 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8666, Japan

Tel: +81-3-3784-8533

E-mail address: yukihiro@med.showa-u.ac.jp

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PLoS One. doi: 10.1371/journal.pone.0232194.r005

Decision Letter 2

Fabio Sallustio

9 Apr 2020

A cross-sectional analysis of clinicopathologic similarities and differences between Henoch-Schönlein purpura nephritis and IgA nephropathy

PONE-D-20-00737R2

Dear Dr. Wada,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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PLoS One. doi: 10.1371/journal.pone.0232194.r006

Acceptance letter

Fabio Sallustio

14 Apr 2020

PONE-D-20-00737R2

A cross-sectional analysis of clinicopathologic similarities and differences between Henoch-Schönlein purpura nephritis and IgA nephropathy

Dear Dr. Wada:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Correlations between g-Gd-IgA1 intensity and s-Gd-IgA1 level.

Scatter plots of correlations between g-Gd-IgA1 positivity and s-Gd-IgA1 levels in patients with HSPN (A) and IgAN (B). Data were statistically analyzed using Spearman correlations.

(PDF)

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S2 Fig. Both types of Gd-IgA1 among HSPN patients with or without steroid therapy at the time of renal biopsy.

(PDF)

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S3 Fig. Serum inflammatory cytokines determined by ELISA among HSPN patients with or without steroid therapy at the time of renal biopsy.

(PDF)

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S4 Fig. Comparisons of both types of Gd-IgA1 among groups based on the Oxford classification of patients with HSPN or IgAN.

(PDF)

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S5 Fig. Serum inflammatory cytokines determined by ELISA among HSPN patients with or without any systemic symptoms other than nephritis.

(PDF)

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(PDF)

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S1 Table. Correlation between both types of Gd-IgA1 and inflammatory cytokines in HSPN patients with or without steroid therapy at the time of renal biopsy.

(RTF)

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S1 Dataset. Original data.

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Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

A cross-sectional analysis of clinicopathologic similarities and differences between Henoch-Schönlein purpura nephritis and IgA nephropathy

Motonori Sugiyama

Yukihiro Wada

Nobuhiro Kanazawa

Shohei Tachibana

Taihei Suzuki

Kei Matsumoto

Masayuki Iyoda

Hirokazu Honda

Takanori Shibata

Roles

Abstract

Introduction

Methods

Results

Conclusions

Introduction

Patients and methods

Study design and participants

Clinical and pathologic parameters

Immunofluorescence staining

ELISA for s-Gd-IgA1

ELISA for inflammatory cytokines

Immunohistochemistry for IgA

Immunohistochemistry for Gd-IgA1

Double immunostaining for CD31 with Gd-IgA1

Statistical analysis

Results

Clinical characteristics

Table 1. Clinical characteristics of patients with HSPN and IgAN.

Histologic findings

Table 2. Comparison of histologic findings between patients with HSPN and IgAN.

Levels of s-Gd-IgA1 and intensity of g-Gd-IgA1 deposition

Fig 1. S-Gd-IgA1 levels determined by ELISA and g-Gd-IgA1 deposition identified by IHC staining between HSPN and IgAN.

Levels of serum inflammatory cytokines

Fig 2. Serum inflammatory cytokines levels determined by ELISA between patients with HSPN or IgAN.

Association of Gd-IgA1 with laboratory parameters or pathologic findings

Table 3. Correlation between both types of Gd-IgA1 and clinicopathologic parameters in patients with HSPN or IgAN.

Association of Gd-IgA1 with inflammatory cytokines

Table 4. Correlation between both types of Gd-IgA1 and inflammatory cytokines in patients with HSPN and IgAN.

Association of inflammatory cytokines with HSPN progression

Fig 3. Comparisons of serum inflammatory cytokines among groups based on the Oxford classification of patients with HSPN.

Distinction between glomerular IgA and Gd-IgA1 deposition in HSPN vs. IgAN

Fig 4. Distinction between glomerular IgA and Gd-IgA1 deposition identified by IHC staining in HSPN vs. IgAN.

Fig 5. Double immunostaining for CD31 with Gd-IgA1 between HSPN and IgAN samples.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

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Decision Letter 1

Fabio Sallustio

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Fabio Sallustio

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Acceptance letter

Fabio Sallustio

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Associated Data

Supplementary Materials

Data Availability Statement

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