**1. Introduction**

Immunoglobulin A nephropathy (IgAN) is the most common primary glomerulonephritis [1]. According to a systematic review of 40 worldwide studies, the incidence of IgAN is reportedly 2.5/100,000/year [2]. If left untreated, IgAN has a poor prognosis, developing into end-stage renal failure in approximately 20% to 40% of cases within 20 years after onset [3].

There are two IgA isotypes in humans, IgA1 and IgA2 [4]. Galactose-deficient IgA1 (Gd-IgA1), which lacks galactose (Gal) in the O-glycan side chains in the hinge region and exposes N-acetylgalactosamine (GalNAc), has been identified as one of the key molecules in the pathogenesis of IgAN and is increased in the sera of patients with IgAN [5]. According to the multi-hit hypothesis [6], Gd-IgA1 is recognized by anti-glycan autoantibodies, resulting in the formation of pathogenic immune complexes. These immune complexes are deposited in the kidneys, activate mesangial cells, and induce glomerular injury.

Serum Gd-IgA1 levels can predict IgAN progression [7,8]. In contrast, most relatives of IgAN patients with abnormal IgA1 glycoforms do not develop IgAN [9], and serum

**Citation:** Fukao, Y.; Suzuki, H.; Kim, J.S.; Jeong, K.H.; Makita, Y.; Kano, T.; Nihei, Y.; Nakayama, M.; Lee, M.; Kato, R.; et al. Galactose-Deficient IgA1 as a Candidate Urinary Marker of IgA Nephropathy. *J. Clin. Med.* **2022**, *11*, 3173. https://doi.org/ 10.3390/jcm11113173

Academic Editors: Christos Argyropoulos and Giacomo Garibotto

Received: 31 March 2022 Accepted: 30 May 2022 Published: 2 June 2022

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Gd-IgA1 levels do not correlate with proteinuria [5], suggesting that serum Gd-IgA1 may not be a useful biomarker.

We previously developed a monoclonal antibody (KM55 mAb) that specifically recognizes Gd-IgA1, and demonstrated that glomerular Gd-IgA1 was specifically detected in IgAN and IgA vasculitis by immunohistochemical analysis using KM55 mAb [10,11].

In a mouse model, injection of purified nephritogenic IgA from IgAN-prone mice led to deposition in the glomeruli in nude mice. Parts of the injected IgA passed through into the bladder, suggesting that some parts of such glomerular IgA were cleared into the urine. In addition, the nephritogenic IgA had strong affinity not only to the glomerular mesangium, but also to the subepithelium [12]. On the other hand, several studies have supported that epithelial cells have the potential to clear matrix material and epithelial deposits into the cavity of Bowman's capsule [13]. Therefore, we hypothesized that urinary Gd-IgA1 could be a disease-specific marker. Indeed, an enzyme-linked immunosorbent assay (ELISA) using Helix aspersa agglutinin (HAA), a GalNAc-specific lectin, could detect urinary Gd-IgA1 and differentiate patients with IgAN from patients with other renal diseases [14]. However, lectin-dependent assays are not suitable for large-scale, multi-specimen testing because of the instability of glycan recognition. Thus, a novel and stable assay is required for the early detection of IgAN.

Many studies have shown that the degree of proteinuria is an outcome predictor in IgAN [15,16]. However, it is difficult to determine whether urinary protein excretion is due to active lesions triggered by glomerular immune deposition or chronic lesions represented by glomerulosclerosis and nephron reduction [17]. Therefore, proteinuria may not always reflect the disease activity, and its assessment is insufficient to determine the indications for treatment.

Several reports have indicated a high remission rate of tonsillectomy combined with steroid pulse therapy in the early stages of IgAN [18,19]. In addition, the renal biopsy findings of 56 patients with hematuria without overt proteinuria revealed that IgAN was common in their pathological diagnoses, and 31% of the patients with IgAN had crescentic lesions [20]. Thus, early diagnosis and treatment of IgAN are important for remission, and a useful biomarker for the indication of renal biopsy is desired.

There are no established disease-specific biomarkers for IgAN. Furthermore, repeated renal biopsies are difficult because of the invasiveness. We established a stable and simple ELISA for Gd-IgA1 using the KM55 mAb in 2015 [11]. In this study, we investigated the usefulness of urinary Gd-IgA1 as a disease-specific marker for IgAN from Japanese cohorts and further verified this using non-Japanese Asian cohorts.

#### **2. Materials and Methods**

#### *2.1. Patients and Samples*

We recruited 338 Japanese adults ( ≥18 years old) with biopsy-proven IgAN and 120 patients with other renal diseases (disease controls) diagnosed at Juntendo University Hospital, Tokyo, Japan from 2015 to 2018. In addition, to validate the use of the urinary Gd-IgA1 level, we recruited 69 Korean and 35 Taiwanese biopsy-proven IgAN patients, as well as 39 Korean disease control patients.

Clinical and laboratory data were collected at the time of the renal biopsy. The laboratory parameters included serum creatinine (Cr) levels, serum Gd-IgA1 levels, urinary protein-to-creatinine ratios (UPCR), and urinary Gd-IgA1 levels.

## *2.2. Pathological Parameters*

The histological samples were classified according to the clinical guidelines for IgAN from the Japanese Society of Nephrology (JSN) [21] or the Oxford classification [22,23]. Briefly, the histological grade (H-grade) of the JSN criteria was defined as 1 (0–24.9%), 2 (25–49.9%), 3 (50–74.9%), and 4 (75–100%) based on the percentage of glomeruli with pathological features, such as crescents, global sclerosis, and segmental sclerosis, which predict the progression to end-stage renal disease.

#### *2.3. Measurement of Gd-IgA1*

Serum and urinary Gd-IgA1 levels were determined using the KM55 mAb according to the manufacturer's instructions (Immuno-Biological Laboratories, Fujioka, Japan), and logarithmically transformed (log10 basis).

#### *2.4. Statistical Analyses*

Data are expressed as mean ± standard error. Comparisons between groups were performed using the Mann–Whitney U test. Spearman's correlation analysis was used to analyze the correlation between two variables. Statistical significance was defined as *p* < 0.05. Statistical analyses were performed using GraphPad Prism software ver.8.0 (GraphPad Software, San Diego, CA, USA).
