**4. Discussion**

A definitive diagnosis of IgAN requires a pathological diagnosis via renal biopsy. In Japan, an annual screening for urinary abnormalities is performed in school-aged children, and asymptomatic patients with microscopic hematuria or mild proteinuria are more likely to undergo renal biopsy than are patients in other countries [24,25]. Indications for renal biopsy vary in each country due to medical insurance. Clinical remission can be achieved with an early diagnosis and treatment [26]. Therefore, a useful biomarker for early detection is required.

Gd-IgA1 is a critical effector molecule in the pathogenesis of IgAN. Glomerular Gd-IgA1 has been specifically detected in patients with IgAN but not in those with other renal diseases [10]. A previous experiment using real-time imaging revealed that injected IgA from the serum of IgAN-prone mice bound to the glomeruli in normal mice, and these IgA deposits cleared over time [12]. Thus, we hypothesized that a fraction of the Gd-IgA1 in glomerular deposits may be excreted into the urine. Previous reports using HAA lectins supported this hypothesis [14]. In this study, we used a stable lectin-independent method with the KM55 mAb to measure the urinary levels of Gd-IgA1. Indeed, the urinary excretion of Gd-IgA1 was elevated in patients with IgAN compared with that in disease controls. This may be due to a mechanism in IgAN in which the greater the amount of Gd-IgA1 deposited, the more glomerular injury occurs, resulting in the increased excretion of urinary Gd-IgA1. As reported in a systematic review [27], the usefulness of serum Gd-IgA1 levels as a tool for assessing disease severity is controversial. In the present study, we found a correlation between urinary Gd-IgA1 levels and the histological severity in IgAN patients. Moreover, the T score of the Oxford classification was associated with urinary Gd-IgA1 levels. Previous reports indicated that the T score is associated with a poor renal outcome [28,29]. Of note, the T score was significantly associated with the renal outcome, independently of clinical data [30]. This suggests that urinary Gd-IgA1 may be useful in determining the disease activity of IgAN.

IgAN is a multifactorial disease with a complex pathogenesis involving genetic and environmental factors. Therefore, we validated the urinary Gd-IgA1 assay in different cohorts of IgAN patients. The levels of urinary Gd-IgA1 were also significantly higher in patients with IgAN than in disease controls in the Korean cohort. Of note, in the Taiwanese and Korean cohorts, urinary Gd-IgA1 levels were much higher than those in the Japanese cohort, which may be due to differences in indications for renal biopsy in these countries, resulting in a higher proportion of advanced-stage cases with a higher urinary protein level and lower eGFR at the time of the renal biopsy.

Clinically, a renal biopsy is rarely performed if proteinuria is negative, even in patients with hematuria. A single-center study in Japan reported that as many as 31% of IgAN patients with hematuria without overt proteinuria (less than 0.3 g/gCr) showed crescents in the renal biopsy findings [20]. Moreover, even in the early stages of IgAN, with normal kidney function and trace proteinuria, long-term renal survival is not always favorable [31]. As early identification and treatment can lead to clinical remission of IgAN [18], biomarkers that can be used for early diagnosis and enable determination of the timing of therapeutic interventions are needed. In the present study, even in patients with IgAN and trace proteinuria (less than 0.3 g/gCr), urinary Gd-IgA1 was detected. Importantly, this suggests that urinary Gd-IgA1 is an early biomarker compared to proteinuria in patients with IgAN.

There are several limitations in the present study. First, we only included Asian patients with IgAN. Large-scale, worldwide studies are needed to elucidate the underlying mechanisms of urinary Gd-IgA1 excretion. Moreover, longitudinal analysis during course of treatment is desired to elucidate disease activity.

In summary, we found higher urinary levels of Gd-IgA1 in patients with IgAN than in patients with other renal diseases. Urinary Gd-IgA1 may be a highly disease-specific marker in several Asian countries. Furthermore, urinary Gd-IgA1 can be detected in patients with trace proteinuria. The present study suggests that urinary Gd-IgA1 is useful not only for the early screening and diagnosis of IgAN but also for determining the disease severity.

**Supplementary Materials:** The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/jcm11113173/s1, Supplementary Figure S1: Levels of urinary Gd-IgA1 in Japanese, Korean and Taiwanese patients with IgAN. Levels of urinary Gd-IgA1 in Korean and Taiwanese patients with IgAN were higher than those in the Japanese patients.

**Author Contributions:** Conceptualization, Y.F. and H.S.; writing, Y.F. and J.S.K.; review and editing, H.S., K.H.J., Y.M., T.K., Y.N., M.N., M.L., R.K., J.-M.C., S.H.L. and Y.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported, in part, by the JSPS KAKENHI, Grant Numbers 15K09274 and 18K08252, and the Practical Research Project for Renal Diseases from the Japan Agency for Medical Research and Development, AMED.

**Institutional Review Board Statement:** The study was conducted in accordance with the Declaration of Helsinki and approved by the ethics committee of Juntendo University Hospital (protocol code 2013058 and 858 and dates of approval 6 October 2013 and 19 June 2015), Kyung Hee University (protocol code 2009-06-301 and date of approval 15 February 2022) and Kaohsiung Medical University (protocol code KMUHIRB-G(I)-20180012 and date of approval 13 July 2018).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.
