**1. Introduction**

IgA nephropathy (IgAN) or Berger's disease is the most common chronic glomerulonephritis worldwide [1].

In Europe, it is diagnosed in 20% of kidney biopsies performed in childhood [2]. The condition is one of the major causes of end-stage renal disease (ESRD) which develops in 20–40% of patients at 20 years after the diagnosis [3].

The clinical presentation of IgAN may vary, reflecting a wide range of histological findings, from no changes on light microscopy to severe necrotizing lesions with crescents [4]. Clinically, IgAN manifests with persistent or periodic erythrocyturia, either isolated or with concomitant proteinuria of varying severity, sometimes accompanied by hypertension.

The gold standard for the diagnosis of IgAN is the evaluation of a kidney biopsy specimen. The disease is diagnosed based on the predominant IgA deposits on histopathological evaluation. The deposits may also include immunoglobulins M or G. In 90% of cases, the complement component C3 is also identified in the kidney biopsy specimen [3,5]. The Oxford classification (MEST-C) used to evaluate kidney biopsies allows the assessment of risk factors for future renal failure [6].

Proteinuria, reduction of the glomerular filtration rate (GFR), hypertension, old age, male sex and the absence of macroscopic hematuria are independent predictors of a poor outcome of the disease [7–11].

The pathophysiology of the disease is not entirely understood. According to the "four-hits" theory, the initial underlying insult is overproduction of abnormal, galactosedeficient immunoglobulin A1 (GdIgA1) which forms polymers (first hit). Then, specific IgA and/or IgG antibodies against the abnormal IgA1 are produced (second hit), combining and forming circulating immune complexes (third hit). These complexes accumulate in the renal mesangium, inducing a chronic inflammatory response by increased cytokine and growth factor production, which leads to cellular proliferation and mesangial matrix expansion (fourth hit) [2,12,13]. Chronic inflammation results in renal parenchymal fibrosis and progressive renal failure.

A key role in the pathogenesis and progression of IgAN is played by the complement system activation [3,14,15]. IgAN-associated processes involve the alternative and lectin pathways. The processes associated with complement activation likely occur systemically, in the circulating IgA-containing immune complexes and the glomeruli [16].

In the immune system, the ultimate effect of the complement system activation is the formation of C5b-9 sequence (membrane attacking complex, MAC) which perforates the cell membranes of pathogens. Mesangial MAC deposits are commonly observed in IgAN, and its presence is identified by the detection of C9 neoantigen corresponding to the C5b-9. Urinary excretion of the soluble form of MAC was found to be increased in patients with IgAN, likely due to complement activation in the urinary space [16].

The aim of the study was to evaluate the influence of the severity of mesangial C3 deposits in kidney biopsy specimens and the serum C3 level on the clinical course and outcomes of IgAN in children.

#### **2. Material and Methods**

The study included 148 children (91 boys and 56 girls) from the 166 patients included in the Polish Pediatric IgAN Registry. The patients included in the study fulfilled the following inclusion criteria: IgAN diagnosed based on kidney biopsy with evaluation by light microscopy and immunofluorescence. Patients without complete clinical and histopathological data, with the glomerular number < 8, with secondary IgAN and IgA vasculitis nephritis (IgAVN, Henoch-Schönlein nephritis) were excluded from the study.

Proteinuria and serum levels of albumin, creatinine, IgA, C3 and C4 were evaluated twice in the study group, at baseline and the end of follow-up.

Nephrotic range proteinuria was defined as ≥50 mg/kg/d, and non-nephrotic range proteinuria as <50 mg/kg/d, and urinary protein was measured by the Exton method. Serum creatinine level, expressed in mg/dL, was measured by the dry chemistry method

(Vitro, Ortho Clinical Diagnostic). GFR (mL/min/1.73 m2) was estimated using the Schwartz formula. Immunoglobulin A and complement component C3 and C4 serum levels were measured by the nephelometric method in 5 centers and by the turbidimetric method in 3 centers. The use of two different methods for assaying immunoglobulin A and C3 and C4 were related to the retrospective nature of the study conducted in various centers. Referring to studies by Denham et al. which indicate good agreemen<sup>t</sup> between methods in determining protein levels, including IgA and C3, we considered it as a limitation of the study, but the age-related reference ranges did not differ significantly between the centers [17].

A diagnostic kidney biopsy was performed on all children in the study group.

The specimens from each kidney biopsy were routinely evaluated using light microscopy, immunofluorescence and electron microscopy by at least two pathologists.

Kidney biopsy specimens were routinely evaluated by light microscopy and immunofluorescence, and categorized using the Oxford classification, with a calculation of the MEST-C score (1—present, 0—absent; M—mesangial hypercellularity; E—endocapillary hypercellularity; S—segmental sclerosis/adhesion; T—tubular atrophy/interstitial fibrosis T0 0–25%, T1 26–50%, T2 > 50%; C—crescents, C0 0%, C1 0–25%, C2 > 25%; with the overall score calculated as the sum of M, E, S, T and C).

When evaluated by immunofluorescence microscopy, the intensity of IgA and C3 deposits were rated from 0 to +4.

The patients received renoprotective therapy (angiotensin-converting enzyme inhibitor [ACEI]/angiotensin receptor blocker [ARB]), glucocorticosteroids (Encorton) or immunosuppressive drugs such as azathioprine, cyclophosphamide, cyclosporin A and mycophenolate mofetil. Drug treatment was categorized as I—immunosuppression, S— steroids, R—renoprotection.

The study endpoint was an abnormal glomerular filtration rate (eGFR <90 mL/min).

The study was approved by the Bioethics Committee at the Medical University of Warsaw (No. KB/147/2017). Informed consent for study participation was obtained from the legal guardians of the study participants.

Flow diagram of the study is shown in Figure 1.

**Figure 1.** Flow diagram of the study.
