**4. Discussion**

In this study, we evaluated the histopathological and clinical implications of obesity in patients with IgAN. Intriguingly, multivariable logistic regression demonstrated that a BMI greater than 25 kg/m<sup>2</sup> was independently associated with high MME in patients with IgAN. Patients with elevated BMI were more hypertensive and had increased proteinuria, but the association of BMI with renal function and proteinuria was modest in IgAN.

Only a few studies have investigated the influence of obesity on histological parameters in IgAN. First, Bonnet et al. suggested that vascular, tubular, and interstitial indices were higher in obese patients than non-obese patients using a semi-quantitative classification scheme, but the glomerular index did not di ffer significantly. A recent cohort analysis of 481 IgAN patients also showed that high BMI was associated with a high risk of IF [22]. These previous studies did not focus and evaluate the association between increased BMI and MME in IgAN. Tanaka et al. [29] also reported that obese patients with IgAN showed significantly increased proteinuria, accompanied by GBM thickening and glomerulomegaly, mimicking obesity-related glomerulopathy. This study showed no significant di fferences in the severity of mesangial proliferation and matrix expansion between obese and non-obese group, but the number of enrolled patients were very small. Our study is the first study to show an independent association between obesity and MME in IgAN. Moreover, in subjects with tubulointerstitial disease, obesity did not show a significant association with MME, which means that the association between obesity and MME in IgAN is not an incidental finding seen in obese patients. Although the precise mechanism by which obesity causes MME remains unclear, obesity can lead to a rise in the concentration of leptin, which stimulates the expression of transforming growth factor-β1 (TGF-β1). TGF-β1 is known to act as the main driver of extracellular matrix accumulation, mesangial cell proliferation, and progressive glomerulosclerosis [30,31]. These in turn may be responsible for the proliferation of mesangial cells and expanded mesangial matrix. MME has been regarded as a step towards glomerular sclerosis [32]. Although FSGS has long been recognized as the hallmark lesion of obesity-related glomerulopathy [13], Serra et al. [33] also found that increased mesangial matrix, podocyte hypertrophy, and mesangial cell proliferation were more frequent in extremely obese patients compared to normal weight controls. Of note, our study supports these findings in that obesity could independently increase MME after adjustment for confounding factors and strengthens our knowledge of the pathogenic role of obesity in mesangial lesions in IgAN.

Obesity-induced subclinical inflammation is responsible for activation of the complement system [34]. C3 and C4 are expressed and secreted by adipose tissue [35], and serum C3 is strongly associated with components of metabolic syndrome, such as insulin resistance, lipid profile, and BMI [36–38]. Furthermore, weight gain increases C3, which decreases upon weight loss [39]. Therefore, C3 has been linked to the etiology of obesity, and to a wide range of obesity sequelae. In this study, serum C3 and C4 levels were increased in patients with higher BMI. Serum C3 level was positively associated with 24 h proteinuria using multivariable analysis. Consistent with our study, some researchers previously reported that serum C3 and C4 levels were positively correlated with increased BMI and severe proteinuria in IgAN patients [19,21]. High serum C4 level was also associated with severe proteinuria and renal pathological damage in IgAN [40]. Altogether, these findings sugges<sup>t</sup> that obesity may activate the complement system and is related to the severity of renal presentation in patients with IgAN.

In this study, mesangial C3 deposition decreased as BMI increased, but multivariable linear regression and logistic regression analyses showed that mesangial C3 deposition was not independently associated with BMI. Additionally, mesangial C3 deposition was not significantly associated with eGFR or 24 h proteinuria. Therefore, we speculate that the e ffect of obesity on the activation of the local complement system is modest in IgAN. Previous studies revealed conflicting results, suggesting an important role of local complement activation in the pathogenesis of IgAN. Mesangial C3c deposition was associated with active inflammation in IgAN [41], and mesangial C3 deposition was an independent risk factor for progression of IgAN [42]. A recent study also reported that mesangial C3 deposition was associated with poor outcome in IgAN; however, eGFR or proteinuria at the time of kidney biopsy did not differ according to the degree of mesangial C3 deposition [43]. The implications of mesangial complement deposition may have differential effects on renal presentation and prognosis, and further analysis is needed to clarify the pathologic role of mesangial complement deposition in IgAN.

Another interesting finding is that obesity was not independently associated with clinical parameters such as renal function and proteinuria in IgAN. In this study, obese patients showed decreased eGFR and increased 24 h proteinuria compared to other groups, but there were no significant relationships between BMI and eGFR or 24-h proteinuria in multivariable analysis. In addition, the histologic classification of IgAN, such as the Oxford classification or WHO classification, was not different between the groups. Previous studies demonstrated similar findings to our results. A prospective cohort study of 331 French IgAN patients showed that patients with elevated BMI had worse renal presentations and outcomes, but that obesity, per se, did not directly affect these outcomes [18]. In a study of 481 Chinese IgAN patients, there was no significant difference in clinical parameters or Oxford classification scores among BMI groups [22]. In a study including a small number of IgAN patients, obesity had no significant impact on serum creatinine levels or pathological severity in multiple linear regression models [44]. These findings sugges<sup>t</sup> that obesity may not be independently related to decreased renal function, increased proteinuria, or severe forms of histologic classification in IgAN, although it likely affects histologic damage. Obese patients may simultaneously have multiple unhealthy lifestyle behaviors, such as smoking, increased energy intake, low physical activity, and various medical conditions including hypertension, diabetes mellitus, and dyslipidemia, which are known risk factors for renal disease progression [45]. In this study, we also showed that obese patients with IgAN had more the presence of hypertension and dyslipidemia, and were more actively treated with supportive managements, such as anti-hypertensive medications, thiazide, and statins, as well as corticosteroids, than underweight or normal/overweight patients. Therefore, increased weight alone may not be enough to induce severe renal parameters and histologic classifications. To elucidate a direct association between obesity and long-term kidney damage, we need to perform further studies involving a larger number of patients with IgAN.

This study has several limitations. First, our findings are limited by the cross-sectional and retrospective design, uncertain causality, and possible influence of confounding factors that could not be captured; in addition, there were no longitudinal follow-up data. Therefore, a prospective, multi-center, large scale study with a longer follow-up period is required to define the histopathological impact of BMI more clearly in IgAN. Second, although this study included eight university hospitals, the evaluation of renal biopsy specimens was conducted by expert renal pathologists in each center. Therefore, there may be interobserver variability. However, each hospital shares the same renal biopsy report form, and the pathologists at each hospital were trained by one pathologist. Thus, observer bias in histologic assessment may be relatively small. To compensate for this limitation, the histologic grading and staging of IgAN were described according to both the Oxford and WHO classifications. Third, the inclusion of only Korean patients may limit the generalizability of our findings to other ethnic populations. Current practice guidelines suggested that obesity is typically defined quite simply as excess body weight for height, but this simple definition of obesity cannot reflect the consideration of age or race/ethnicity. Therefore, further studies may be needed to elucidate the association with obesity and clinicopathologic parameters in various ethnic groups of IgAN. Fourth, we did not measure the anthropometric parameters associated with metabolic syndrome, such as waist circumference, skin fold thickness and waist-to-hip ratio, and the index of insulin resistance in this study. Further studies will be required to determine whether these markers may be more reliable measures than BMI in IgAN.
