Introduction
The most common vasculitis in children is Henoch–Schönlein purpura (HSP) [
1]. Its typical symptoms manifest in the skin, gastro-intestinal tract, joints, and kidneys, and the severity of the renal symptoms determines the long-term outcome. Of HSP patients, on average, 34% develop renal symptoms [
2]. The symptoms are typically self-limiting proteinuria or hematuria, or both, but some patients develop severe proteinuria, and these patients are at greater risk for chronic kidney disease [
3,
4].
For Henoch–Schönlein nephritis (HSN), the most common histologic classification is from the International Study of Kidney Disease in Children (ISKDC) [
5]. Although it also considers mesangial proliferation, ISKDC classification is largely based on the percentage of glomeruli with crescents, and treatment modality selection is based on ISKDC grade. The prognostic value of the ISKDC classification is, however, questionable, giving rise to a need for a new classification [
6,
7]. The Oxford classification for IgA nephropathy (IgAN), a disease with similar renal histology to HSN, appeared in 2009 [
8]. The updated version of the Oxford classification (MEST-C) now considers five histologic parameters: mesangial hypercellullarity (M), endocapillary proliferation (E), segmental glomerulosclerosis (S), tubular atrophy and/or interstitial fibrosis (T), and crescents (C) [
9]. However, in pediatric HSN, the feasibility of MEST-C requires still further research [
7]. Furthermore, we recently introduced a semiquantitative classification (SQC) for histologic evaluation of HSN [
10].
In this study, we evaluated ISKDC, SQC, and MEST-C scores in sequential renal biopsy specimens. Additionally, from diagnostic kidney biopsies, we analyzed immunohistochemically the expression of pro-fibrotic and inflammatory cell markers. Alpha-smooth muscle actin (α-SMA) and vimentin are pro-fibrotic markers, whereas P-selectin glycoprotein ligand-1 (PSGL-1) is a marker for inflammatory cells; early detection of the processes featuring these markers could prove important in enabling early treatment before the development of fibrosis. The aim of our study was to evaluate the prognostic value of (1) the ISKDC, SQC, and MEST-C classifications and (2) the expression of pro-fibrotic and inflammatory markers. Our hypothesis was that expression of these markers in the diagnostic kidney biopsies correlates with the findings in the follow-up renal biopsy, and that their expression is higher in HSN patients than in control samples from normal kidneys.
Discussion
The aim of our study was to evaluate the prognostic value of three histologic classifications: ISKDC, SQC, and MEST-C. We also evaluated whether pro-fibrotic and inflammatory markers in diagnostic renal biopsy specimens predicted clinical outcomes and lesions in follow-up renal biopsies. Between the diagnostic and follow-up biopsy, active changes decreased and chronic changes increased regardless of the clinical symptoms. Nonetheless, patients with proteinuria at the follow-up biopsy (group II) had higher SQC activity scores in the diagnostic biopsy and higher activity and chronicity scores at the follow-up biopsy than patients without proteinuria (group I). The studied pro-fibrotic and inflammatory markers were higher in HSN biopsy specimens than in controls, but showed no value as predictors of prolonged proteinuria.
We divided the patients according to proteinuria at the follow-up biopsy, since prolonged proteinuria is associated with worsened prognosis [
15,
16]. In accordance with these reports, all five patients with poor outcome in our study had prolonged proteinuria. In most patients, regardless of the proteinuria, activity scores decreased and chronicity scores increased from diagnostic biopsy to follow-up biopsy. This was also evident in MEST-C scores: between the biopsies E1 decreased, whereas S1, T1–2, and T > 0% increased. Regarding these changes in activity and chronicity scores in serial HSN biopsies, previous studies have shown comparable results [
17‐
19]. Consistent with the findings of Shin et al. [
17], the follow-up biopsies of 21 patients with favorable outcome contained chronic histologic changes. This suggests that even with favorable therapeutic outcome, HSN patients require long-term follow-up since the increased histological chronicity may impact the long-term prognosis. Furthermore, since most of our patients were treated with immunosuppressive drugs and angiotensin-converting enzyme inhibitors, progression of the chronicity signs can be considered as an insufficient response to the treatment.
Based on our results, MEST-C parameters in pediatric HSN seem feasible. MEST-C originates from adult IgAN patient data and thus some modifications to the classification may prove necessary for the evaluation of pediatric HSN. For example, tubulointerstitial lesions of MEST-C (i.e., T1 and 2) in our cohort were rare. They were, however, more common after inclusion of biopsies with less than 25% of affected area. Progression of tubulointerstitial lesions associates with a longer duration of proteinuria [
20], and it could therefore prove important to consider all lesions affecting the tubulointerstitium and not only those exceeding 25% of the renal cortex area.
C1–2 were present in both groups’ diagnostic biopsies, but were only seen in the follow-up biopsies in group II. The persistence of crescents thus seems to associate with prolonged proteinuria, supporting results from other studies that treatment should be commenced before fibrotization of crescents [
19]. Active treatment is also supported by the finding that active lesions in the diagnostic biopsy predicted chronic lesions in the follow-up biopsy. M1 was similarly present in both biopsies. Similar results were observed in adult IgAN patients after a 6-month treatment with mycophenolate mofetil and prednisone [
21]. M1 thus seem not to be a flexible sign of clinical improvement and may suggest that the underlying pathogenetic event is still present. In HSN, the clinical meaning (and whether they need further therapy) of the persistent M1 and increased S1/T1-2 findings is unknown. It is possible that the renal damage continues despite minimal clinical signs and that the eGFR has remained stable due to the insufficient follow-up.
Oxford classification has been validated to predict outcomes in IgAN, and in a recent study, M1, S1, and T1–2 were predictive of progression decades after the renal biopsy [
22]. Previous studies on the feasibility of MEST-C in pediatric HSN have suggested that the chronic lesions (S1 and T1–2) correlate with poor outcome. Xu et al. reported that segmental glomerulosclerosis (S1) was associated with a poor renal outcome in univariate analysis, whereas no significant association existed with other parameters. T1 and 2 lesions were, however, negatively associated with the risk of proteinuria remission while other lesions showed no significant association. Interestingly, the number of biopsies with especially E1 and T1 and 2 in the study by Xu et al. [
23] differs from ours. This may reflect inter-observer variation between pathologists or differences in patient material and biopsy timing. T1–2 and S1 associated with worsened renal survival also in a recent study of 75 biopsied pediatric HSN patients [
24]. In two studies with adult HSN patients, E1 also predicted outcome, with or without S1 [
25,
26]. In our study, marked differences between groups I and II occurred in C1–2 and E1 in the follow-up biopsy, although no MEST-C parameter differed significantly. Significant differences existed, however, in the SQC subclasses as activity indices in both biopsies and chronicity index in the follow-up biopsy were higher in proteinuric patients (group II) than in non-proteinuric patients (group I). This may indicate that the evaluation of several histologic variables together could be more advantageous than the assessment of single separate variables.
Vimentin and α-SMA are markers of fibroblast activation [
27], whereas PSGL-1 is an inflammatory cell marker [
28]. Vimentin and α-SMA associate with disease progression in IgAN [
29], but reports on the expression of these molecules in HSN are scarce. Kawasaki et al. studied the expression of α-SMA and c-MET in HSN. Hepatocyte growth factor, which has anti-fibrogenetic effects, uses c-MET as a receptor. In the study by Kawasaki et al., the expression of α-SMA in the first renal biopsy correlated with the chronicity index of the second biopsy in patients with crescents (ISKDC ≥ III) in the first renal biopsy. c-MET showed no significant correlation with activity or chronicity indices [
18]. Our results are consistent with those of Kawasaki et al. concerning α-SMA since in our study, tubulointerstitial α-SMA had significant positive correlation with the tubulointerstitial index—which reflects mainly chronic changes—of the follow-up biopsy. Nonetheless, α-SMA showed no significant correlation with the chronicity index itself. Tubulointerstitial scores for α-SMA, vimentin, and PSGL-1 were surprisingly higher in patients without proteinuria at the follow-up biopsy, but all three markers showed great overlap between the HSN groups (Fig.
2). We were not able to analyze the expression of α-SMA and vimentin from glomeruli. This was due to heavy staining, which precluded any quantitative or semiquantitative analysis.
Our study has limitations. First, most patients had received therapy, which had most likely altered the disease course and possibly hampered the prognostic significance of renal biopsies overall [
30]. Second, the low number of patients with unfavorable outcome limits the interpretation of results from comparison between patients with favorable and unfavorable outcome. These two limitations are probably due to the active treatment of severe HSN in Finland [
31]. Third, histologic evaluation is always subject to inter-rater variability. The inter-rater variability of SQC, assessed in our previous study, showed fair to good reproducibility in a randomly chosen subset of ten biopsies [
10].
Our study has strengths. First, we were able to analyze follow-up biopsies from patients with and without proteinuria at the follow-up biopsy. This allowed us to evaluate the biopsies in relation to the patients’ clinical status. Second, data on the feasibility of the MEST-C in pediatric HSN is scarce, and in our study, the follow-up biopsies also underwent evaluation with MEST-C classification. Third, the median follow-up time in our study was 8.6 years.
Our results suggest that from diagnostic to follow-up renal biopsy, active signs decrease and chronic signs increase regardless of the clinical status, and thus follow-up biopsies provide limited additive information to the ongoing clinical symptoms (especially severe proteinuria) in HSN outcome prediction. Nonetheless, all severe HSN patients, even after a good therapeutic result, require long-term follow-up. Compared to control specimens, HSN biopsies showed over-expression of the pro-fibrotic and inflammatory markers, but none were associated with prolonged proteinuria.
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