Discussion
In 2019, Sethi et al reported that only LMN exhibited positive EXT1/2 expression in renal tissue, and patients testing positive for EXT1/2 appeared to have a better prognosis.9 10 However, studies on the expression of EXT1/2 and their clinical significance in both confirmed and suspected LMN are limited. Recent research suggests that EXT1/EXT2-positive MN may help identify LMN, and no individual case demonstrated positivity for just a single EXT marker.20 Furthermore, EXT1 exhibited a stronger positive intensity in glomeruli compared with EXT2.21 Therefore, we chose EXT1 as the representative marker for detection.
According to the study reported in 2021, EXT1/2 staining was observed in autoimmune diseases, such as membranous lupus nephritis and mixed connective tissue disease. In positive cases, EXT1/2 displayed granular deposition along the GBM.21 However, we observed that mixed class V LN exhibited a higher rate of EXT1-positive cases compared with pure class V LN. Moreover, EXT1 showed a higher deposition rate in the mesangium area than in the GBM for pure V LMN. We believe the results were not falsely positive, as none of the PLA2R-positive MN exhibited mesangium EXT1 deposition, and EXT1-positive area was verified by IF double-staining and IEM. EXTs, similar to other glycosyltransferases, are secreted in a truncated form into the extracellular medium, including the GBM.22 23 The mesangial area is rich in extracellular matrix components, like collagen and fibronectin, which might retain chemicals such as EXTs.24 25 EXT1/EXT2, in conjunction with heparan sulfates, may coat immune complexes, preventing these deposits from initiating an inflammatory response.26 27 It remains unclear whether the mesangial deposition of EXT1 plays a pathogenetic role, acts as a standby mechanism or accelerates the healing process. Overall, the impact of the deposition region of EXT1 on LN biological behaviour warrants further investigation.
Choung et al reported that some cases with lupus-like features were positive for EXT1.28 They speculated that lupus-like MN might be significantly associated with an underlying autoimmune disease. In our study, we observed similar clinicopathological differences, particularly in renal outcomes, between EXT1-positive and EXT1-negative cases in both confirmed and suspected LMN. During follow-up, one patient with positive EXT1 in suspected LMN developed SLE, whereas no patient with negative EXT1 met the diagnostic criteria for SLE. We speculate that EXT1-positive cases might have a pathogenesis similar to LN, which is consistent with Choung et al, even if they initially presented atypically. However, EXT1-positive patients in confirmed LMN demonstrated more severe renal damage, higher SCR, lower eGFR levels and more active lesions like increased crescent frequency, endothelial cell proliferation and higher AI scores compared with suspected LMN. The mechanism remains unclear, but we hypothesise it might relate to the intensity and deposition area of EXT1 to a certain extent, since confirmed LMN exhibited less EXT1 expression in the GBM and a lower frequency of multisite positivity compared with suspected LMN.
Ravindran A et al reported that the EXT1/2-positive patients presented more frequently with proteinuria ≥3.5 g/day.21 Our study showed similar result in EXT1-positive LMN, although the total 24-hour proteinuria had no significant difference. At the same time, our study showed that EXT1-positive patients in the confirmed LMN group had shorter duration of lupus and nephropathy than EXT1-negative patients. This result suggests that LN with EXT1 deposits probably lead to an earlier onset of proteinuria and diagnosis of LN, and EXT1 deposits may act as a cofactor for renal disease manifestations detected during screening patients with SLE for LN. Furthermore, compared with the EXT1-negative group, the EXT1-positive group was reported to have slower chronic kidney progression and a better prognosis.20 21 Saidi et al found that EXT-negative patients had significantly more repeat biopsies with proliferative class 3 or 4 lupus nephritis since the diagnosis of SLE-MN. Multivariable analysis suggested that the EXT status independently predicted clinical remission at the end of follow-up, which was similar to our results.29 It is worth noting that, despite EXT1-positive patients in the confirmed LMN group displaying more active and severe characteristics, the renal outcome was notably better after immunosuppressive therapy compared with the EXT1-negative patients. We speculate that active lesions might be more responsive to immunosuppressive therapy, leading to improved outcomes. EXTs are present in the podocyte Golgi apparatus, where they are responsible for the glycosylation of heparan sulfates that are eventually transported to the GBM. Heparan sulfates can act as clearance receptors for aberrant extracellular proteins, thus facilitating the removal of immune complexes and proteins and associating with a favourable prognosis clinically.30 In our study, we found EXT1-positive patients had a higher CR rate in the whole study cohort than EXT1-negative patients, consistent with a previous study.20 However, there was no significant difference when subdividing cases into confirmed and suspected LMN groups, possibly due to the limited number of cases. The suspected LMN group was clinically heterogeneous, as we observed 2 cases combined with colon adenoma, 1 with liver cirrhosis and 11 that developed other systemic autoimmune diseases like antiphospholipid antibody syndrome, mixed connective tissue disease, rheumatoid arthritis, Sjögren syndrome and autoimmune hepatitis during follow-up. This diversity might explain the low EXT1 positivity rate and unclear aetiology. Yet, EXT1-positive cases might have some innate similarities, especially in renal outcomes across both groups. Therefore, it is noteworthy that suspected LMN with positive EXT1 should be considered as a potential autoimmune disease, as it might later develop into LN.
The limitation of this study is that it was a single-centre, retrospective study with a limited number of renal biopsy samples, which causes bias in comparing clinicopathological data between subgroups and adds some difficulty in the interpretation of some data. Further research would benefit from multicentre collaboration and a larger database.
In conclusion, this study highlighted the importance of EXT1 staining in LMN, particularly emphasising the significance of EXT1 positivity in suspected LMN. Future studies should explore the pathogenic role and potential mechanisms of deposition area of EXT in LMN.