Endothelial cells regulate mesangial cells through the Dll4/Notch3 axis to participate in glomerular injury in lupus nephritis.

INTRODUCTION: Increasing evidence suggests that microvascular dysregulation mediates mesangial cell (MC) alteration in the pathogenesis of LN (Lupus nephritis). However, the heterogeneity of endothelial cells (ECs) in LN and their regulatory mechanisms on MCs have not been thoroughly investigated. OBJECTIVE: To elucidate the regulatory role and mechanism of ECs in the development and progression of LN. METHODS: We analyzed EC heterogeneity and cell-cell interactions in the kidneys of LN mice using single-nucleus RNA sequencing (snRNA-seq). In vitro, MCs were treated with Dll4 protein or the Notch3 inhibitor (Tarextumab) to detect the effects of Dll4/Notch3 axis on MCs. Then, in an EC and MC co-culture system, we interfered Dll4 in ECs or added Tarextumab in MCs to confirm that ECs can secret Dll4 to regulate MCs by Notch3 pathway after LPS stimulation. ELISA and immunohistochemical staining were performed on clinical blood samples and renal biopsies, respectively, to compare Dll4 expression levels between systemic lupus erythematosus (SLE) patients without LN (non-renal SLE) and those with LN. RESULTS: We found that the number of ECs was increased during LN process and ECs exhibited cellular heterogeneity, among which EC subcluster EC-1 was identified as a potential regulator of MCs, possibly through the Dll4/Notch3 axis. In vitro experiments showed that Dll4 can promote the proliferation and migration of MC through Notch3. Interfering Dll4 in ECs or administering a Notch3 inhibitor (Tarextumab) in MC can significantly inhibit the effects on MC. Clinically, we analyzed peripheral blood and kidney tissues from non-renal SLE and LN patients and confirmed that Dll4 expression correlated with the disease activity of LN. CONCLUSIONS: Our findings suggest that abnormal EC activation may be associated with LN progression, potentially through promoting MC migration and proliferation via the Dll4/Notch3 axis. This study reveals a candidate molecular axis in the progression of LN and provides preliminary preclinical insights for future investigation into therapeutic strategies in LN.