Background/Purpose: Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease that leads to damage to several tissues and organs. Inflammation of the kidney is one of the most severe manifestattions of SLE that leads to lupus nephritis (LN). Podocytes are critical for the maintenance of the glomerular filtration barrier and are injured in many kidney diseases including LN. The pathogenesis of LN involves the activation of the complement system and deposition of immune complexes. While the established pathways of complement activation are well-known, recent studies have revealed that complement component C3 can undergo activation within the cell. Specifically, proteases belonging to the cathepsin family have been shown to cleave C3 and C5, thereby introducing a novel pathway for complement activation. In this study, we investigated the production and activation of complement within the podocytes in LN, focusing on the role of cathepsin inhibition as a potential therapeutic tool to reduce intracellular complement activation

Methods: Human immortalized podocytes were cultured on engineered cell-derived decellularized matrix (DCM) coated plates (Adv. Funct. Mater. 2020, 30(44):1908752). Podocytes were then exposed to IgG from patients with LN or to hypoxia. Cathepsin was inhibited in vitro and in vivo using nanoparticles (NP) loaded with E64d, a cathepsin inhibitor, and tagged with a nephrin antibody. MRLlpr, lupus-prone mice, and MRLMpJ control mice, received E64d or empty NP intravenously weekly starting at ages between weeks 8 and 12, followed by tissue harvesting at week 19. Differences at the levels of specific proteins, including C3, C3d, C5b9, and IgG, between E64d-treated and empty NP groups were analyzed. Due to non-normality of residuals, the Wilcoxon-Mann-Whitney test was used for statistical comparison. A p-value < 0.05 was considered significant.

Results: Human podocytes exposed to LN IgG or to hypoxia displayed increased production of C3, C4, C5b9, and C3 activation products. In parallel, podocytes produced cathepsins, and cathepsin inhibition using E64d NP curbed the intracellular C3 activation. There were no significant differences observed in C3 deposition between E64d-treated vs empty NP-treated mice, in either MRLlpr or MRLMpJ mice. The production though of C3d and the deposition of IgG were significantly decreased in the MRLlpr and MRLMpJ mice treated with E64d NP (p< 0.05).

Conclusion: Targeted inhibition of cathepsins in podocytes using E64d-loaded and nephrin antibody-tagged NP effectively reduced intracellular C3 activation in human podocytes exposed to LN IgG or hypoxia, as well as in MRLlpr and MRLMpJ mice. Furthermore, E64d-loaded NP treatment showed a significant decrease in IgG deposition in the mice. This study sheds light on the production of complement components by podocytes and their cathepsin-dependent intracellular activation. More importantly, our experiments indicate that targeted inhibition of complement activation in podocytes averts the deposition of IgG.Our findings have implications for the development of novel treatment strategies for LN and related autoimmune kidney diseases.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/targeted-inhibition-of-cathepsins-limits-the-intracellular-complement-activation-in-lupus-nephritis-podocytes/