Elevated Levels of IL-6 in IgA Nephropathy Patients Are Induced by an Epigenetically Driven Mechanism Modulated by Viral and Bacterial RNA

. Background: Immunoglobulin A nephropathy (IgAN) is the most frequent primary glomerulonephritis characterized by the presence of IgA immune complexes in the glomeruli. Newly, the role of IL-6 in pathogenesis is becoming increasingly important but reason why levels of IL-6 are elevated in IgAN patients is not well understood. One attainable hypothesis comes out from our recent whole genome DNA methylation screening in IgAN patients, that identified, among others, a hypermethylated region comprising Vault RNA 2-1 (VTRNA2-1), a non-coding RNA. Methods: Total RNA were isolated from PBMCs of IgAN patients, transplanted IgAN patients (TP-IgAN), non-IgAN transplanted patients (TP) and healthy subjects (HS). VTRNA2-1, CREB and PKR transcripts were evaluated by RT-PCR. Total and phosphorylated PKR, CREB and Il-6 proteins were evaluated by ELISA. Poly (I:C), a synthetic analogue of dsRNA, and Pfizer-BioNTech COVID-19 vaccine were used to transfect patient PBMCs. PKR inhibitor imoxin (C16) 1 µ M was used to stimulate patient PBMCs. Results: Here we confirm that VTRNA2-1 is low expressed in IgAN subjects compared to HS and we found that also in transplanted IgAN patients (TP-IgAN), compared to non-IgAN transplanted patients (TP), the VTRNA2-1 transcript was expressed at level very low. We found that in IgAN patients with downregulated VTRNA2-1, PKR is overactivated, coherently with the role of the VTRNA2-1 that binds to PKR and inhibits its phosphorylation. The loss of the VTRNA2-1 natural restrain caused the activation of CREB by PKR, a classical cAMP-inducible CRE-binding factor interacting with a region of the IL-6 promoter and leading to IL-6 production, both in IgAN and in TP-IgAN patients. PKR is normally activated by bacterial and viral RNA and we found that both the RNA poly(I:C) and the COVID-19 vaccine stimulation significantly increase the IL-6 levels in PBMCs from HS but had an opposite effect in those from IgAN patients. Conclusions: In conclusion, the discovery of the upregulated VTRNA2-1/PKR/ CREB/IL-6 pathway in IgAN patients may provide novel approach to treat the disease and may be useful for development of precision nephrology and personalized therapy, possibly by checking the VTRNA2-1 methylation level in IgAN patients. Funding: Private Foundation Support

Background: To develop targeted treatments for immune complex diseases such as lupus nephritis (LN) we need to understand immune and renal cell interactions.The advent of spatial sub-cellular resolution whole transcriptome technologies offers the potential to study cell distribution within glomeruli, but requires new mathematical approaches.Conventional 'fixed-window' approaches bin data across spots, discarding the granularity of a high-resolution platform and missing small or rare cells such as immune infiltrates.
Methods: Murine kidneys treated with topical imiquimod or vehicle were prepared for single nuclei RNA sequencing (snRNA-Seq, 10x Genomics) and 200nm spot spatial transcriptomics (STOmics, Beijing Genomics Institute) and analysed (Seurat, RCTD).We developed topological automatic cell type identification (TopACT), combined with multiparameter persistent homology (MPH) to quantify multiscale spatial cell organization.TopACT independently classifies and annotates spot level cell type using a dynamic local neighbourhood.
Results: On synthetic data imputed from renal snRNA-Seq, TopACT produced high accuracy spot-level cell type annotations in comparison to fixed-window approaches.In murine kidney, TopACT spatially resolved individual immune cells in LN, enriched within glomeruli, where the average MPH landscape indicated large loops of immune cells (Fig 1A).This leads to the prediction, driven by spatial data, and confirmed by CD45 immunofluorescence, of a peripheral ring structure of glomerular immune cells in LN (Fig 1B and C).
Conclusions: Our multiscale method for topological automatic cell classification improves accuracy of cell-type information for subcellular resolution spatial transcriptomics, and detects the spatial arrangement of glomerular immune cells in LN.TopACT is generalisable, flexible and has potential for further application to 3D or spatiotemporal data.
Funding: Commercial Support -Funding from UKRI/ Medical Research Council and Kidney Research UK.Beijing Genomics Institute collaborated on the research -no direct financial support /funding to research team., Government Support -Non-U.S. Background: ACE is a well-known enzyme to regulate blood pressure and inflammation, and structurally having two enzymatic domains: N-domain and C-domain.Its C-domain regulates blood pressure by producing angiotensin II in renin-angiotensin system.We recently reported a novel function of ACE: ACE overexpressed neutrophils have a protective role in immune complex (IC)-mediated crescentic glomerulonephritis (GN) via complement C3b-CR1/2 axis, efficiently removing IC deposits.Here, we show that ACE C-terminal catalytic domain is responsible for the renoprotective role in myeloid cells in crescentic GN via lectin pathway.

TH-PO534 Poster Thursday
Methods: We induced the nephrotoxic serum nephritis (NTN) in C57Bl/6 (WT), Jwt mice that overexpressing ACE in myeloid cells (neutrophils and monocytes/ macrophages), Jcko mice that overexpressing C-domain knockout ACE in myeloid cells, and Jnko mice that overexpressing N-domain knockout ACE in myeloid cells, and evaluated renal function and histology (crescent formation and fibrinoid necrosis).In addition, we examined complement pathway activation in vitro and blood angiotensin II level.
Results: 7 days after induction of NTN, Jwt mice showed less severe proteinuria and mild histological glomerular damages, showing overexpressed ACE in neutrophils and monocytes/macrophages has the protective role in IC-mediated crescentic GN.When we induced NTN in Jcko mice and Jnko mice, Jcko mice lost the renoprotective effects and Jnko mice still showed the severe glomerular damage.These data clearly showed that ACE C-domain, but not N-domain, has the renoprotective role.Regarding complement activation, recombinant ACE activated lectin pathway, but not classical and alternative pathways.Given the normal blood pressure level and the normal plasma angiotensin II level in all these mouse strains, the ACE C-domain mediated renoprotective effect is independent of angiotensin II.
Conclusions: ACE C-domain in granulocytes has a protective role in crescentic glomerulonephritis via complement lectin pathway independent of Angiotensin II.

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Glomerular Diseases: From Inflammation to Fibrosis -I Glomerular Diseases: From Inflammation to Fibrosis -I ACE in Granulocytes Has a Protective Role in Crescentic Glomerulonephritis via Complement Lectin Pathway Independent of Angiotensin II Suguru Saito, Narihito Tatsumoto, Michifumi Yamashita.Cedars-Sinai Medical Center, Los Angeles, CA.