#5648 RIPK3 MEDIATES KIDNEY INJURY INDUCED BY A CYTOKINE STORM
Authors: Mauvecín Juan Guerrero , Sánchez Diego Martín , Sanz Ana Belen , Ortiz Alberto 
Published: June 2023

Abstract

Background and Aims
Receptor-interacting protein kinase 3 (RIPK3) is traditionally involved in necroptosis, a regulated necrosis pathway which has been observed in diseases associated to cell death and inflammation. However, RIPK3 has been also associated to inflammatory responses independent of necroptosis. In previous studies we demonstrated that RIPK3 deficiency does not prevent renal injury but protects from inflammation in folic acid-induced AKI (FA-AKI), suggesting a proinflammatory role independently of cell death. In the present work, we aim to explore the role of RIPK3 in kidney injury and inflammation induced by a cytokine storm.

Method
Lipopolysaccharide (LPS) was used to induce cytokine storm-AKI in mice. For this, female 12- to 14-week-old wild type (WT), RIPK3-KO, MLKL-KO or NRLP3-KO C57BL/6J mice received a single intraperitoneal (i.p) injection of LPS 5 mg/kg or vehicle and were sacrificed 1h, 4h and 24 hours later. To generate chimera mice, WT and Ripk3-KO receptor mice were irradiated to deplete the autologous bone marrow (BM). BM was extracted from the femur and tibia of donor WT or Ripk3-KO mice, and 107 cells were transferred to irradiated receptor mice by intravenous injection. After 1 month, LPS-AKI was induced. Plasma was collected to assess kidney function. Kidneys were collected for RNA, protein studies, and histologic studies. Additionally, a WT group received 1.65 mg/kg of the necroptosis inhibitor Necrostatin-1 (Nec1) i.p. prior to LPS to evaluate the impact of necroptosis pathway. Liver, lungs and heart were also collected to assess the systemic inflammatory response. Additionally, cultured murine immortalized tubular MCT cells, and primary tubular cells, bone marrow dendritic cells (BMDC) and bone marrow derived macrophages (BMDM) isolated from WT and Ripk3-KO mice were studied. Cells were stimulated with 100 ng/ml LPS for 6h and RNA was studied. To analyze the impact of the inflammatory response on tubular cells, supernatants from LPS-stimulated BMDM and BMDC were collected and used to stimulate MCT cells. RNA expression was studied by RT-PCR and protein expression by Western Blotting.

Results
The kidney expression of RIPK3 mRNA and protein was upregulated in cytokine storm-AKI in mice, while Ripk3 deficiency improved survival and renal function, with less expression of proinflammatory cytokines and inflammatory infiltrate. Necroptosis did not seem to be implicated in cytokine storm-AKI, since neither Nec1 nor genetic MLKL ablation offered protection on renal function. Systemic inflammation in non-kidney organs was also milder with Ripk3 deficiency. In addition, inflammasome-related proteins were upregulated in cytokine storm-AKI, and this was reduced in Ripk3-KO mice. However, Nlrp3-deficient mice developed kidney injury and inflammation after LPS injection. Next, we explored the role of RIPK3 in BM-derived cells in cytokine storm-AKI by generating chimera mice. In this context, WT mice with Ripk3 deficient BM exhibited less inflammation and better renal function. This supports that RIPK3 from BM cells mediates kidney inflammation and injury during cytokine storm-AKI. In cultured cells, LPS induced RIPK3 expression in BMDMs but not in tubular cells, and RIPK3 mediated IL-6 expression in BMDMs but not in tubular cells. Moreover, IL-6 and conditioned media from LPS-exposed WT macrophages promoted proinflammatory responses in cultured tubular cells, that was partially ameliorated for RIPK3-KO LPS-macrophage conditioned medium.

Conclusion
In conclusion, RIPK3 mediates kidney injury and systemic inflammation induced by a cytokine storm independently of the necroptosis pathway. These results identify RIPK3 as a therapeutic target for renal inflammatory diseases.