Dapagliflozin Alleviates Renal Fibrosis by Inhibiting RIP1-RIP3-MLKL-Mediated Necroinflammation in Unilateral Ureteral Obstruction

Dapagliflozin, a sodium-glucose cotransporter-2 inhibitor, offers renoprotection in diabetes. However, potential for use in nondiabetic kidney disease remains unknown. Herein, we assessed whether dapagliflozin alleviates renal fibrosis by interfering with necroinflammation in a rat model of unilateral ureteral obstruction (UUO) and in vitro. After induction of UUO, rats were administered dapagliflozin daily for seven consecutive days. UUO induced significant renal tubular necrosis and overexpression of RIP1-RIP3-MLKL axis proteins; these coincided with NLRP3 inflammasome activation, and subsequent development of renal fibrosis. Oxidative stress caused by UUO is tightly associated with endoplasmic reticulum stress and mitochondrial dysfunction, leading to apoptotic cell death through Wnt3α/β-catenin/GSK-3β signaling; all of which were abolished by both dapagliflozin and specific RIP inhibitors (necrostatin-1 and GSK872). In H2O2-treated HK-2 cells, dapagliflozin and RIP inhibitors suppressed overexpression of RIP1-RIP3-MLKL proteins and pyroptosis-related cytokines, decreased intracellular reactive oxygen species production and apoptotic cell death, whereas cell viability was improved. Moreover, activated Wnt3α/β-catenin/GSK-3β signaling was inhibited by dapagliflozin and Wnt/β-catenin inhibitor ICG-001. Our findings suggest that dapagliflozin ameliorates renal fibrosis by inhibiting RIP1-RIP3-MLKL-mediated necroinflammation via Wnt3α/β-catenin/GSK-3β signaling in UUO.

Kidney-Draining Lymph Node Fibrosis Following Unilateral Ureteral Obstruction

Although the primary organ has been the subject of intense investigation in the field of organ fibrosis over the past several decades, the presence of lymph node fibrosis due to persistent activation of the immune response in its partner organ remains largely unknown. Previously, we demonstrated that activation of the immune response following ischemia-reperfusion injury (IRI) and crescentic glomerulonephritis (CGN) in the kidney was associated with extracellular matrix (ECM) production by fibroblastic reticular cells (FRCs) of the kidney-draining lymph node (KLN). Here, we sought to determine whether FRCs in the KLN become similarly fibrogenic following unilateral ureteral obstruction (UUO) of the kidney. We subjected 6–8-week-old C57BL/6J mice to UUO for 2, 7, and 14 days. We examined the microarchitecture of the kidney and KLN by immunofluorescence staining at each timepoint, and we quantified immune cell populations in the KLN by flow cytometry. The contralateral kidney unaffected by UUO and its partner KLN were used as controls. We found through immunofluorescence staining that FRCs increased production of ECM fibers and remodeled the microarchitecture of the UUO KLN, contributing to fibrosis that mirrored the changes in the kidney. We also observed by flow cytometry that the populations of CD11b+ antigen-presenting cells, CD11c+ dendritic cells, and activated CD4+ and CD8+ T cells were significantly higher in the UUO KLN than the KLN draining the unaffected contralateral kidney. Expression of the TGFβ/TGFβR signaling pathway was upregulated and colocalized with FRCs in the UUO KLNs, suggesting a possible mechanism behind the fibrosis. Both release of ureteral ligation at 2 days following UUO and depletion of FRCs at the time of injury onset halted the progression of fibrosis in both the kidney and the KLN. These findings for the first time highlight the association between fibrosis both in the kidney and the KLN during UUO, and they lay the groundwork for future studies that will investigate more deeply the mechanisms behind the connection between FRCs and KLN fibrosis.

15-Lipoxygenase Worsens Renal Fibrosis, Inflammation, and Metabolism in a Murine Model of Ureteral Obstruction

INTRODUCTION: 15-Lipoxygenase (15-LO) is a non-heme iron-containing dioxygenase that has both pro- and anti-inflammatory roles in many tissues and disease states. 15-LO is thought to influence macrophage phenotype; and silencing 15-LO reduces fibrosis after acute inflammatory triggers. The goal of this study was to determine if altering 15-LO expression influences inflammation and fibrogenesis in a murine model of unilateral ureteral obstruction (UUO). METHODS: C57BL/6J mice, 15-lipoxygenase knockout (Alox15-/-) mice, and 15-lipoxygenase transgenic overexpressing mice (15LOTG) were subjected UUO and kidneys were analyzed at 3, 10, and 14-days post injury. Histology for fibrosis, cytokine quantification, flow cytometry, and metabolomics were performed on injured tissues and controls. PD146176, a specific 15-LO inhibitor, was used to complement studies involving knockout animals. RESULTS: Compared to WT animals undergoing UUO, Alox15-/- mouse kidneys had less pro-inflammatory, pro-fibrotic message along with less fibrosis. PD146176 inhibited 15-LO, and resulted in reduced fibrosis similar to Alox15-/- mice. Flow cytometry revealed that Alox15-/- UUO-injured kidneys had a dynamic change in macrophage phenotype, with an early blunting of CD11bHiLy6CHi "M1" macrophages and increase in anti-inflammatory CD11bHiLy6CInt "M2c" macrophages and reduced expression of the fractalkine receptor, CX3CR1. Many of these findings were reversed when UUO was performed on 15LOTG mice. Metabolomics analysis revealed that WT kidneys developed a glycolytic shift post-injury, while Alox15-/- kidneys exhibited increased oxidative phosphorylation. CONCLUSIONS: 15-LO manipulation by genetic or pharmacologic means induces dynamic changes in the inflammatory microenvironment in the UUO-model and appears to be critical in the progression of UUO-induced fibrosis.