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Effect of the SYK Inhibitor Entospletinib on Renal Inflammation and the Transition from Acute Kidney Injury to Chronic Kidney Disease Induced by Ischemia-Reperfusion Injury

ABSTRACT: We used single-cell RNA sequencing with the Evercode™ WT kit (Parse Biosciences) to profile kidney immune cells in a mouse model of ischemia-reperfusion injury (IRI), with or without treatment with the SYK inhibitor Entospletinib. The dataset captures immune dynamics at early and late stages of injury and was used to identify leukocyte populations associated with inflammation and its resolution, as well as the immunomodulatory effects of SYK inhibition in vivo.

ORGANISM(S): Mus musculus

PROVIDER: GSE299409 | GEO | 2025/07/07

REPOSITORIES: GEO

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Project description:Fibroblasts are present in every organ. While the role fibroblasts in chronic diseases such as fibrosis or tumor expression has been extensively explored, little is known about their physiological role. The kidney possesses a unique capacity to recover from even severe acute injury. We study molecular mechanisms of this intrinsic repair capacity in the mouse model of ischemia-reperfusion (IR). In this model, the renal artery and vein are clamped for 45 min, leading to acute kidney injury. The kidney spontaneously recovers from such IR injury within 14 days. IR kidney injury is associated with a transient accumulation of fibroblasts in the diseased tissue. We hypothesized that fibroblasts aid the repair of acute IR injury in the kidney. To elucidate how FSP1+ fibroblasts may contribute to the repair of kidney injury, we undertook a global unbiased approach to compare gene expression profiles of fibroblasts isolated from kidneys post-IRI and from control kidneys by FACS sorting. To investigate the role fibroblasts may play in the repair of kidney inhury, we performed ischemia reperfusion injury surgery on transgenic mice in which the FSP-1 promoter drives EGFP expression. Kidney injury peaks at day 3 post-IRI, followed by spontaneous regeration that restores nearly perfect kidney architecture and health by day 10. Fibroblasts are thought to possibly play a role in this process, as they are normally rare in the healthy kidney, acute kidney injury is associated with a transient accumulation of interstitial fibroblasts, but whether they may help repair the acute kidney injury or in fact could contribute to the damage is not known. To compare the gene expression profiles of normal fibroblasts and those from post-ischemic kidneys, we sacrificed control FSP1-GFP mice and the FSP1-GFP mice three days post-IRI. We generated single-cell suspensions from both the post-IRI and control kidneys, and then isolated FSP1-GFP+ cells by FACS sorting that, when cultured on plastic, displayed typical fibroblast morphology. Total RNA was immediately extracted from the sorted cells and amplified to produce enough for a array. We biotinylated five of the samples from post-ischemic kidneys and three of the control (non-ischemic) kidneys and used Affymetrix 3' Arrays to examine differences in gene expression profiles between the two groups that may she some light on what role, if any, fibroblasts play in the spontaneous healing of the kidney after acute kidney injury. We performed ischemia reperfusion surgery in FSP1-GFP mice, and at day 3, we sacrificed the mice, isolated FSP1-GFP positive cells from both IR and normal control kidneys by FACS sorting, extracted total RNA from the isolated FSP1-GFP cells and used Affymetrix Mouse Expression Array 430 2.0 microarrays to perform gene expression profiling of the samples. All told, we performed the FACS Sorting, RNA extration, and hybridization with 5 ischemic kidneys and 3 normal kidneys. Fibroblasts, acute kidney injury, repair, comparative gene expression profiling, microarrays, FACS sorting, role in healing

Project description:Background: Bromodomain and extra-terminal domain (BET) proteins and the spleen tyrosine kinase (SYK) represent promising targets in Diffuse large B-cell (DLBCL) and Burkitt’s lymphoma (BL). We evaluated the anti-lymphoma activity of the isoform specific bivalent BET inhibitor AZD5153 (AZD) and the pan-BET inhibitor I-BET151 (I-BET) as single agents and in combination with SYK inhibitor Entospletinib in vitro. Methods: Single agent exposures were evaluated on two DLBCL and two BL cell lines analyzing cell proliferation and metabolic activity. Proliferation, metabolic activity, apoptosis, cell cycle and morphology were investigated after combined AZD or I-BET and Ento exposure. RNAseq of combined AZD+Ento exposure was characterized in SU-DHL-4. Background: Bromodomain and extra-terminal domain (BET) proteins and the spleen tyrosine kinase (SYK) represent promising targets in Diffuse large B-cell (DLBCL) and Burkitt’s lymphoma (BL). We evaluated the anti-lymphoma activity of the isoform specific bivalent BET inhibitor AZD5153 (AZD) and the pan-BET inhibitor I-BET151 (I-BET) as single agents and in combination with SYK inhibitor Entospletinib in vitro. Methods: Single agent exposures were evaluated on two DLBCL and two BL cell lines analyzing cell proliferation and metabolic activity. Proliferation, metabolic activity, apoptosis, cell cycle and morphology were investigated after combined AZD or I-BET and Ento exposure. RNAseq of combined AZD+Ento exposure was characterized in SU-DHL-4. Results: Both BET inhibitors reduced cell proliferation/metabolic activity dose and time de-pendently. Combined BET and SYK inhibition enhanced the anti-proliferative effect and induc-ing a G0/G1 cell cycle arrest. SU-DHL-4 demonstrated a pronounced modulation of gene expres-sion by AZD, which was markedly increased by additional SYK inhibition. Functional enrich-ment analyses identified combination-specific GO terms related to cell division, transport and DNA replication. Genes such as PLEKHH3, MYB, SLC8A1, PARP9, HSPB1 and S100A4 were iden-tified as the presumable key regulators. Conclusion: Simultaneous inhibition of BET and SYK enhanced the anti-proliferative effects, and especially the combination-specific gene expression signature.

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Effect of the SYK Inhibitor Entospletinib on Renal Inflammation and the Transition from Acute Kidney Injury to Chronic Kidney Disease Induced by Ischemia-Reperfusion Injury

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