sphingosine-kinase and Necrosis

Renal ischemia-reperfusion is a main cause of acute kidney injury (AKI), which is associated with high mortality. Here we show that extracellular vesicles (EVs) secreted from hiPSC-MSCs play a critical role in protection against renal I/R injury. hiPSC-MSCs-EVs can fuse with renal cells and deliver SP1 into target cells, subsequently active SK1 expression and increase S1P formation. Chromatin immunoprecipitation (ChIP) analyses and luciferase assay were used to confirm SP1 binds directly to the SK1 promoter region and promote promoter activity. Moreover, SP1 inhibition (MIT) or SK1 inhibition (SKI-II) completely abolished the renal protective effect of hiPSC-MSCs-EVs in rat I/R injury mode. However, pre-treatment of necroptosis inhibitor Nec-1 showed no difference with the administration of hiPSC-MSCs-EVs only. We then generated an SP1 knockout hiPSC-MSC cell line by CRISPR/Cas9 system and found that SP1 knockout failed to show the protective effect of hiPSC-MSCs-EVs unless restoring the level of SP1 by Ad-SP1 in vitro and in vivo. In conclusion, this study describes an anti-necroptosis effect of hiPSC-MSCs-EVs against renal I/R injury via delivering SP1 into target renal cells and intracellular activating the expression of SK1 and the generation of S1P. These findings suggest a novel mechanism for renal protection against I/R injury, and indicate a potential therapeutic approach for a variety of renal diseases and renal transplantation.

Topics: Acute Kidney Injury; Animals; Apoptosis; Cell Differentiation; Cell Line, Transformed; Epithelial Cells; Extracellular Vesicles; Gene Expression Regulation; Humans; Induced Pluripotent Stem Cells; Kidney; Lysophospholipids; Male; Mesenchymal Stem Cells; Necrosis; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Sprague-Dawley; Reperfusion Injury; Signal Transduction; Sp1 Transcription Factor; Sphingosine

Endothelial dysfunction is a major clinical problem affecting virtually every patient requiring critical care. Volatile anesthetics are frequently used during the perioperative period and protect the heart and kidney against ischemia and reperfusion injury. We aimed to determine whether isoflurane, the most commonly used volatile anesthetic in the USA, protects against endothelial apoptosis and necrosis and the mechanisms involved in this protection. Human endothelial EA.hy926 cells were pretreated with isoflurane or carrier gas (95% room air + 5% CO(2)) then subjected to apoptosis with tumor necrosis factor-α or to necrosis with hydrogen peroxide. DNA laddering and in situ Terminal Deoxynucleotidyl Transferase Biotin-dUTP Nick-End Labeling (TUNEL) staining determined EA.hy926 cell apoptosis and percent LDH released determined necrosis. We also determined whether isoflurane modulates the expression and activity of sphingosine kinase-1 (SK1) and induces the phosphorylation of extracellular signal regulated kinase (ERK MAPK) as both enzymes are known to protect against cell death. Isoflurane pretreatment significantly decreased apoptosis in EA.hy926 cells as evidenced by reduced TUNEL staining and DNA laddering without affecting necrosis. Mechanistically, isoflurane induces the phosphorylation of ERK MAPK and increased SK1 expression and activity in EA.hy926 cells. Finally, selective blockade of SK1 (with SKI-II) or S1P(1) receptor (with W146) abolished the anti-apoptotic effects of isoflurane. Taken together, we demonstrate that isoflurane, in addition to its potent analgesic and anesthetic properties, protects against endothelial apoptosis most likely via SK1 and ERK MAPK activation. Our findings have significant clinical implication for protection of endothelial cells during the perioperative period and patients requiring critical care.

Topics: Anesthetics, Inhalation; Apoptosis; Cell Line; DNA Fragmentation; Endothelial Cells; Extracellular Signal-Regulated MAP Kinases; Flavonoids; Humans; Hydrogen Peroxide; Isoflurane; Necrosis; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Tumor Necrosis Factor-alpha

Acute kidney injury (AKI) frequently leads to systemic inflammation and extrarenal organ dysfunction. Volatile anesthetics are potent anti-inflammatory agents and protect against renal ischemia-reperfusion injury. Here, we sought to determine whether isoflurane, a commonly used volatile anesthetic, protects against AKI-induced liver and intestinal injury, the mechanisms involved in this protection, and whether this protection was independent of the degree of renal injury. Bilateral nephrectomy-induced AKI under pentobarbital sodium anesthesia led to severe hepatic and intestinal injury with periportal hepatocyte vacuolization, small intestinal necrosis, apoptosis, and proinflammatory mRNA upregulation. In contrast, isoflurane anesthesia reduced hepatic and intestinal injury after bilateral nephrectomy. Mechanistically, isoflurane anesthesia upregulated and induced small intestinal crypt sphingosine kinase-1 (SK1) as SK1 mRNA, protein, and enzyme activity increased with isoflurane treatment. Furthermore, isoflurane failed to protect mice treated with a selective SK inhibitor (SKI-II) or mice deficient in the SK1 enzyme against hepatic and intestinal dysfunction after bilateral nephrectomy, demonstrating the key role of SK1. Therefore, in addition to its potent anesthetic properties, isoflurane protects against AKI-induced liver and intestine injury via activation of small intestinal SK1 independently of the effects on the kidney. These findings may help to elucidate the cellular signaling pathways underlying volatile anesthetic-mediated hepatic and intestinal protection and result in novel clinical applications of volatile anesthetics to attenuate perioperative complications arising from AKI.

Topics: Acute Kidney Injury; Alanine Transaminase; Anesthetics, Inhalation; Animals; Apoptosis; Chemical and Drug Induced Liver Injury; Enzyme Activation; Intestine, Small; Isoflurane; Liver; Male; Mice; Mice, Inbred C57BL; Necrosis; Nephrectomy; Phosphotransferases (Alcohol Group Acceptor)

The roles of sphingosine kinases SK1 and SK2 in ischemia-reperfusion injury have not been fully elucidated since studies have found beneficial effects of SK1 while others showed no role in this injury. To help resolve this, we used SK1 or SK2 knockout mice and confirmed that renal ischemia-reperfusion injury induced SK1, but not SK2, in the kidneys. Furthermore, knockout or pharmacological inhibition of SK1 increased injury after renal ischemia-reperfusion injury. In contrast, lack of SK2 conferred renal protection following injury. In addition, we used lentiviral gene delivery to selectively express enhanced green fluorescent protein (EGFP) or human SK1 coexpressed with EGFP (EGFP-huSK1) in the kidney. Mice with kidney-specific overexpression of EGFP-huSK1 had significantly improved renal function with lower plasma creatinine, renal necrosis, apoptosis, and inflammation. Moreover, EGFP-huSK1 overexpression in cultured human proximal tubule (HK-2) cells protected against peroxide-induced necrosis. Selective overexpression of EGFP-huSK1 led to increased HSP27 mRNA and protein expression in vivo and in vitro. Functional protection as well as induction of HSP27 with EGFP-huSK1 overexpression in vivo was blocked with sphingosine-1-phosphate-1 receptor(1) (S1P(1)) antagonism. Thus, our findings suggest that SK1 is renoprotective by S1P(1) activation and perhaps HSP27 induction. Kidney-specific expression of SK1 through lentiviral delivery may be a viable therapeutic option to attenuate renal ischemia-reperfusion injury.

Topics: Animals; Apoptosis; Biomarkers; Cell Line; Creatinine; Disease Models, Animal; Gene Transfer Techniques; Genetic Vectors; Heat-Shock Proteins; HSP27 Heat-Shock Proteins; Humans; Inflammation Mediators; Kidney; Lentivirus; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Molecular Chaperones; Necrosis; Neutrophil Infiltration; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase Inhibitors; Receptors, Lysosphingolipid; Recombinant Fusion Proteins; Reperfusion Injury; Sphingosine-1-Phosphate Receptors; Time Factors

We previously showed that the inhalational anesthetic isoflurane protects against renal ischemia reperfusion injury in part via sphingosine kinase (SK)-mediated synthesis of sphingosine-1-phosphate (S1P). In this study, we tested the hypothesis that isoflurane directly targets renal proximal tubule cells via SK activation, S1P synthesis and activation of S1P receptors to initiate cytoprotective signaling.. Isoflurane-mediated phosphorylation of extracellular signal-regulated kinase (ERK) and Akt and induction of HSP70 in human kidney proximal tubule (HK-2) cells were inhibited by dimethylsphingosine (DMS), an SK inhibitor, and VPC23019, an S1P(1/3) receptor selective antagonist, in HK-2 cells. A selective S1P(1) receptor agonist, SEW2781, mimicked isoflurane-induced phosphorylation of ERK and Akt and induction of HSP70. Moreover, isoflurane-mediated protection against H(2)O(2)-induced necrosis of HK-2 cells was significantly attenuated by an S1P(1/3) receptor antagonist, VPC23019, and by SK inhibitors DMS or 4-[[4- (4-chlorophenyl)-2-thiazolyl]amino]phenol. Finally, overexpression of the SK1 enzyme in HK-2 cells protected against H(2)O(2)-induced necrosis.. Collectively, our study demonstrates that S1P released via isoflurane-mediated SK1 stimulation produces direct anti-necrotic effects probably via S1P(1) receptor-mediated cytoprotective signaling (ERK/Akt phosphorylation and HSP70 induction) in HK-2 cells. Our findings may help to unravel the cellular signaling pathways of volatile anesthetic-mediated renal protection and lead to new therapeutic applications of volatile anesthetics during the perioperative period.

Topics: Anesthetics, Inhalation; Cells, Cultured; Humans; Isoflurane; Kidney Tubules, Proximal; Lysophospholipids; Necrosis; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid produced by sphingosine kinase (SphK1 and 2). We previously showed that S1P receptors (S1P1, S1P2, and S1P3) are expressed in hepatic myofibroblasts (hMF), a population of cells that triggers matrix remodeling during liver injury. Here we investigated the function of these receptors in the wound healing response to acute liver injury elicited by carbon tetrachloride, a process that associates hepatocyte proliferation and matrix remodeling. Acute liver injury was associated with the induction of S1P2, S1P3, SphK1, and SphK2 mRNAs and increased SphK activity, with no change in S1P1 expression. Necrosis, inflammation, and hepatocyte regeneration were similar in S1P2-/- and wild-type (WT) mice. However, compared with WT mice, S1P2-/- mice displayed reduced accumulation of hMF, as shown by lower induction of smooth muscle alpha-actin mRNA and lower induction of TIMP-1, TGF-beta1, and PDGF-BB mRNAs, overall reflecting reduced activation of remodeling in response to liver injury. The wound healing response was similar in S1P3-/- and WT mice. In vitro, S1P enhanced proliferation of cultured WT hMF, and PDGF-BB further enhanced the mitogenic effect of S1P. In keeping with these findings, PDGF-BB up-regulated S1P2 and SphK1 mRNAs, increased SphK activity, and S1P2 induced PDGF-BB mRNA. These effects were blunted in S1P2-/- cells, and S1P2-/- hMF exhibited reduced mitogenic and comitogenic responses to S1P. These results unravel a novel major role of S1P2 in the wound healing response to acute liver injury by a mechanism involving enhanced proliferation of hMF.

Topics: Acute Disease; Animals; Becaplermin; Carbon Tetrachloride Poisoning; Cell Division; Cells, Cultured; Chemical and Drug Induced Liver Injury; DNA Replication; Enzyme Induction; Extracellular Matrix; Fibroblasts; Gene Expression Regulation; Liver Regeneration; Lysophospholipids; Mice; Mice, Inbred C57BL; Mice, Knockout; Myoblasts, Smooth Muscle; Necrosis; Phosphotransferases (Alcohol Group Acceptor); Platelet-Derived Growth Factor; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-sis; Receptors, Lysosphingolipid; Sphingosine; Sphingosine-1-Phosphate Receptors; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta1

The inhalational anesthetic isoflurane has been shown to protect against renal ischemia-reperfusion (IR) injury. Previous studies demonstrated that isoflurane modulates sphingolipid metabolism in renal proximal tubule cells. We sought to determine whether isoflurane stimulates sphingosine kinase (SK) activity and synthesis of sphingosine-1-phosphate (S1P) in renal proximal tubule cells to mediate renal protection via the S1P signaling pathway. Isoflurane anesthesia reduced the degree of renal failure and necrosis in a murine model of renal IR injury. This protection with isoflurane was reversed by SK inhibitors (DMS and SKI-II) as well as an S1P(1) receptor antagonist (VPC23019). In addition, mice deficient in SK1 enzyme were not protected from IR injury with isoflurane. SK activity as well as SK1 mRNA expression increased in both cultured human proximal tubule cells (HK-2) and mouse kidneys after exposure to isoflurane. Finally, isoflurane increased the generation of S1P in HK-2 cells. Taken together, our findings indicate that isoflurane activates SK in renal tubule cells and initiates S1P-->S1P(1) receptor signaling to mediate the renal protective effects. Our findings may help to unravel the cellular signaling pathways of volatile anesthetic-mediated renal protection and lead to new therapeutic applications of inhalational anesthetics during the perioperative period.

Topics: Anesthetics, Inhalation; Animals; Cell Line; Creatinine; Enzyme Inhibitors; Humans; Isoflurane; Kidney Diseases; Kidney Tubules; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Necrosis; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Sphingosine

Owing to its ability to induce growth arrest and differentiation of keratinocytes, 1alpha,25-dihydroxyvitamin D3 and its analogs are useful for the treatment of hyperproliferative skin diseases, such as psoriasis vulgaris. It has been implicated that the 1alpha,25-dihydroxyvitamin D3-induced differentiation of keratinocytes is mediated, at least in part, by the formation of ceramides; however, ceramides have also been identified to induce apoptosis in many cells, including keratinocytes. Therefore, it was of interest to investigate the influence of 1alpha,25-dihydroxyvitamin D3 on apoptosis in keratinocytes. Most interestingly, physiological concentrations of 1alpha,25-dihydroxyvitamin D3 did not induce apoptosis in keratinocytes, despite the formation of ceramides. Moreover, 1alpha,25-dihydroxyvitamin D3 appeared cytoprotective and made keratinocytes resistant to apoptosis induced by ceramides, ultraviolet irradiation, or tumor necrosis factor-alpha. The cytoprotective effect was accompanied by the formation of the sphingolipid breakdown product sphingosine-1-phosphate, which prevented apoptosis in analogy to 1alpha,25-dihydroxyvitamin D3. The effect of 1alpha,25-dihydroxyvitamin D3 was specific as the almost inactive precursor cholecalciferol neither induced sphingosine-1-phosphate formation nor prevented cells from apoptosis. Besides this, the cytoprotective aptitude of 1alpha,25-dihydroxyvitamin D3 was completely abolished by the sphingosine kinase inhibitor N,N-dimethylsphingosine, which blocked sphingosine-1-phosphate formation. Moreover, sphingosine-1-phosphate was able to restore the cytoprotective effect of 1alpha,25-dihydroxyvitamin D3 in the presence of N,N-dimethylsphingosine. Taken together, here we report for the first time that 1alpha,25-dihydroxyvitamin D3 protects keratinocytes from apoptosis and additionally this cytoprotection is mediated via the formation of sphingosine-1-phosphate.

Topics: Apoptosis; Calcitriol; Cell Division; Cell Survival; Cells, Cultured; Ceramides; Cytoprotection; Humans; Hydroxycholecalciferols; Keratinocytes; Lysophospholipids; Necrosis; Phosphotransferases (Alcohol Group Acceptor); Proto-Oncogene Proteins c-bcl-2; Sphingosine; Tumor Necrosis Factor-alpha; Ultraviolet Rays