Macrophage-derived Osteopontin (SPP1) Protects From Nonalcoholic Steatohepatitis.

BACKGROUND & AIMS: Nonalcoholic steatohepatitis (NASH) is characterized by steatosis, lobular inflammation, hepatocyte ballooning degeneration, and fibrosis, all of which increase the risk of progression to end-stage liver disease. Osteopontin (OPN, SPP1) plays an important role in macrophage (MF) biology, but whether MF-derived OPN affects NASH progression is unknown. METHODS: We analyzed publicly available transcriptomic datasets from patients with NASH, and used mice with conditional overexpression or ablation of Spp1 in myeloid cells and liver MFs, and fed them a high-fat, fructose, and cholesterol diet mimicking the Western diet, to induce NASH. RESULTS: This study demonstrated that MFs with high expression of SPP1 are enriched in patients and mice with nonalcoholic fatty liver disease (NAFLD), and show metabolic but not pro-inflammatory properties. Conditional knockin of Spp1 in myeloid cells (Spp1KI Mye) or in hepatic macrophages (Spp1KI LvMF) conferred protection, whereas conditional knockout of Spp1 in myeloid cells (Spp1ΔMye) worsened NASH. The protective effect was mediated by induction of arginase-2 (ARG2), which enhanced fatty acid oxidation (FAO) in hepatocytes. Induction of ARG2 stemmed from enhanced production of oncostatin-M (OSM) in MFs from Spp1KI Mye mice. OSM activated STAT3 signaling, which upregulated ARG2. In addition to hepatic effects, Spp1KI Mye also protected through sex-specific extrahepatic mechanisms. CONCLUSION: MF-derived OPN protects from NASH, by upregulating OSM, which increases ARG2 through STAT3 signaling. Further, the ARG2-mediated increase in FAO reduces steatosis. Therefore, enhancing the OPN-OSM-ARG2 crosstalk between MFs and hepatocytes may be beneficial for patients with NASH.

Arginase 2 Promotes Cisplatin-Induced Acute Kidney Injury by the Inflammatory Response of Macrophages.

Acute kidney injury (AKI) is a complex clinical syndrome with a rapid decrease in renal function caused by several different etiologies, including sepsis, ischemia, and the administration of nephrotoxic drugs. Tubular arginase 2 (ARG2), an arginine-metabolic enzyme, is a potential therapeutic target for AKI, but it has not been confirmed under various AKI conditions. The aim of this study was to investigate ARG2 as a therapeutic target for cisplatin-induced AKI. Cisplatin-treated mice with a genetic deficiency in Arg2 had significant amelioration of renal dysfunction, characterized by decreased acute tubular damage and apoptosis. In contrast, cisplatin-induced tubular toxicity was not ameliorated in proximal tubule cells derived from Arg2-deficient mice. Immunohistochemical analysis demonstrated the increased infiltration of ARG2-positive macrophages in kidneys damaged by cisplatin. Importantly, cisplatin-treated Arg2 knockout mice exhibited a significant reduction in kidney inflammation, characterized by the decreased infiltration of inflammatory macrophages and reduced gene expression of interleukin (IL)-6 and IL-1ß. The secretion of IL-6 and IL-1ß induced by lipopolysaccharides was decreased in bone marrow-derived macrophages isolated from Arg2-deficient mice. Furthermore, the lipopolysaccharide-induced elevation of mitochondrial membrane potential and production of reactive oxygen species were reduced in bone marrow-derived macrophages lacking Arg2. These findings indicate that ARG2 promotes the inflammatory responses of macrophages through mitochondrial reactive oxygen species, resulting in the exacerbation of AKI. Therefore, targeting ARG2 in macrophages may constitute a promising therapeutic approach for AKI.

Extensive sequence and structural evolution of Arginase 2 inhibitory antibodies enabled by an unbiased approach to affinity maturation.

Affinity maturation is a powerful technique in antibody engineering for the in vitro evolution of antigen binding interactions. Key to the success of this process is the expansion of sequence and combinatorial diversity to increase the structural repertoire from which superior binding variants may be selected. However, conventional strategies are often restrictive and only focus on small regions of the antibody at a time. In this study, we used a method that combined antibody chain shuffling and a staggered-extension process to produce unbiased libraries, which recombined beneficial mutations from all six complementarity-determining regions (CDRs) in the affinity maturation of an inhibitory antibody to Arginase 2 (ARG2). We made use of the vast display capacity of ribosome display to accommodate the sequence space required for the diverse library builds. Further diversity was introduced through pool maturation to optimize seven leads of interest simultaneously. This resulted in antibodies with substantial improvements in binding properties and inhibition potency. The extensive sequence changes resulting from this approach were translated into striking structural changes for parent and affinity-matured antibodies bound to ARG2, with a large reorientation of the binding paratope facilitating increases in contact surface and shape complementarity to the antigen. The considerable gains in therapeutic properties seen from extensive sequence and structural evolution of the parent ARG2 inhibitory antibody clearly illustrate the advantages of the unbiased approach developed, which was key to the identification of high-affinity antibodies with the desired inhibitory potency and specificity.

Arginine-dependent immune responses.

A growing body of evidence indicates that, over the course of evolution of the immune system, arginine has been selected as a node for the regulation of immune responses. An appropriate supply of arginine has long been associated with the improvement of immune responses. In addition to being a building block for protein synthesis, arginine serves as a substrate for distinct metabolic pathways that profoundly affect immune cell biology; especially macrophage, dendritic cell and T cell immunobiology. Arginine availability, synthesis, and catabolism are highly interrelated aspects of immune responses and their fine-tuning can dictate divergent pro-inflammatory or anti-inflammatory immune outcomes. Here, we review the organismal pathways of arginine metabolism in humans and rodents, as essential modulators of the availability of this semi-essential amino acid for immune cells. We subsequently review well-established and novel findings on the functional impact of arginine biosynthetic and catabolic pathways on the main immune cell lineages. Finally, as arginine has emerged as a molecule impacting on a plethora of immune functions, we integrate key notions on how the disruption or perversion of arginine metabolism is implicated in pathologies ranging from infectious diseases to autoimmunity and cancer.

Arginase 2 negatively regulates sorafenib-induced cell death by mediating ferroptosis in melanoma.

Ferroptosis, a newly defined and iron-dependent cell death, morphologically and biochemically differs from other cell deaths. Melanoma is a serious type of skin cancer, and the poor efficacy of current therapies causes a major increase in mortality. Sorafenib, a multiple kinase inhibitor, has been evaluated in clinical phase trials of melanoma patients, which shows modest efficacy. Emerging evidence has demonstrated that arginase 2 (Arg2), type 2 of arginase, is elevated in various types of cancers including melanoma. To investigate the role and underlying mechanism of Arg2 in sorafenib-induced ferroptosis in melanoma, reverse transcriptase-quantitative polymerase chain reaction, western blot analysis, adenovirus and lentivirus transduction, and in vivo tumor homograft model experiments were conducted. In this study, we show that sorafenib treatment leads to melanoma cell death and a decrease in Arg2 at both the mRNA and protein levels. Knockdown of Arg2 increases lipid peroxidation, which contributes to ferroptosis, and decreases the phosphorylation of Akt. In contrast, overexpression of Arg2 rescues sorafenib-induced ferroptosis, which is prevented by an Akt inhibitor. In addition, genetic and pharmacological suppression of Arg2 is able to ameliorate the anticancer activity of sorafenib in melanoma cells in vitro and in tumor homograft models. We also show that Arg2 suppresses ferroptosis by activating the Akt/GPX4 signaling pathway, negatively regulating sorafenib-induced cell death in melanoma cells. Our study not only uncovers a novel mechanism of ferroptosis in melanoma but also provides a new strategy for the clinical applications of sorafenib in melanoma treatment.

Hydroxyurea improves nitric oxide bioavailability in humanized sickle cell mice.

Despite advancements in disease management, sickle cell nephropathy, a major contributor to mortality and morbidity in patients, has limited therapeutic options. Previous studies indicate hydroxyurea, a commonly prescribed therapy for sickle cell disease (SCD), can reduce renal injury in SCD but the mechanisms are uncertain. Because SCD is associated with reduced nitric oxide (NO) bioavailability, we hypothesized that hydroxyurea treatment would improve NO bioavailability in the humanized sickle cell mouse. Humanized male 12-wk-old sickle (HbSS) and genetic control (HbAA) mice were treated with hydroxyurea or regular tap water for 2 wk before renal and systemic NO bioavailability as well as renal injury were assessed. Untreated HbSS mice exhibited increased proteinuria, elevated plasma endothelin-1 (ET-1), and reduced urine concentrating ability compared with HbAA mice. Hydroxyurea reduced proteinuria and plasma ET-1 levels in HbSS mice. Untreated HbSS mice had reduced plasma nitrite and elevated plasma arginase concentrations compared with HbAA mice. Hydroxyurea treatment augmented plasma nitrite and attenuated plasma arginase in HbSS mice. Renal vessels isolated from HbSS mice also had elevated nitric oxide synthase 3 (NOS3) and arginase 2 expression compared with untreated HbAA mice. Hydroxyurea treatment did not alter renal vascular NOS3, however, renal vascular arginase 2 expression was significantly reduced. These data support the hypothesis that hydroxyurea treatment augments renal and systemic NO bioavailability by reducing arginase activity as a potential mechanism for the improvement on renal injury seen in SCD mice.

Single Nucleotide Polymorphisms in the Arginase 1 and 2 Genes Are Differentially Associated with Circulating l-Arginine Concentration in Unsupplemented and l-Arginine-Supplemented Adults.

BACKGROUND: Genetic variation in arginase may underlie variability in whole blood l-arginine concentrations in unsupplemented and l-arginine-supplemented adults. OBJECTIVES: We aimed to study whether single nucleotide polymorphisms (SNPs) in the arginase 1 (ARG1) and arginase 2 (ARG2) genes are associated with blood l-arginine concentrations in unsupplemented and l-arginine-supplemented individuals. METHODS: In 374 adults (mean ± SD age: 59.6 ± 14.6 y; 180 males), we analyzed SNPs in the ARG1 (rs2246012 and rs2781667) and ARG2 genes (rs3742879 and rs2759757) and their associations with blood l-arginine concentrations. We analyzed associations of haplotypes for the ARG1 gene and for the ARG1 and ARG2 genes combined with blood l-arginine concentrations in supplement users and unsupplemented participants. RESULTS: Of study participants, 120 had low (<42 µmol/L), 133 had medium (42-114 µmol/L), and 121 had high blood l-arginine concentrations (>114 µmol/L); 58 individuals were current l-arginine supplement users. We found a significantly higher prevalence of the minor allele of ARG1 rs2246012 in supplement users with higher blood l-arginine concentrations (P = 0.03). Mean ± SEM l-arginine concentration was 263 ± 9.76 µmol/L in supplement users homozygous for the minor allele of ARG1 rs2246012 (P = 0.004); it was 70.4 ± 25.6 µmol/L in unsupplemented participants homozygous for the minor allele of ARG2 rs3759757 (P = 0.03). The ARG1 haplotype was significantly associated with blood l-arginine concentrations in supplement users (P = 0.046), whereas the combined ARG1/ARG2 haplotype was significantly associated with blood l-arginine concentrations in the cohort as a whole (P = 0.012). CONCLUSIONS: Genetic variability in the ARG1 and ARG2 genes is associated with blood l-arginine concentrations in humans: ARG1 is associated with blood l-arginine concentrations in l-arginine supplement users, whereas ARG2 is associated with blood l-arginine concentrations in unsupplemented participants. Our study is the first to describe a possible functional relation between ARG1 and ARG2 SNPs and blood l-arginine concentrations; genetic variability in ARG1 may explain variation in blood l-arginine concentrations during supplement use and discrepant study results.

Arginase 2 and Polyamines in Human Pancreatic Beta Cells: Possible Role in the Pathogenesis of Type 2 Diabetes.

Arginase 2 (ARG2) is a manganese metalloenzyme involved in several tissue specific processes, from physiology to pathophysiology. It is variably expressed in extra-hepatic tissues and is located in the mitochondria. In human pancreatic beta cells, ARG2 is downregulated in type 2 diabetes. The enzyme regulates the synthesis of polyamines, that are involved in pancreas development and regulation of beta cell function. Here, we discuss several features of ARG2 and polyamines, which can be relevant to the pathophysiology of type 2 diabetes.

Arginase 2 is a mediator of ischemia-reperfusion injury in the kidney through regulation of nitrosative stress.

Kidney ischemia-reperfusion injury is a major cause of acute kidney injury (AKI). Following reduced kidney perfusion, the pathological overproduction of reactive oxygen and reactive nitrogen species play a substantial role in the development of kidney ischemia-reperfusion injury. Arginase 2 (ARG2) competes with nitric oxide synthase for the same substrate, L-arginine, and is implicated in the regulation of reactive nitrogen species. Therefore, we investigated the role of ARG2 in kidney ischemia-reperfusion injury using human proximal tubule cells (HK-2) and a mouse model of kidney ischemia-reperfusion injury. ARG2 was predominantly expressed in kidney tubules of the cortex, which was increased after ischemia-reperfusion injury. In HK-2 cells, ARG2 was expressed in punctate form in the cytoplasm and upregulated after hypoxia-reoxygenation. ARG2 knockdown reduced the level of reactive oxygen species and 3-nitrotyrosine after hypoxia-reoxygenation injury compared with control siRNA. Consistent with these results, in Arg2 knockout mice, abnormal kidney function and the increased acute tubular necrosis score induced by ischemia-reperfusion injury was significantly reduced without any obvious blood pressure changes. Additionally, an accumulation of 3-nitrotyrosine and apoptosis of renal tubule cells were attenuated in Arg2 knockout mice compared with wild-type mice. Inhibition of arginase by Nω-hydroxy-nor-L-arginine alleviated kidney ischemia-reperfusion injury like the results found in Arg2 knockout mice. Thus, ARG2 plays a pivotal role in ischemia-reperfusion-induced AKI by means of nitrosative stress. Hence, an ARG2-specific inhibitor may effectively treat kidney ischemia-reperfusion injury.

Arginine bioavailability and endothelin-1 system in the regulation of vascular function of umbilical vein endothelial cells from intrauterine growth restricted newborns.

BACKGROUND AND AIMS: Intrauterine growth restriction (IUGR) is a major risk factor for perinatal morbidity and mortality, leading to long-term adverse cardiovascular outcomes. The present study aimed to investigate the potential mechanisms in IUGR-associated vascular endothelial dysfunction. METHODS AND RESULTS: Human umbilical vein endothelial cells (HUVECs) were derived from IUGR or normal newborns. We found that the proliferation of IUGR-derived HUVECs was accelerated compared to those from normal subjects. Gene profiles related to vascular function including vasomotion, oxidative stress, and angiogenesis were dysregulated in IUGR-HUVECs. Compared with HUVECs from normal newborns, nitric oxide (NO) production was reduced, with imbalance between endothelial nitric oxide synthase (eNOS) and arginase-2 (Arg-2) in IUGR. Meanwhile, intracellular asymmetric dimethylarginine (ADMA) level was elevated with diminished dimethylarginine dimethylaminohydrolase 1 (DDAH1) expression in IUGR-HUVECs. Furthermore, endothelin-1 (ET-1) and hypoxia-inducible factor 1α (HIF-1α) expression were increased, and endothelin receptor type-B (ETBR) was reduced in the IUGR group. IUGR-HUVECs exposed to hypoxia increased the ratio of ADMA to l-arginine, HIF-1α and protein arginine methyltransferase 1 (PRMT1) expression compared to controls. CONCLUSIONS: The present study demonstrated that the reduction of NO bioavailability and release results from elevated Arg-2, accumulation of intracellular ADMA, and imbalance of ET-1 and ETBR, further leading to IUGR-associated vascular endothelial dysfunction. Our study provides novel evidence on the mechanism underlying fetal programming associated with IUGR, which will serve as potential therapeutic targets in the prevention of adverse cardiovascular consequences in adulthood.

MicroRNA-613 is downregulated in HCMV-positive glioblastoma and inhibits tumour progression by targeting arginase-2.

Glioblastoma is the most common and malignant tumour that occurs primarily in nervous system and has a high morbidity. Research on glioblastoma has recently focused on human cytomegalovirus, belonging to the beta subfamily of Herpesviridae that plays crucial roles in cancer development and progression. This study aimed to investigate the role of human cytomegalovirus-associated microRNA-613 in glioblastoma. In this study, we demonstrate that microRNA-613 expression was frequently reduced in human cytomegalovirus-positive glioblastoma specimens/cells compared with human cytomegalovirus-negative glioblastoma tissue/cells, and a significant correlation was observed between the reduction in microRNA-613 expression and the presence of unfavourable variables, including tumour size (p = 0.0118), World Health Organization stage (p = 0.0169), the overall survival (p = 0.0107) and disease-free (p = 0.0159) survival of patients. Overexpression of microRNA-613 in the glioblastoma cell lines U87 and U251 retarded cell growth and induced cell apoptosis. Upregulation of microRNA-613 inhibited glioblastoma cell clone formation, invasion and migration. Furthermore, we demonstrated that arginase-2 was directly regulated by microRNA-613 and played an essential role in mediating the biological effects of microRNA-613 in glioblastoma. Re-expression of arginase-2 markedly reversed the inhibitory properties of microRNA-613 in glioblastoma cells. Taken together, our data provide compelling evidence that human cytomegalovirus reduced the level of microRNA-613 which functions as an anti-onco-miRNA in glioblastoma, primarily by downregulating the expression of arginase-2.

Arginase 2 deficiency results in spontaneous steatohepatitis: a novel link between innate immune activation and hepatic de novo lipogenesis.

BACKGROUND & AIMS: Innate immune activation has been postulated as a central mechanism for disease progression from hepatic steatosis to steatohepatitis in obesity-related fatty liver disease. Arginase 2 competes with inducible nitric oxide synthase (iNOS) for its substrate and the balance between these two enzymes plays a crucial role in regulating immune responses and macrophage activation. Our aim was to test the hypothesis that arginase 2 deficiency in mice favours progression from isolated hepatic steatosis, induced by high fat feeding, to steatohepatitis. METHODS: Arginase 2-knockout (Arg2(-/-)) mice were studied for changes in liver histology and metabolic phenotype at baseline and after a short term course (7 week) feeding with a high fat (HFAT) diet. In additional experiments, Arg2(-/-) mice received tail vein injections of liposome-encapsulated clodronate (CLOD) over a three-week period to selectively deplete liver macrophages. RESULTS: Unexpectedly, Arg2(-/-) mice showed profound changes in their livers at baseline, characterized by significant steatosis as demonstrated with histological and biochemical analysis. These changes were independent of systemic metabolic parameters and associated with marked mRNA level increases of genes involved in hepatic de novo lipogenesis. Liver injury and inflammation were present with elevated serum ALT, marked infiltration of F4/80 positive cells, and increased mRNA levels of inflammatory genes. HFAT feeding exacerbated these changes. Macrophage depletion after CLOD injection significantly attenuated lipid deposition and normalized lipogenic mRNA profile of livers from Arg2(-/-) mice. CONCLUSIONS: This study identifies arginase 2 as a novel link between innate immune responses, hepatic lipid deposition, and liver injury.

Poly(ADP-ribose)polymerase 1 inhibition protects against age-dependent endothelial dysfunction.

Age-related endothelial dysfunction is closely associated with the local production of reactive oxygen species (ROS) within and in the vicinity of the vascular endothelium. Oxidant-induced DNA damage can activate the nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP-1), leading to endothelial dysfunction in various pathophysiological conditions. The present study aimed to investigate the role of PARP-1 in age-dependent changes in endothelial cell function and its underlying mechanism. Wild-type (WT) and PARP-1(-/-) mice were divided into young (2 months) and old (12 months) groups. Isolated aortic rings were suspended to record isometric tension to assess endothelial function. Nitric oxide (NO) production and content in plasma were detected by spectrophotometry. Superoxide (O2(-) production was detected by dihydroethidium. Expression of PARP-1, endothelial nitric oxide synthase (eNOS), induced nitric oxide synthase (iNOS), and arginase-2 (Arg2) was assessed by western blot analysis. Endothelium-dependent relaxation in response to acetylcholine was lost in old WT, but not PARP-1(-/-), mice. Endothelium-independent vasodilation was not impaired in aging mice. Production of O2(-) was greater in aging WT mice than young or aging PARP-1(-/-) mice. eNOS expression was not affected by aging in WT or PARP-1(-/-) mice, but p-eNOS expression decreased and iNOS and Arg2 levels were upregulated only in aging WT mice. In conclusion, PARP-1 inhibition may protect against age-dependent endothelial dysfunction, potentially by regulating NO bioavailability via iNOS. Inhibition of PARP-1 may help in vascular aging prevention.

Liriodendrin exerts protective effects against chronic endometritis in rats by modulating gut microbiota composition and the arginine/nitric oxide metabolic pathway.

BACKGROUND: Chronic endometritis (CE), a gynecological disease, is characterized by inflammation. Liriodendrin is reported to exhibit anti-inflammatory properties. However, the therapeutic effects of liriodendrin on CE and the underlying molecular mechanisms have not been elucidated. This study aimed to investigate the therapeutic effects of liriodendrin on CE in rats and the underlying mechanisms. METHODS: A CE rat model was established and administered with liriodendrin for 21 days. The serum levels of inflammatory cytokines were examined using enzyme-linked immunosorbent assay. The uterine mRNA levels of cytokines were examined using quantitative real-time polymerase chain reaction analysis. The activation of the Toll-like receptor 4 (TLR4)/NF-κB pathway was investigated using western blotting analysis. The effects of liriodendrin on intestinal flora and serum metabolites were examined using 16S rRNA sequencing and untargeted serum metabolomics, respectively. The protein and mRNA levels of arginase-2 (Arg-2) and the nitric oxide (NO) metabolic pathway-related factors were assessed. Molecular docking was performed to explore the interaction between liriodendrin and Arg-2. RESULTS: Liriodendrin alleviated the CE-induced pathological changes in the uterus, modulated the serum levels of inflammatory cytokines, and downregulated the mRNA and protein levels of TLR4/NF-κB pathway-related factors. Treatment with liriodendrin mitigated the CE-induced upregulation of Firmicutes/Bacteroidetes ratio and Lachnospiraceae abundance and downregulation of Ruminococcaceae abundance. Serum metabolomic analysis revealed that liriodendrin regulated the biosynthesis of choline metabolism pathway-related factors. Liriodendrin suppressed the CE-induced upregulation of Arg-2 and downregulation of inducible nitric oxide synthase (iNOS) expression, and NO levels by directly binding to the amino acid residues of Arg-2 through hydroxyl bonds. CONCLUSIONS: Liriodendrin exerted therapeutic effects on CE in rats through the alleviation of inflammation by modulating the gut microbiota structure, directly downregulating Arg-2, and regulating the arginine/NO metabolic pathway.

Lactate-upregulated ARG2 expression induces cellular senescence in fibroblast-like synoviocytes of osteoarthritis via activating the mTOR/S6K1 signaling pathway.

Cellular senescence was implicated in the pathogenesis of age-related diseases such as osteoarthritis (OA). Increasing evidence suggests that alterations in the OA joint microenvironment play a crucial role in the pathogenesis of OA. This study aims to establish a clear link between the impact of accumulated lactate on the senescence of fibroblast-like synoviocytes (FLS) within the OA microenvironment. OA models and models with intra-articular injection of lactate were established in rat models, histological analyses were performed. Human OA-FLS treated with lactate was analyzed by mRNA sequencing, senescence related experiments and underlying signaling pathway activation were comprehensively evaluated. This study confirmed that OA models and lactate-injection models exhibited higher synovitis scores. Enrichment analyses indicated dysregulated cell cycle and cellular senescence pathways in OA-FLS treated with lactate. Lactate significantly up-regulated arginase 2 (ARG2) expression and promoted OA-FLS senescence, including G1/S arrest, increased reactive oxygen species and ß-galactosidase production, high expression of senescence-associated secretory phenotype factors, which could be attenuated by siRNA-Arg2. The ARG2-mTOR/S6K1 axis was identified as a potential signaling for lactate-induced OA-FLS senescence, and activated mTOR/S6K1 signaling could be reduced by siRNA-Arg2, rapamycin (mTOR inhibitor), and LY294002 (PI3K inhibitor). Our study provides novel targets and insights for OA therapies.

Arginase 2 attenuates ulcerative colitis by antioxidant effects of spermidine.

BACKGROUND: Spermidine suppress oxidative stress and is involved in various disease pathogenesis including ulcerative colitis (UC). Arginase 2 (ARG2) plays a central role in the synthesis of spermidine. This study aimed to clarify the effect of endogenously produced spermidine on colitis. METHODS: The physiological role of ARG2 and spermidine was investigated using Arg2-deficient mice with reduced spermidine. Immunohistochemical staining of the rectum was used to analyze ARG2 expression and spermidine levels in healthy controls and UC patients. RESULTS: In mice with dextran sulfate sodium-induced colitis, ARG2 and spermidine levels were increased in the rectal epithelium. Spermidine protects colonic epithelial cells from oxidative stress and Arg2 knockdown cells reduced antioxidant activity. Organoids cultured from the small intestine and colon of Arg2-deficient mice both were more susceptible to oxidative stress. Colitis was exacerbated in Arg2-deficient mice compared to wild-type mice. Supplementation with spermidine result in comparable severity of colitis in both wild-type and Arg2-deficient mice. In the active phase of UC, rectal ARG2 expression and spermidine accumulation were increased compared to remission. ARG2 and spermidine levels were similar in healthy controls and UC remission patients. CONCLUSIONS: ARG2 produces spermidine endogenously in the intestinal epithelium and has a palliative effect on ulcerative colitis. ARG2 and spermidine are potential novel therapeutic targets for UC.

Multi-dimensional metabolomic profiling reveals dysregulated ornithine metabolism hallmarks associated with a severe acute pancreatitis phenotype.

To reveal dysregulated metabolism hallmark that was associated with a severe acute pancreatitis (SAP) phenotype. In this study, LC-MS/MS-based targeted metabolomics was used to analyze plasma samples from 106 acute pancreatitis (AP) patients (34 mild, 38 moderate, and 34 severe) admitted within 48 hours from abdominal pain onset and 41 healthy controls. Temporal metabolic profiling was performed on days 1, 3, and 7 after admission. A random forest (RF) was performed to significantly determine metabolite differences between SAP and non-SAP (NSAP) groups. Mass spectrometry imaging (MSI) and immunohistochemistry were conducted for the examination of pancreatic metabolite and metabolic enzyme alterations, respectively, on necrosis and paracancerous tissues. Simultaneously determination of serum and pancreatic tissue metabolic alterations using an L-ornithine-induced AP model to discover metabolic commonalities. Twenty-two significant differential metabolites screened by RF were selected to build an accurate model for the prediction of SAP from NSAP (AUC = 0.955). Six of 22 markers were found by MSI with significant alterations in pancreatic lesions, reduced ornithine-related metabolites were also identified. The abnormally expressed arginase2 and ornithine transcarboxylase were further discovered in combination with time-course metabolic profiling in the SAP animal models, the decreased ornithine catabolites were found at a late stage of inflammation, but ornithine-associated metabolic enzymes were activated during the inflammatory process. The plasma metabolome of AP patients is distinctive, which shows promise for early SAP diagnosis. AP aggravation is linked to the activated ornithine metabolic pathway and its inadequate levels of catabolites in in-situ lesion.

Layer-by-Layer Assembly of Renal-Targeted Polymeric Nanoparticles for Robust Arginase-2 Knockdown and Contrast-Induced Acute Kidney Injury Prevention.

The mitochondrial enzyme arginase-2 (Arg-2) is implicated in the pathophysiology of contrast-induced acute kidney injury (CI-AKI). Therefore, Arg-2 represents a candid target for CI-AKI prevention. Here, layer-by-layer (LbL) assembled renal-targeting polymeric nanoparticles are developed to efficiently deliver small interfering RNA (siRNA), knockdown Arg-2 expression in renal tubules, and prevention of CI-AKI is evaluated. First, near-infrared dye-loaded poly(lactic-co-glycolic acid) (PLGA) anionic cores are electrostatically coated with cationic chitosan (CS) to facilitate the adsorption and stabilization of Arg-2 siRNA. Next, nanoparticles are coated with anionic hyaluronan (HA) to provide protection against siRNA leakage and shielding against early clearance. Sequential electrostatic layering of CS and HA improves loading capacity of Arg-2 siRNA and yields LbL-assembled nanoparticles. Renal targeting and accumulation is enhanced by modifying the outermost layer of HA with a kidney targeting peptide (HA-KTP). The resultant kidney-targeting and siRNA loaded nanoparticles (PLGA/CS/HA-KTP siRNA) exhibit proprietary accumulation in kidneys and proximal tubular cells at 24 h post-tail vein injection. In iohexol-induced in vitro and in vivo CI-AKI models, PLGA/CS/HA-KTP siRNA delivery alleviates oxidative and nitrification stress, and rescues mitochondrial dysfunction while reducing apoptosis, thereby demonstrating a robust and satisfactory therapeutic effect. Thus, PLGA/CS/HA-KTP siRNA nanoparticles offer a promising candidate therapy to protect against CI-AKI.

Rapid profiling of fish cell nitrogen metabolism with single-cell Raman spectroscopy: Unveiling enzyme's role in ammonia detoxification.

Ammonia is a prevalent aquatic pollutant that disrupts cellular functions and energy metabolism in fish, posing significant environmental and health threats. This research investigates the critical role of arginase 2 (ARG2) in mitigating ammonia toxicity in fish cells and its implications in adapting to nitrogen metabolism under high ammonia exposure. Through a CRISPR-Cas9 engineered ARG2 knockdown (KD) in the Epithelioma Papulosum Cyprini (EPC) cell line, we first investigated the biochemical responses of ARG2 KD and wild-type (WT) EPC cells to ammonia stress (NH4Cl treatment), showing diminished urea production and decreased cell viability in ARG2 KD cells. Subsequently, single-cell Raman spectroscopy analysis revealed that ARG2 KD cells exhibited profound metabolic shifts, including changes in protein, nucleic acids, lipid and sugar levels, showing the adjusting role of ARG2 in the balance of carbohydrate and nitrogen metabolism. Furthermore, the upregulated responses of various amino acids, such as glutamine, arginine, alanine, glutamic acid, glycine, histidine, phenylalanine and valine, in WT cells after NH4Cl treatment diminished in ARG2 KD cells except for the decrease in aspartic acid, indicating a switching effect of ARG2 in nitrogen metabolism under ammonia stress. This study highlights ARG2's essential role in ammonia detoxification and emphasizes ARG2's protective function and its importance in metabolism, shedding light on the adaptive mechanisms fish cells deploy against high ammonia environments. These insights contribute to deep understanding of aquatic organisms' molecular responses to environmental ammonia pollution, offering potential strategies for their protection.

The Role of the L-Arginine-Nitric Oxide Molecular Pathway in Autosomal Dominant Polycystic Kidney Disease.

Recently, arginine has been proven to play an important role in ADPKD physiopathology. Arginine auxotrophy in ADPKD induces cell hyperproliferation, blocking the normal differentiation of renal tube cells and causing cyst formation. We explored the L-arginine (Arg)-nitric oxide (NO) molecular pathway in ADPKD, a multisystemic arginine auxotrophe disease. We developed a prospective case-control study that included a group of 62 ADPKD subjects with an estimated filtration rate over 60 mL/min/1.73 mp, 26 subjects with chronic kidney disease with an eGFR > 60 mL/min/1.73 mp, and a group of 37 healthy subjects. The laboratory determinations were the serum level of arginine, the enzymatic activity of arginase 2 and inducible nitric oxide synthase, the serum levels of the stable metabolites of nitric oxide (nitrate, direct nitrite, and total nitrite), and the endogenous inhibitors of nitric oxide synthesis (asymmetric dimethylarginine and symmetric dimethylarginine). In the ADPKD group, the levels of the arginine and nitric oxide metabolites were low, while the levels of the metabolization enzymes were higher compared to the control group. Statistical analysis of the correlations showed a positive association between the serum levels of Arg and the eGFR and a negative association between Arg and albuminuria. ADPKD is a metabolic kidney disease that is auxotrophic for arginine. Exploring arginine reprogramming and L-Arg-NO pathways could be an important element in the understanding of the pathogenesis and progression of ADPKD.