Symmetric Dimethylarginine Research Articles

PS-BPC11-9: ADMA/SDMA RATIO AND ITS RELATIONS TO URIC ACID AND ALBUMIN IN CONTINUOUS AMBULATORY PERITONEAL DIALYSIS PATIENTS IN INDONESIA

Background and Aims: Asymmetric Dimethylarginine (ADMA) and Symmetric Dimethylarginine (SDMA), rarely reported together, are markers of endothel dysfunction and kidney function. ADMA was inversely related to albumin, while SDMA was positively correlated to albumin. Meanwhile, uric acid was positively correlated to both SDMA and ADMA. By using ADMA and SDMA as ratio, we postulated that their ratio can more precisely predict activity dimethylarginine dimethylaminohydrolase (DDAH), a key enzyme that metabolized ADMA, and not SDMA, and can be used as marker of NO activity. Low NO activity is related to high vascular tonus and peripheral resistance, hypertension and low Sodium excretion and cardiac output. Method: This was a cross-sectional study conducted in adults underwent chronic Continuous Ambulatory Peritoneal Dialysis (CAPD) in tertiary hospital in Jakarta, Indonesia. Uric acid, albumin, ADMA and SDMA levels were measured at the same time patients controlled to outpatient clinic after washout from hyperuricemia drug for 2 to 4 weeks. Analysis performed using Pearson for normal distribution data, Spearman for nonparametric analysis and linier regression test and ROC curve with SPSS v20. Results and Discussion: Total of 55 subjects (83%) were included, male 63.6%, mean age of 40.71 ± 14,4 year old. Median of CAPD use was 27 months. Causes of CKD were glomerulonephritis (47.3%), diabetes mellitus (25.5%), hypertension (25.5%) and polycystic kidney disease (1.8%). At the time of research, 90.9%, were uncontrolled hypertension patients. Median level of UA was 7.30 ± 1.59 mg/dl and 60% had UA levels of 7 mg/dl or higher. Albumin level lower than 3.5 g/dl (mean 3.55 ± 0.49 g/dl) were 24 patients (43.6%). Median ADMA level was 87.5 ± 16.64 ng/ml and SDMA level was 633.73 ± 231.54 ng/mL. Median ADMA/SDMA ratio was 0.13 + 0,08 ng/ml. ADMA, SDMA, ADMA/SDMA ratio were correlated with uric acid with p = 0.027 (95%CI 0.175–0.471), p < 0.001 (95%CI 0.688–0.928) and p < 0.001 (95%CI -14,96 to -5,03) with sensitivity and specificity 91% and 95%, 97% and 68% and 79% and 96%, respectively. ADMA, SDMA, ADMA/SDMA ratio were not statistically significantly correlated with albumin with p = 0.26, p = 0.14 and p = 0.23, respectively. Albumin level was caused by many factors that are not in the scope of this research, this might explain this result. Conclusion: ADMA/SDMA ratio had comparable sensitivity and specificity to ADMA or SDMA alone when correlated with uric acid in CAPD patients. Albumin might not correlated with ADMA, SDMA, or ADMA/SDMA ratio in CAPD patients.

Diagnostic assessment of two-dimensional shear wave elastography in relation to dimethyl arginine levels in dogs with chronic kidney disease.

In veterinary medicine, previous studies regarding the diagnostic performance of shear wave elastography (SWE) in chronic kidney disease (CKD) are not consistent with each other. Moreover, there has been no study evaluating the relationship between symmetric dimethyl arginine (SDMA) concentration and renal shear wave velocity (SWV) using two-dimensional SWE (2D SWE) in dogs with CKD. This study aimed to evaluate the diagnostic capability of 2D SWE in dogs with CKD and to assess the relationship between renal SWV and SDMA concentration. Dogs with healthy kidneys and dogs with CKD underwent 2D SWE and SDMA assay. Renal stiffness was estimated as renal SWV in m/s. SDMA concentration had a weak positive correlation with the left (r = 0.338, p = 0.022) and right renal SWV (r = 0.337, p = 0.044). Renal SWV was not significantly different between healthy kidney and CKD groups in the left (p = 0.085) and right (p = 0.171) kidneys. 2D SWE may could not distinguish between dogs with healthy kidney and dogs with early stage of CKD, but it would be useful for assessing the serial change of renal function in dogs.

Renal resistive index in obese and non‑obese cats.

This study aimed to compare renal function between obese and normal‑weight healthy cats, using intrarenal resistive index (RI), serum symmetric dimethylarginine (SDMA), and serum creatinine, and to identify the variables that might influence intrarenal RI. Thirty crossbred client‑owned cats met the inclusion criteria and were allocated into two groups: Control and Obese. Body weight, body mass index (BMI), body condition score (BCS), SAP, serum SDMA, urea, and creatinine were evaluated. B‑mode and Doppler ultrasound of the kidneys were done. RI evaluation was in the interlobar artery. SDMA and intrarenal RI were compared between groups, also considering the gender of the cats. A correlation analysis between intrarenal RI with the other parameters was performed. SDMA was higher in the Obese group. Intrarenal RI was higher in females than males in the Obese group. Obese females presented higher RI and SDMA than Control females. A positive correlation was observed between RI, age, body weight, and BMI. Six obese cats (40%) showed increased RI. The increase in body weight, BCS, and BMI resulted in a simultaneous increase in RI and SDMA. The RI may assist in monitoring renal function, and may be associated with preclinical kidney changes in obese cats.

Asymmetric Dimethylarginine in an NMU-induced Rat Mammary Tumor Model.

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) production and a newly discovered risk factor involved in endothelial dysfunction and adverse cardiovascular events. Recently, both NO and ADMA have also emerged as molecules of interest in carcinogenesis and tumor growth progression. Our earlier studies have confirmed elevated plasma ADMA levels in patients with hematological malignancies. However, the cause of elevated ADMA was unclear. The aim of this study was to assess the concentrations of ADMA, symmetric dimethylarginine (SDMA) and L-arginine in rats exposed to N-nitroso-N-methylurea (NMU) for the induction of mammary tumors. A total of 95 female rats of the Sprague-Dawley strain were used in the study. Plasma concentrations of ADMA, SDMA and L-arginine were quantified and statistically analyzed. Mean ADMA levels were higher in the tumor-bearing group compared to the control group. Mean plasma levels of SDMA and L-Arginine were not significantly different between the groups. The L-ARG/ADMA ratio was lower in rats with tumors compared to controls. Histological assessment confirmed expression of ADMA within the tumor cells, which strongly suggests that these tumor cells were the source of ADMA. Other studies are warranted to further explain the role of ADMA in neoplastic diseases.

Asymmetric and symmetric dimethylarginine gingival crevicular fluid levels in periodontitis.

This study aimed to evaluate the level of ADMA (asymmetric dimethylarginine), SDMA (symmetric dimethylarginine), and IL-1β (Interleukin-1β) in gingival crevicular fluid (GCF) from periodontitis patients and control subjects. ADMA and SDMA are potentially hazardous non-proteinogenic amino acids that limit nitric oxide (NO) synthesis and have many functions in various human disorders. ADMA causes a structural change in nitric oxide synthase, while SDMA blocks arginine cell uptake. Increased plasma ADMA has been widely recognized as a "trigger" initiating impaired NO bioavailability and vascular dysfunction, which ultimately leads to oxidative stress. Twenty-five patients with periodontitis (P) (Stage III, Grade C, n=25) and 20 control (C) subjects were included in the study. The IL-1β level of GCF was measured by enzyme immunoassay (ELISA) and ADMA and SDMA by liquid chromatography-mass spectrometry (LC-MS/MS). Periodontitis patients had higher clinical parameters than controls (p < .001). Levels of IL-1β, ADMA and SDMA GCF were statistically significantly higher in group P than in group C (respectively; p=.003, p < .0001, p < .0001). There was no difference in the ADMA/SDMA ratio (p=.312) between the groups. There were significant positive correlations between clinical periodontal parameters and IL-1β, ADMA, and SDMA levels (p < .05). ADMA and SDMA levels were significantly correlated with IL-1β (p < .05). These findings suggest that ADMA and SDMA may be involved in the pathogenesis of the periodontal disease.

Association of serum ADMA, SDMA and L-NMMA concentrations with disease progression in COVID-19 patients.

This study determines and compares the concentrations of arginine and methylated arginine products ((asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), n-monomethyl-1-arginine (L-NMMA) and homoarginine (HA)) for assessment of their association with disease severity in serum samples of COVID-19 patients. Serum arginine and methylated arginine products of 57 mild-moderate and 29 severe (N = 86) COVID-19 patients and 21 controls were determined by tandem mass spectrometry. Moreover, the concentrations of some of the routine clinical laboratory parameters -neutrophil lymphocyte ratio (NLR), C-reactive protein, ferritin, D-dimer, and fibrinogen measured during COVID-19 follow-up were also taken into consideration and compared with the concentrations of arginine and methylated arginine products. Serum ADMA, SDMA and L-NMMA were found to be significantly higher in severe COVID-19 patients, than in both mild-moderate patients and the control group (P < 0.001 for each). In addition, multiple logistic regression analysis indicated L-NMMA (cut-off =120 nmol/L OR = 34, 95% confidence interval (CI) = 3.5-302.0, P= 0.002), CRP (cut-off = 32 mg/L, OR = 37, 95% CI = 4.8-287.0, P < 0.001), and NLR (cut-off = 7, OR = 22, 95% CI = 1.4-335.0, P = 0.020) as independent risk factors for identification of severe patients. The concentration of methylated arginine metabolites are significantly altered in COVID-19 disease. The results of this study indicate a significant correlation between the severity of COVID-19 disease and concentrations of CRP, NLR and L-NMMA.

PRMT1 and PRMT5: on the road of homologous recombination and non-homologous end joining.

DNA double-strand breaks (DSBs) are widely accepted to be the most deleterious form of DNA lesions that pose a severe threat to genome integrity. Two predominant pathways are responsible for repair of DSBs, homologous recombination (HR) and non-homologous end-joining (NHEJ). HR relies on a template to faithfully repair breaks, while NHEJ is a template-independent and error-prone repair mechanism. Multiple layers of regulation have been documented to dictate the balance between HR and NHEJ, such as cell cycle and post-translational modifications (PTMs). Arginine methylation is one of the most common PTMs, which is catalyzed by protein arginine methyltransferases (PRMTs). PRMT1 and PRMT5 are the predominate PRMTs that promote asymmetric dimethylarginine and symmetric dimethylarginine, respectively. They have emerged to be crucial regulators of DNA damage repair. In this review, we summarize current understanding and unaddressed questions of PRMT1 and PRMT5 in regulation of HR and NHEJ, providing insights into their roles in DSB repair pathway choice and the potential of targeting them for cancer therapy.

Do arginine metabolites have a role in periodontitis due to smoking? A new perspective.

Cigarette consumption is common around the world and besides its negative effects on health, and its effects on periodontitis draw attention. Arginine metabolites are involved in the pathogenesis of several systemic inflammatory diseases' including cardiovascular diseases. Our aim was to determine periodontitis and healthy individuals' arginine metabolites and IL-6 levels in saliva and serum and to evaluate those according to smoking status. The study consisted of four groups: healthy individuals (control [C]; n=20), smokers with healthy periodontium (S-C; n=20), nonsmokers with Stage-III Grade-B generalized periodontitis (P; n=20) and smokers with Stage-III Grade-C generalized periodontitis (S-P; n=18). Periodontal parameters were measured. Analysis of methylated arginine metabolites was performed by LC-MS/MS, and IL-6 levels were determined by ELISA kits. In nonsmokers, salivary concentrations of asymmetric dimethylarginine (ADMA) and symmetrical dimethylarginine (SDMA) were higher in the periodontitis than control (p < 0.001, p=0.010). Smokers with periodontitis exhibited higher ADMA (p=0.033, p < 0.001) and arginine (p=0.030, p=0.001) saliva concentrations than smoking and nonsmoking controls. Our results demonstrated that salivary concentrations of ADMA and SDMA were associated with periodontitis. Smoking increased ADMA, SDMA and NG -monomethyl L-arginine (L-NMMA) levels in serum only in periodontitis patients.

Outcome of Acquired Fanconi Syndrome Associated with Ingestion of Jerky Treats in 30 Dogs.

Acquired canine proximal renal tubulopathy (Fanconi syndrome) related to excessive ingestion of jerky treats has been recognized since 2007. This study aimed to improve knowledge about the syndrome's characteristics, especially long-term outcome. By reaching out to veterinarians and dog owners, dogs suspected of jerky induced Fanconi syndrome were identified. The dog's medical records were reviewed, and owners interviewed. Data was analyzed using linear mixed models (p &lt; 0.05 was considered statistically significant) and descriptive statistics are reported. Thirty dogs, median body weight 6.8 (range 1.2-59) kg and age 6.5 (0.5-14) years, were enrolled as suspected cases based on history of jerkey ingestion and confirmed normoglycemic/hypoglycemic glycosuria. Clinical signs included polydipsia (23/30), polyuria (21/30), lethargy (19/30), weight loss (15/30), hyporexia (11/30), vomiting (7/30), diarrhea (7/30) and no clinical signs (2/30). Para-clinical findings included azotemia (6/28), hypophosphatemia (9/25), metabolic acidosis (3/8), hypokalemia (6/20), proteinuria (13/26), aminoaciduria (4/4), hematuria (22/29) and ketonuria (7/27). Clinical signs resolved in 22/28 within 11 (0.3-52) weeks and glycosuria resolved in 28/30 within 6.5 (1-31) weeks. There were no associations between serum creatinine and urea and the amount/duration of jerky ingestion. Serum symmetric dimethylarginine concentrations were only available for a few dogs, therefore no conclusion was achieved on a possible association with duration of jerky ingestion. Apart from a larger percentage of dogs achieving complete recovery, the current findings are in agreement with previous reports.

Renoprotective effects of docosahexaenoic acid in cats with early chronic kidney disease due to polycystic kidney disease: a pilot study.

Lipids containing n-3 fatty acids have been reported to have protective effects on renal function, with docosahexaenoic acid (DHA) expected to be particularly effective. However, no reports have demonstrated the renoprotective effects of DHA-enriched lipids in cats with chronic kidney disease (CKD). Therefore, the aim of this pilot study was to examine the renoprotective effects of DHA-enriched fish oil in cats. Five healthy cats and five cats with early non-azotaemic CKD due to autosomal dominant polycystic kidney disease (PKD) were orally administered DHA-enriched fish oil in liquid form (250 or 500 mg/kg body weight [BW] and 250 mg/kg BW of DHA, respectively) for 28 days. Inappropriately dilute urine and markedly increased urinary N-acetyl-d-glucosamine (NAG) index were detected in cats with PKD before DHA-enriched fish oil administration. Changes in the fatty acid composition ratio in the blood of all 10 cats were assessed after orally administering 250 mg/kg of DHA. Post-administration, no adverse clinical effects were observed, and blood and urine tests were within the reference intervals in healthy cats. Cats with PKD showed significantly decreased serum symmetric dimethylarginine (SDMA), urine protein:creatinine ratio (UPC) and urinary NAG index at post-administration. Furthermore, oral administration of DHA-enriched fish oils significantly decreased the blood concentration ratio of arachidonic acid (AA) in cats with PKD post-administration. Furthermore, the concentration ratio of DHA in the blood significantly increased in both healthy cats and cats with PKD, and the DHA:AA ratio also increased. Oral administration of DHA-enriched fish oils for 28 days significantly decreased blood AA levels and significantly increased DHA concentration and DHA:AA ratios in cats with PKD, and improved the SDMA, UPC and urinary NAG index, suggesting its potential for renoprotective effects in cats with early non-azotaemic CKD due to PKD.

Constitutive PRMT5 Expression Drives Spontaneous Lymphoblastic Lymphoma In Vivo

Protein arginine methyltransferase 5 (PRMT5) is a highly conserved enzyme that catalyzes monomethylation and symmetric dimethylation of arginine residues on many target proteins, including histones and non-histone proteins (P53, P65/NFκB, E2F1, AKT and spliceosome proteins). (Shailesh, 2018) PRMT5 is overexpressed in numerous solid and hematologic malignancies. (Yang, 2013) It is associated with several oncogenic mechanisms, from driving the malignant phenotype to promoting tumor cell survival and invasiveness. While preliminary data has shown PRMT5 to be upregulated in T-ALL/LBL, the mechanism by which PRMT5 promotes T-ALL/LBL has yet to be established. (Mei, 2019) Preclinical transgenic models of leukemia and lymphoma driven by a variety of oncogenes (MYC, NOTCH) have identified PRMT5 activity as a critical component for the driver phenotype. (Li, 2015) To test the hypothesis that overexpression of this enzyme is capable of malignant driver activity, we developed a transgenic (Tg) mouse model designed to over-express PRMT5 in the lymphoid compartment. (Figure 1) Tg mice were created by injecting a linearized pBH-Eμ-PRMT5 vector into FVB/N pronuclei and implanted into pseudo-pregnant FVB/N mice. We obtained five founder lines crossed with wild-type FVB/N mice to obtain the F1 generation. RT-PCR for PRMT5 showed preferential overexpression in Tg B lymphocytes compared to control FVB/N mice (p<0.01) but also overexpression in T and NK cell compartments due to leaky Eμ phenotype. We observed a high percentage of F1 progeny from all five founder lines to develop spontaneous lymphoproliferative disease compared to control mice (p<0.001). Immunophenotypic and pathologic evaluation was performed by flow cytometry, immunohistochemistry, and clonality by IgH and TCR Vβ PCR analysis. Pathologic features showed a range of lymphoid neoplasms, with pre-T lymphoblastic immunophenotype being the most common. Tumor-bearing animals demonstrated diffuse involvement of all organs, peripheral blood, and bone marrow. Pre-T lymphoblastic lymphomas (LBL) showed expression of cytoplasmic CD3, surface CD4 (dim), TdT, and variable Ki67 with constitutive overexpression of PRMT5, MYC, CYCLIN D1, Ki67, and CD99. We developed a T-LBL cell line (Tg813) derived from a thymic tumor that displayed clonal TCR Vβ.17 gene rearrangement, intracellular TdT and CD3 expression with dim surface CD4 expression, immunophenotypic characteristics consistent with T-ALL/LBL. This cell line was maintained in culture and successfully passaged in immune-competent FVB mice, which led to disseminated disease, suggesting that PRMT5 drives an immune escape program in vivo. Preliminary data demonstrate that tumor-bearing animals display a prominent population of normal and neoplastic T-cell subsets with strong PD1 expression. PD1 expression in T-ALL/LBL has been linked with oncogenic function (Yang, 2019) and, in normal T-cell subsets, is a marker for exhaustion. (Lee, 2015) In addition, treatment of our Tg813 in vitro model with PRMT5 knockdown (CRISPR CAS9) and selective inhibition with a SAM-competitive inhibitor (C220 and PRT382, Prelude Therapeutics) showed a reduction of global symmetric dimethyl arginine, loss of the PRMT5 epigenetic mark H4R3(Sme2) and decreased proliferation with cell cycle arrest by day 6, findings consistent with PRMT5-dependent driver activity. Engraftment of Tg813 into SCID and immunocompetent FVB/N mice led to disseminated lymphomas (mean: 21 days). Tg813 engrafted animals treated with C220 (10mg/kg) showed improved survival compared to vehicle-treated mice (p<0.001). This preclinical model supports the hypothesis that PRMT5 over-expression provides cancer driver activity and a unique opportunity to study the role of this oncogene in an immune-competent, in vivo model system. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

PRMT5 Inhibition Enhances Elimination of FLT3-ITD AML Stem Cells in Combination with TKI Treatment

Introduction: The FMS-like tyrosine kinase-3 (FLT3) internal tandem duplication (ITD) is a commonly observed molecular abnormality in AML and is associated with increased risk of relapse and poor survival. FLT3 tyrosine kinase inhibitors (TKIs) have shown benefit in the treatment of FLT3-ITD AML but do not eliminate primitive leukemia stem cells and are limited by frequent relapse and resistance. There is considerable interest in understanding mechanisms of FLT3-ITD AML stem cell persistence to develop strategies to enhance their elimination. Here we evaluated the role of epigenetic regulators in maintenance of human FLT3-ITD AML cells. Methods: We performed an epigenetic screen using a well-curated collection of epigenetic probes (Epigenetics Probes Collection, Structural Genomics Consortium, n=41). FLT3-ITD (MOLM-13 and MV4-11), and FLT3-WT AML cell lines (OCI-AML3) were exposed to inhibitors for 7 days and CellTiter-Glo and Annexin V labeling were used to evaluate response. We further studied the effects of the PRMT5 inhibitors, GSK-591and LLY-283, with and without FLT3 TKIs, Quizartinib and Giltertinib, on FLT3-ITD+ and FLT3-WT AML cell lines. Synergy between PRMT5 inhibitors and TKIs was analyzed with Calcusyn software using the median effects method of Chou and Talalay. We performed in vivo analysis of the effects of PRMT5 and FLT3 kinase inhibition in a FLT3-ITD Tet2 deleted genetic mouse model of FLT3-ITD AML. Finally, we studied in vitro and in vivo effects of PRMT5 and FLT3 inhibition on primary CD34+ cells from FLT3-ITD AML patients. Results: Screening with epigenetic probes identified PRMT5 arginine methyl transferase inhibitors, GSK591 and LLY-283, amongst the probes with the greatest inhibitory effects on growth and survival of FLT3-ITD MOLM-13 and MV4-11 AML cells. In contrast, inhibitors of PRMT1, PRMT3. PRMT4 and PRMT7 were associated with significantly less growth inhibition of FLT3-ITD AML cell lines. FLT3-WT OCI-AML3 AML cells were also sensitive to PRMT5 inhibition. However, ectopic FLT3-ITD expression in FLT3-WT THP1 cells led to enhanced sensitivity to PRMT5 inhibition. The combination of GSK-591 or LLY-283 with either of the FLT3 TKIs Quizartinib or Giltertinib resulted in synergistically enhanced inhibition of FLT3-ITD+ MOLM-13 and MV4-11 AML cells, compared to TKI or PRMT5 inhibitor alone. On the other hand, FLT3-WT cells were insensitive to TKI alone, and the combination of PRMT5 inhibitor and TKI did not lead to synergistic enhancement of inhibition. We next evaluated the effect of PRMT5 inhibition in a genetic mouse model of FLT3-ITD AML. AML mice were treated with vehicle (control), Giltertinib, LLY283 or the combination of Giltertinib and LLY283 for 2 weeks. AML cells from LLY-283 treated mice showed significantly reduced symmetric dimethylarginine in SmB protein. Significant inhibition of mature AML cells and of AML stem and progenitor cells was seen in the bone marrow and spleen of mice treated with LLY283, and significantly enhanced inhibition was seen with combination of LLY283 and Giltertinib (Figure 1a). Finally, we evaluated the effect of TKI (Giltertinib or Quizartinib), LLY283 or the combination on primary FLT3-ITD AML CD34+ cells with diverse co-mutations (n=8). LLY283 significantly inhibited FLT3-ITD AML CD34+ cell growth and survival in vitro by itself, and the combination of TKI and LLY283 resulted in significantly increased inhibition compared to TKI alone, including both CD34+CD38- and CD34+CD38+ cell populations (Figure 1b). In addition, in vivo treatment of immunodeficient NRGS mice xenografted with human FLT3-ITD AML cells with LLY283 or the combination of Giltertinib and LLY283 led to significant depletion of AML cells in BM and spleen. Conclusion: An epigenetic probe screen identified PRMT5 as a key regulator of FLT3-ITD AML cell viability. We show an important role for PRMT5 in maintenance of murine and human FLT3-ITD AML stem cells and in their persistence following FLT3 TKI treatment. Treatment with a PRMT5 inhibitor in combination with a FLT3 TKI could be a promising approach to enhance elimination of FLT3-ITD AML stem cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

EXTH-103. DISCOVERY AND PHARMACOLOGICAL CHARACTERIZATION OF SKL27969, A NOVEL BRAIN-PENETRATING PRMT5 INHIBITOR WITH STRONG ANTITUMOR ACTIVITY, IN GLIOBLASTOMA AND BRAIN METASTASIS PRECLINICAL MODELS

Abstract Protein arginine methyltransferase 5 (PRMT5), a type II methyltransferase, regulates cellular processes such as survival, proliferation, and apoptosis by inducing symmetric arginine dimethylation of multiple cellular proteins involved in the regulation of cellular transcription and RNA splicing. Elevated tumor PRMT5 protein level has recently been correlated with poor prognosis and poor patient survival in various human cancers, including glioblastoma multiforme (GBM), lung cancer, breast cancer, and ovarian cancer. SKL27969 is a highly selective and potent PRMT5 inhibitor that is designed for brain penetration. It strongly binds to S-adenosylmethionine (SAM)-bound PRMT5/MEP50 (methylosome protein 50), leading to potent cellular symmetric dimethylarginine (SDMA) inhibition with an IC50 of 3.4 nM. Cell line panel profiling of SKL27969 revealed a broad spectrum of anti-proliferative activity in various cancer cell types, including CNS tumors, solid cancers prone to spread to brain, other solid cancers, and hematologic cancers. SKL27969 showed excellent brain exposure, with total and unbound partition coefficient (Kp and Kpuu) values of 6.57 and 1.08, respectively, and high tumor-to-plasma ratio, which resulted in significant survival benefit in orthotopic GBM xenograft models and metastatic brain cancer models such as intracranial xenograft models of non-small cell lung cancer (NSCLC) or triple-negative breast cancer (TNBC). Administration of SKL27969 showed strong tumor growth suppression in subcutaneous xenograft models of GBM, NSCLC, or TNBC. High correlation between tumor and plasma SDMA levels after SKL27969 treatment were observed in the preclinical models. Furthermore, SKL27969 showed potentiation of DNA damage when used in combination with DNA-damaging agents, which may suggest possible benefit as a combination therapy. Given the therapeutic target potential of PRMT5 in glioblastoma and other brain metastatic cancers, and the preclinical efficacy of SKL27969, these results have enabled the initiation of Phase 1/2 open-label, multicenter dose-finding study in patients with advanced solid tumors (NCT05388435).

Effects of tofacitinib therapy on arginine and methionine metabolites in association with vascular pathophysiology in rheumatoid arthritis: A metabolomic approach.

Rheumatoid arthritis (RA) has been associated with changes in lipid, arginine and NO metabolism with increased cardiovascular (CV) risk. The aim of this study is to evaluate the effect of tofacitinib, a Janus kinase (JAK) inhibitor, on arginine and methionine metabolism in correlation with inflammation, functional and pathological vascular changes during one-year treatment of patients with RA. Thirty RA patients with active disease were treated with either 5 mg bid or 10 mg bid tofacitinib for 12 months. We determined DAS28, CRP, IgM rheumatoid factor (RF) and anti-cyclic citrullinated peptide (CCP) levels. We assessed brachial artery flow-mediated vasodilation (FMD), carotid intima-media thickness (IMT) and pulse-wave velocity (PWV) by ultrasound at baseline and after 6 and 12 months. We also determined plasma L-arginine, L-citrulline, L-ornithine, inducible nitric oxide synthase (iNOS), asymmetric (ADMA) and symmetric dimethylarginine (SDMA), L-N-monomethyl-arginine (L-NMMA), cysteine, homocysteine, and methionine levels at these time points. Twenty-six patients (13 on each arm) completed the study. CRP, ESR and DAS28 decreased significantly during one-year treatment with tofacitinib. Arginine and ADMA showed a negative univariate correlation with CRP but not with FMD, PWV or IMT. Tofacitinib at 10 mg bid significantly increased L-arginine, L-ornithine, iNOS and methionine levels after 12 months. ADMA and SDMA levels did not change in our study. Methionine showed negative correlation with FMD at baseline and positive correlation with PWV after 12 months. No change was observed in FMD and PWV but a significant increase was measured in IMT at 6 and 12 months. Multivariate analysis indicated variable correlations of L-arginine, L-citrulline, ADMA, L-NMMA, homocysteine and methionine with DAS28, CRP, ESR and RF but not with anti-CCP after one-year treatment. With respect to vascular pathophysiology, only PWV and methionine correlated with each other. One-year tofacitinib treatment suppressed systemic inflammation and improved functional status in RA. FMD, PWV have not been affected by one-year tofacitinib treatment., while IMT increased further despite treatment. Increased arginine and methionine might contribute to the anti-inflammatory effects of tofacitinib. Increased arginine availability with no changing ADMA may protect FMD and PWV from deterioration. The increase of IMT in the anti-inflammatory environment cannot be explained by arginine or methionine metabolism in this study.

Do Antiepileptic Drugs Change the Levels of Arginine Derivatives in Epileptic Children Treated with Polytherapy? The Results of a Case-Control Study.

Previously, a relation between therapy with antiepileptic drugs (AEDs) and the levels of biochemical parameters was observed in adult patients suffering from epilepsy. Among these biochemical factors, arginine derivatives are often analyzed, i.e., asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), and homoarginine (hArg) as they may be linked with increased risk for cardiovascular disease (CVD). Since the levels of arginine derivatives may increase during therapy, and the treatment of epilepsy often lasts many years, patients may experience CVD faster. The aim of the present study was to analyze the levels of arginine derivatives in children with epilepsy who were treated with multiple AEDs to answer the question whether pediatric patients may be at increased risk of CVD in the future. We prospectively analyzed 21 children suffering from epilepsy who took ≥2 AEDs for at least 6 months and 22 children without epilepsy (reference group). The levels of the arginine derivatives, e.g., ADMA, SDMA, and hArg, were determined in the blood serum using the HPLC method. No differences in both the mean levels of ADMA and SDMA, as well as in the mean values of the arginine derivative ratios, were observed between the groups. The tendency toward a lower level of hArg was found in epileptic patients more than in the reference group (p = 0.091). Epileptic children receiving three or more AEDs had significantly lower concentrations of hArg and values of the hArg/ADMA ratio than the reference group (p = 0.023 and p = 0.006, respectively). In turn, the mean hArg/ADMA ratio was lower in children receiving three or more AEDs compared to children receiving two AEDs (p = 0.002). There was also a positive correlation between the hArg and ADMA concentrations in children with epilepsy taking two AEDs; the higher the level of hArg, the greater the level of ADMA on average (r = 0.650, p = 0.022). Taking three or more AEDs by epileptic children resulted in lower levels of both hArg and the value of the hArg/ADMA ratio.

Asymmetric and Symmetric Dimethylarginines as Renal Function Parameters in Paediatric Kidney Diseases: A Literature Review from 2003 to 2022.

Asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA), inhibitors of nitric oxide synthase, play important roles in many processes in the body. Most data in the literature concern their importance in adult chronic kidney disease (CKD). According to them, SDMA well reflects the glomerular filtration rate (GFR), and higher ADMA concentrations are associated with hypertension and higher mortality. In addition, both substances are recognised cardiovascular risk factors in CKD. The purpose of this review was to summarise the studies on dimethylarginines in renal diseases in children, about which we have much fewer data. The review focuses specifically on dimethylarginine's relation to routinely used renal function parameters. Finally, we analysed 21 of the 55 articles published between 2003 and 2022 on dimethylarginines in kidney diseases in children (from birth to 18 years of age), obtained by searching PubMed/MEDLINE (search terms: "dimethylarginine" and "kidney").

Efficacy of domperidone plus renal diet in slowing the progression of chronic kidney disease in dogs with leishmaniosis

BackgroundChronic kidney disease (CKD) represents the main cause of mortality in dogs with leishmaniosis. Domperidone has recently been reported to improve kidney function in leishmaniotic dogs affected by CKD. Serum symmetric dimethylarginine (sSDMA) has also been shown to be a useful biomarker for earlier detection of decreased kidney function when compared to serum creatinine (sCr). This study aimed to assess the efficacy of domperidone plus renal diet in slowing the progression of nephropathy in leishmaniotic dogs with CKD, evaluating sSDMA and sCr as markers of kidney function.MethodsThis study was a therapeutic, prospective, randomized, controlled, 11-month-long field trial. Dogs were recruited if classified as “exposed” to or “infected” with Leishmania infantum and affected by CKD at early stages. After enrolment (T0), dogs were randomized into groups T (treatment) and C (control). All dogs were fed a renal diet and then followed up at 90 (T1), 210 (T2), and 330 (T3) days after inclusion in the study. At T1 and T2, dogs in group T received an oral suspension of domperidone (1 ml/10 kg once a day for up to 28 days).ResultsTwenty-two dogs (i.e., n = 12 in group T and n = 10 in group C) completed the study. At T0, the entire population of enrolled dogs presented a mean sSDMA value of 16.5 ± 3.4 μg/dl. At T1 (i.e., after 3 months of renal diet), sSDMA was significantly decreased in both groups, with an sSDMA of 13.1 ± 4.4 μg/dl for the entire population involved. From T1 to T3, sSDMA gradually increased in group C, while remaining stable in group T, which continued to show a significantly lower value of sSDMA at T3 than at T0. Regarding sCr, at T0 and T1, the mean values of the entire population of dogs were 1.1 ± 0.3 and 1.0 ± 0.4 mg/dl, respectively, with no statistical differences between groups T and C. In group T, sCr decreased significantly from T0 to T1, while returning at T3 to values similar to T0.ConclusionsIn this study, domperidone plus renal diet reduced the progression of kidney disease in leishmaniotic dogs affected by CKD.Graphical

Stocking density-induced changes in growth performance, blood parameters, meat quality traits, and welfare of broiler chickens reared under semi-arid subtropical conditions.

Broiler production in semi-arid tropics must contend with high levels of heat stress, which have implications on stocking density, bird welfare, and profitability. Under these conditions, optimal stocking densities are likely to be lower than expected, thus must be experimentally determined. Therefore, this study investigated growth performance, haematology, serum biochemistry, carcass and meat quality, sizes of internal organs, and stress biomarkers in response to different stocking densities in broilers reared under semi-arid subtropical conditions. Five hundred, day-old Ross 308 broilers (44.0 ± 5.24 g live-weights) were randomly distributed to 25 replicate pens (1.32 m2 floor space each) to create five stocking densities: 1) 10 birds/pen (SD10); 2) 15 birds/pen (SD15); 3) 20 birds/pen (SD20); 4) 25 birds/pen (SD25); and 5) 30 birds/pen (SD30). There was a linear decrease (P < 0.05) in overall feed intake and weight gain in weeks 2 and 3 as stocking density increased. However, weight gain showed positive and negative quadratic responses (P < 0.05) in weeks 5 and 6, respectively, as stocking density increased. No linear or quadratic effects (P ˃ 0.05) were observed for overall feed conversion ratio, haematological parameters, and meat quality traits in response to stocking density. Symmetric dimethylarginine, alanine transaminase, and albumin levels quadratically increased (P < 0.05) in response to increasing stocking densities. Serum glucose and thigh weight were not affected (P < 0.05) while final body, drumstick, breast, and wing weights linearly declined with stocking density. Increasing stocking density linearly reduced (P < 0.05) the weights of gizzard, proventriculus, caecum, and colon. Stocking density had no effect (P ˃ 0.05) on latency-to-lie. It was concluded that higher stocking densities compromised feed intake, resulting in poor weight gains. Based on weight gain trends observed in week 5, it was determined that Ross 308 broilers should be reared at no more than 20 birds/pen (~15 birds/m2 or 27.27 kg/m2) under the experimental ambient conditions compared to the much higher globally accepted industry standard of 20 birds/m2.

Features of the Maternal Metabolom in Fetal Growth Delay

Цель. Изучить особенности метаболома у беременных с задержкой роста плода.Материалы и методы. В ходе исследования изучены образцы плазмы крови 41 женщины. Беременные были разделены на группы: 1-ю группу составила 21 беременная с задержкой роста плода, 2-ю группу – 20 женщин с физиологическим течением беременности. Анализ образцов был осуществлен с помощью высокоэффективной жидкостной хроматографии. Статистическая обработка полученных данных осуществлялась с использованием пакета программ Statistica 10.0 (SN – AXAR207F394425FA-Q).Результаты. У пациенток с задержкой роста плода в плазме крови выявлено статистически значимое повышение 14 аминокислот и их производных по сравнению с контрольной группой: цистеиновой кислоты (СА), фосфосерина (PSer), аспарагина (Asn), серина (Ser), треонина (Thr), 1-метилгистидина (1MHis), 3-метилгистидина (1MHis), аргинина (Arg), симметричного диметиларгинина (SDMA), α-аминомасляной кислоты (αАВА), β-аминомасляной кислоты (βАВА), метионина (Met), триптофана (Trp), лейцина (Leu). Выявлена умеренная корреляционная связь массы плода с уровнем цистеиновой кислоты (СА), фосфосерина (PSer), серина (Ser), 3-метилгистидина (3MHis), треонина (Thr), аланина (Ala), таурина (Tau), симметричного диметиларгинина (SDMA), α-аминомасляной кислоты (aABA), β-аминомасляной кислоты (βАВА), триптофана (Trp); сильная корреляционная связь определена с аспарагином (Asn).Заключение. Полученные результаты обосновывают перспективность изучения метаболомики в качестве диагностических маркеров задержки роста плода. Purpose. To study the features of the metabolome in pregnant women with fetal growth retardation.Materials and methods. During the study, blood plasma samples from 41 women were studied. The pregnant women were divided into groups: the 1st group consisted of 21 pregnant women with fetal growth retardation, the 2nd group – 20 women with a physiological course of pregnancy. Samples were analyzed by high performance liquid chromatography. Statistical processing of the obtained data was carried out using the Statistica 10.0 software package (SN – AXAR207F394425FA-Q).Results. In patients with fetal growth retardation in blood plasma, a statistically significant increase in 14 amino acids and their derivatives was revealed compared to the control group: cysteic acid (CA), phosphoserine (PSer), asparagine (Asn), serine (Ser), threonine (Thr), 1-methylhistidine (1MHis), 3-methylhistidine (1MHis), arginine (Arg), symmetrical dimethylarginine (SDMA), α-aminobutyric acid (αABA), β-aminobutyric acid (βABA), methionine (Met), tryptophan (Trp), leucine (Leu). A moderate correlation was found between fetal weight and the level of cysteic acid (CA), phosphoserine (PSer), serine (Ser), 3-methylhistidine (3MHis), threonine (Thr), alanine (Ala), taurine (Tau), symmetrical dimethylarginine (SDMA), α-aminobutyric acid (aABA), β-aminobutyric acid (βABA), tryptophan (Trp); a strong correlation was determined with asparagine (Asn).Conclusion. The results obtained substantiate the prospects of studying metabolomics as diagnostic markers of fetal growth retardation.

PSXI-2 Circulating Mirnas as non-Invasive Biomarkers of Chronic Kidney Disease and Calcium Oxalate Uroliths in Felines

Abstract Chronic kidney disease (CKD), characterized by progressive loss of kidney function, is a major cause of mortality in cats. Another complication is the formation of calcium oxalate (CaOx) uroliths in the bladder and kidneys of cats, where inflammation has a role in its pathogenesis. MicroRNAs (miRNAs) are small non-coding RNAs which bind to the target mRNA to reduce its function. Depending on the target mRNA, they regulate transcription of several pro-inflammatory genes. We assessed the expression profiles of circulating miRNAs in blood, collected at the natural end-of-life in colony cats, in a retrospective study, in healthy (n=10; 5.9-16 yr), CaOx (n=10, 8.6-13.4 yr), and CKD (n=10; 8.3-15.4 yr). Circulating concentrations of creatinine, symmetric dimethylarginine, and blood urea nitrogen were significantly greater in CaOx and CKD cats compared with controls, indicating kidney dysfunction. Expression of miRNAs, measured by qRT-PCR, was analyzed using the ΔΔCt method and normalized to U6. There was a significant upregulation of miR-155 in CaOx stone formers compared with controls (p&amp;lt; 0.05). miR-155 is increased in the serum and urine of human patients with nephrolithiasis. miR-155 can also promote oxalate-induced renal oxidative stress by suppressing MGP expression. In humans, miR-155 concentrations are negatively correlated with cytokines including IL-1β, IL-6, and TNF-α, suggesting the involvement of inflammation in nephrolithiasis. However, miR-155 was downregulated in CKD compared with controls. miR-16 was also downregulated in CKD cats. Consistent with this, there is downregulation of both the miRNAs in human patients with Stage 4 CKD. Further, miRNAs downregulated in CaOx, but not in CKD, included miR-17, miR-19b, miR-27a, and miR-27b, whereas let-7a, miR-15a, miR-16, miR-29b, and miR-200a were downregulated only in CKD cats. Taken together, the profile of circulating miRNAs in CaOx and CKD cats may serve as important biomarkers in the initiation and progression of renal dysfunction in cats.