Testosterone Enhances KV Currents and Airway Smooth Muscle Relaxation Induced by ATP and UTP through P2Y4 Receptors and Adenylyl Cyclase Pathway.

Numerous studies suggest the involvement of adenosine-5'-triphosphate (ATP) and similar nucleotides in the pathophysiology of asthma. Androgens, such as testosterone (TES), are proposed to alleviate asthma symptoms in young men. ATP and uridine-5'-triphosphate (UTP) relax the airway smooth muscle (ASM) via purinergic P2Y2 and P2Y4 receptors and K+ channel opening. We previously demonstrated that TES increased the expression of voltage-dependent K+ (KV) channels in ASM. This study investigates how TES may potentiate ASM relaxation induced by ATP and UTP. Tracheal tissues treated with or without TES (control group) from young male guinea pigs were used. In organ baths, tracheas exposed to TES (40 nM for 48 h) showed enhanced ATP- and UTP-evoked relaxation. Tetraethylammonium, a K+ channel blocker, annulled this effect. Patch-clamp experiments in tracheal myocytes showed that TES also increased ATP- and UTP-induced K+ currents, and this effect was abolished with flutamide (an androgen receptor antagonist). KV channels were involved in this phenomenon, which was demonstrated by inhibition with 4-aminopyridine. RB2 (an antagonist of almost all P2Y receptors except for P2Y2), as well as N-ethylmaleimide and SQ 22,536 (inhibitors of G proteins and adenylyl cyclase, respectively), attenuated the enhancement of the K+ currents induced by TES. Immunofluorescence and immunohistochemistry studies revealed that TES did not modify the expression of P2Y4 receptors or COX-1 and COX-2, while we have demonstrated that this androgen augmented the expression of KV1.2 and KV1.5 channels in ASM. Thus, TES leads to the upregulation of P2Y4 signaling and KV channels in guinea pig ASM, enhancing ATP and UTP relaxation responses, which likely limits the severity of bronchospasm in young males.

Bioprinting of human dermal microtissues precursors as building blocks for endogenousin vitroconnective tissue manufacturing.

The advent of 3D bioprinting technologies in tissue engineering has unlocked the potential to fabricatein vitrotissue models, overcoming the constraints associated with the shape limitations of preformed scaffolds. However, achieving an accurate mimicry of complex tissue microenvironments, encompassing cellular and biochemical components, and orchestrating their supramolecular assembly to form hierarchical structures while maintaining control over tissue formation, is crucial for gaining deeper insights into tissue repair and regeneration. Building upon our expertise in developing competent three-dimensional tissue equivalents (e.g. skin, gut, cervix), we established a two-step bottom-up approach involving the dynamic assembly of microtissue precursors (µTPs) to generate macroscopic functional tissue composed of cell-secreted extracellular matrix (ECM). To enhance precision and scalability, we integrated extrusion-based bioprinting technology into our established paradigm to automate, control and guide the coherent assembly ofµTPs into predefined shapes. Compared to cell-aggregated bioink, ourµTPs represent a functional unit where cells are embedded in their specific ECM.µTPs were derived from human dermal fibroblasts dynamically seeded onto gelatin-based microbeads. After 9 days,µTPs were suspended (50% v/v) in Pluronic-F127 (30% w/v) (µTP:P30), and the obtained formulation was loaded as bioink into the syringe of the Dr.INVIVO-4D6 extrusion based bioprinter.µTP:P30 bioink showed shear-thinning behavior and temperature-dependent viscosity (gel atT> 30 °C), ensuringµTPs homogenous dispersion within the gel and optimal printability. The bioprinting involved extruding several geometries (line, circle, and square) into Pluronic-F127 (40% w/v) (P40) support bath, leveraging its shear-recovery property. P40 effectively held the bioink throughout and after the bioprinting procedure, untilµTPs fused into a continuous connective tissue.µTPs fusion dynamics was studied over 8 days of culture, while the resulting endogenous construct underwent 28 days culture. Histological, immunofluorescence analysis, and second harmonic generation reconstruction revealed an increase in endogenous collagen and fibronectin production within the bioprinted construct, closely resembling the composition of the native connective tissues.

Pyrimidines maintain mitochondrial pyruvate oxidation to support de novo lipogenesis.

Cellular purines, particularly adenosine 5'-triphosphate (ATP), fuel many metabolic reactions, but less is known about the direct effects of pyrimidines on cellular metabolism. We found that pyrimidines, but not purines, maintain pyruvate oxidation and the tricarboxylic citric acid (TCA) cycle by regulating pyruvate dehydrogenase (PDH) activity. PDH activity requires sufficient substrates and cofactors, including thiamine pyrophosphate (TPP). Depletion of cellular pyrimidines decreased TPP synthesis, a reaction carried out by TPP kinase 1 (TPK1), which reportedly uses ATP to phosphorylate thiamine (vitamin B1). We found that uridine 5'-triphosphate (UTP) acts as the preferred substrate for TPK1, enabling cellular TPP synthesis, PDH activity, TCA-cycle activity, lipogenesis, and adipocyte differentiation. Thus, UTP is required for vitamin B1 utilization to maintain pyruvate oxidation and lipogenesis.

EVALUATION OF COMPONENTS OF THE EXTRACELLULAR PURINERGIC SIGNALING SYSTEM IN HUMAN SEPSIS.

ABSTRACT: Objective: Extracellular purines such as adenosine triphosphate (ATP), uridine triphosphate (UTP), and uridine diphosphate (UDP) and the ATP degradation product adenosine are biologically active signaling molecules, which accumulate at sites of metabolic stress in sepsis. They have potent immunomodulatory effects by binding to and activating P1 or adenosine and P2 receptors on the surface of leukocytes. Here we assessed the levels of extracellular purines, their receptors, metabolic enzymes, and cellular transporters in leukocytes of septic patients. Methods: Peripheral blood mononuclear cells (PBMCs), neutrophils, and plasma were isolated from blood obtained from septic patients and healthy control subjects. Ribonucleic acid was isolated from cells, and mRNA levels for purinergic receptors, enzymes, and transporters were measured. Adenosine triphosphate, UTP, UDP, and adenosine levels were evaluated in plasma. Results: Adenosine triphosphate levels were lower in septic patients than in healthy individuals, and levels of the other purines were comparable between the two groups. Levels of P1 and P2 receptors did not differ between the two patient groups. mRNA levels of ectonucleoside triphosphate diphosphohydrolase (NTPDase) 1 or CD39 increased, whereas those of NTPDase2, 3, and 8 decreased in PBMCs of septic patients when compared with healthy controls. CD73 mRNA was lower in PBMCs of septic than in healthy individuals. Equilibrative nucleoside transporter (ENT) 1 mRNA concentrations were higher and ENT2, 3, and 4 mRNA concentrations were lower in PBMCs of septic subjects when compared with healthy subjects. Concentrative nucleoside transporter (CNT) 1 mRNA levels were higher in PBMCs of septic versus healthy subjects, whereas the mRNA levels of CNT2, 3, and 4 did not differ. We failed to detect differences in mRNA levels of purinergic receptors, enzymes, and transporters in neutrophils of septic versus healthy subjects. Conclusion: Because CD39 degrades ATP to adenosine monophosphate (AMP), the lower ATP levels in septic individuals may be the result of increased CD39 expression. This increased degradation of ATP did not lead to increased adenosine levels, which may be explained by the decreased expression of CD73, which converts AMP to adenosine. Altogether, our results demonstrate differential regulation of components of the purinergic system in PBMCs during human sepsis.

[Mechanism of Zhenwu Decoction in improving renal inflammatory injury in mice with DN of spleen-kidney Yang deficiency syndrome by regulating ROCK/IKK/NF-κB pathway].

To investigate the intervention effect and mechanism of Zhenwu Decoction on diabetic nephropathy(DN) mice of spleen-kidney Yang deficiency syndrome based on the Rho-associated coiled-coil kinase(ROCK)/IκB kinase(IKK)/nuclear factor-κB(NF-κB) pathway. Ninety-five 7-week-old db/db male mice and 25 7-week-old db/m male mice were fed adaptively for one week. The DN model of spleen-kidney Yang deficiency syndrome was induced by Dahuang Decoction combined with hydrocortisone by gavage, and then the model was evaluated. After modeling, they were randomly divided into a model group, high-dose, medium-dose, and low-dose Zhenwu Decoction groups(33.8, 16.9, and 8.45 g·kg~(-1)·d~(-1)), and an irbesartan group(25 mg·kg~(-1)·d~(-1)), with at least 15 animals in each group. The intervention lasted for eight weeks. After the intervention, body weight and food intake were measured. Serum crea-tinine(Scr), blood urea nitrogen(BUN), fasting blood glucose(FBG), urinary albumin(uALb), and urine creatinine(Ucr) were determined. The uALb/Ucr ratio(ACR) and 24 h urinary protein(UTP) were calculated. Renal pathological morphology was evaluated by HE staining and Masson staining. The levels of key molecular proteins in the ROCK/IKK/NF-κB pathway were detected by Western blot. Enzyme-linked immunosorbent assay(ELISA) was used to detect interleukin-1ß(IL-1ß), interleukin-6(IL-6), interleukin-8(IL-8), interleukin-10(IL-10), and tumor necrosis factor-α(TNF-α). Compared with the blank group, the model group showed increased content of BUN, uALb, and SCr, increased values of 24 h UTP and ACR, decreased content of Ucr(P<0.05), enlarged glomeruli, thickened basement membrane, mesangial matrix proliferation, inflammatory cell infiltration, and collagen fiber deposition. The protein expression of ROCK1, ROCK2, IKK, NF-κB, phosphorylated IKK(p-IKK), phosphorylated NF-κB(p-NF-κB), and phosphorylated inhibitor of NF-κB(p-IκB) increased(P<0.05), while the protein expression of inhibitor of NF-κB(IκB) decreased(P<0.05). The levels of inflammatory factors IL-1ß, IL-6, IL-8, and TNF-α increased(P<0.05), while the level of IL-10 decreased(P<0.05). Compared with the model group, the groups with drug treatment showed decreased levels of BUN, uALb, SCr, 24 h UTP, and ACR, increased level of Ucr(P<0.05), and improved renal pathological status to varying degrees. The high-and medium-dose Zhenwu Decoction groups and the irbesartan group showed reduced protein expression of ROCK1, ROCK2, IKK, NF-κB, p-IKK, p-NF-κB, and p-IκB in the kidneys(P<0.05), increased protein expression of IκB(P<0.05), decreased levels of serum inflammatory factors IL-1ß, IL-6, IL-8, and TNF-α(P<0.05), and increased level of IL-10(P<0.05). Zhenwu Decoction can significantly improve renal function and renal pathological damage in DN mice of spleen-kidney Yang deficiency syndrome, and its specific mechanism may be related to the inhibition of inflammatory response by down-regulating the expression of key molecules in the ROCK/IKK/NF-κB pathway in the kidney.

Red ginseng aqueous extract improves mucociliary transport dysfunction and histopathology in CF rat airways.

BACKGROUND: We previously discovered that Korean red ginseng aqueous extract (RGAE) potentiates the TMEM16A channel, improved mucociliary transport (MCT) parameters in CF nasal epithelia in vitro, and thus could serve as a therapeutic strategy to rescue the MCT defect in cystic fibrosis (CF) airways. The hypothesis of this study is that RGAE can improve epithelial Cl- secretion, MCT, and histopathology in an in-vivo CF rat model. METHODS: Seventeen 4-month old CFTR-/- rats were randomly assigned to receive daily oral control (saline, n = 9) or RGAE (Ginsenosides 0.4mg/kg/daily, n = 8) for 4 weeks. Outcomes included nasal Cl- secretion measured with the nasal potential difference (NPD), functional microanatomy of the trachea using micro-optical coherence tomography, histopathology, and immunohistochemical staining for TMEM16a. RESULTS: RGAE-treated CF rats had greater mean NPD polarization with UTP (control = -5.48 +/- 2.87 mV, RGAE = -9.49 +/- 2.99 mV, p < 0.05), indicating, at least in part, potentiation of UTP-mediated Cl- secretion through TMEM16A. All measured tracheal MCT parameters (airway surface liquid, periciliary liquid, ciliary beat frequency, MCT) were significantly increased in RGAE-treated CF rats with MCT exhibiting a 3-fold increase (control, 0.45+/-0.31 vs. RGAE, 1.45+/-0.66 mm/min, p < 0.01). Maxillary mucosa histopathology was markedly improved in RGAE-treated cohort (reduced intracellular mucus, goblet cells with no distention, and shorter epithelial height). TMEM16A expression was similar between groups. CONCLUSION: RGAE improves TMEM16A-mediated transepithelial Cl- secretion, functional microanatomy, and histopathology in CF rats. Therapeutic strategies utilizing TMEM16A potentiators to treat CF airway disease are appropriate and provide a new avenue for mutation-independent therapies.

The Anion Channel TMEM16a/Ano1 Modulates CFTR Activity, but Does Not Function as an Apical Anion Channel in Colonic Epithelium from Cystic Fibrosis Patients and Healthy Individuals.

Studies in human colonic cell lines and murine intestine suggest the presence of a Ca2+-activated anion channel, presumably TMEM16a. Is there a potential for fluid secretion in patients with severe cystic fibrosis transmembrane conductance regulator (CFTR) mutations by activating this alternative pathway? Two-dimensional nondifferentiated colonoid-myofibroblast cocultures resembling transit amplifying/progenitor (TA/PE) cells, as well as differentiated monolayer (DM) cultures resembling near-surface cells, were established from both healthy controls (HLs) and patients with severe functional defects in the CFTR gene (PwCF). F508del mutant and CFTR knockout (null) mice ileal and colonic mucosa was also studied. HL TA/PE monolayers displayed a robust short-circuit current response (ΔIeq) to UTP (100 µM), forskolin (Fsk, 10 µM) and carbachol (CCH, 100 µM), while ΔIeq was much smaller in differentiated monolayers. The selective TMEM16a inhibitor Ani9 (up to 30 µM) did not alter the response to luminal UTP, significantly decreased Fsk-induced ΔIeq, and significantly increased CCH-induced ΔIeq in HL TA/PE colonoid monolayers. The PwCF TA/PE and the PwCF differentiated monolayers displayed negligible agonist-induced ΔIeq, without a significant effect of Ani9. When TMEM16a was localized in intracellular structures, a staining in the apical membrane was not detected. TMEM16a is highly expressed in human colonoid monolayers resembling transit amplifying cells of the colonic cryptal neck zone, from both HL and PwCF. While it may play a role in modulating agonist-induced CFTR-mediated anion currents, it is not localized in the apical membrane, and it has no function as an apical anion channel in cystic fibrosis (CF) and healthy human colonic epithelium.

PDZ Peptide of the ZO-1 Protein Significantly Increases UTP-Induced MUC8 Anti-Inflammatory Mucin Overproduction in Human Airway Epithelial Cells.

Mucus hyperproduction and hypersecretion are observed often in respiratory diseases. MUC8 is a glycoprotein synthesized by epithelial cells and generally expressed in the respiratory track. However, the physiological mechanism by which extracellular nucleotides induce MUC8 gene expression in human airway epithelial cells is unclear. Here, we show that UTP could induce MUC8 gene expression through P2Y2-PLCß3-Ca2+ activation. Because the full-length cDNA sequence of MUC8 has not been identified, a specific siRNA-MUC8 was designed based on the partial cDNA sequence of MUC8. siRNA-MUC8 significantly increased TNF-α production and decreased IL-1Ra production, suggesting that MUC8 may downregulate UTP/P2Y2-induced airway inflammation. Interestingly, the PDZ peptide of ZO-1 protein strongly abolished UTP-induced TNF-α production and increased IL-1Ra production and MUC8 gene expression. In addition, the PDZ peptide dramatically increased the levels of UTP-induced ZO proteins and TEER (trans-epithelial electrical resistance). These results show that the anti-inflammatory mucin MUC8 may contribute to homeostasis, and the PDZ peptide can be a novel therapeutic candidate for UTP-induced airway inflammation.

The Chinese patent medicine Tongfengding capsule for gout in adults: a systematic review of safety and effectiveness.

BACKGROUND: Gout is a common inflammatory arthritis caused by increased serum uric acid levels. Untreated or insufficiently treated gout can lead to deposition of monosodium urate crystals in joints, cartilage, and kidneys. Although Tongfengding capsules, a Chinese patent medicine, have long been used to treat gout, their effects and safety have not been reviewed systematically. This study evaluated its efficacy and safety for gout in adults. METHODS: Randomized controlled trials involving Tongfengding capsule for gout in adults were searched from PubMed, EMBASE, Cochrane Central Register of Controlled Trials, CBM, CNKI, and VIP databases, and analyzed using the Cochrane Handbook criteria. The primary outcome measures were the total effective rate. The secondary outcome measures including the blood uric acid (BUA), 24-h urinary total protein (24-h UTP), blood urea nitrogen (BUN), interleukin (IL)-6, IL-8, tumor necrosis factor-alpha (TNF-α) and adverse effects. The risk of bias was evaluated in all included studies. RevMan ver. 5.3.5 and GRADE profiler was used for data analysis and assessing the quality of evidence, respectively. RESULTS: Six studies (n = 607 Chinese participants) were included. Tongfengding capsules plus conventional treatment significantly increased the total effective rate (RR 1.21, 95% CI 1.11-1.33), while reducing the BUA (MD - 66.05 µmol/L, 95% CI - 81.26 to - 50.84), 24-h UTP (MD - 0.83 g/24 h, 95% CI - 0.96 to - 0.70), BUN (MD - 0.90 mmol/L, 95% CI - 1.60 to - 0.20), IL-6 (MD - 6.99 ng/L, 95% CI - 13.22 to - 0.75), IL-8 (MD - 12.17 ng/L, 95% CI - 18.07 to - 6.27), TNF-α (MD - 8.50 ng/L, 95% CI - 15.50 to - 1.51), and adverse effects (RR 0.21, 95% CI 0.04-0.95). CONCLUSION: Tongfengding capsules plus conventional treatment is safe and beneficial for adults with gout compared with conventional treatment.

Adherence to Mediterranean Diet and Response to an Exercise Program to Prevent Hospitalization-Associated Disability in Older Adults: A Secondary Analysis from a Randomized Controlled Trial.

OBJECTIVES: To investigate the relationship between Mediterranean diet (MedDiet) adherence and response to an exercise and health education program to prevent hospitalization-associated disability (HAD) in acutely hospitalized older adults. DESIGN: Randomized controlled trial. SETTING AND PARTICIPANTS: Secondary analysis of a subset of 109 participants from AGECAR-PLUS study with available data on MedDiet adherence (mean age 87, and range 75-98). INTERVENTION: Participants were randomized into the control group (n = 46, usual care) or the intervention group (n = 63, supervised exercise and health education) at admission. MEASUREMENTS: MedDiet adherence was measured with MEDAS and through urinary total polyphenols (UTP). Functional status was assessed with the Barthel Index. RESULTS: At discharge, patients in the intervention group who had low levels of MedDiet or UTP showed an increase in functional status [adjusted mean (95% CI) = 77.8 (70.8-84.8) points, p = 0.005, and adjusted mean (95% CI) = 78.0 (68.3-87.7) points, p = 0.020, respectively]. CONCLUSION: Older individuals over age 75 with low MedDiet adherence were likely to benefit more from a physical exercise and health education intervention.

[Effect of multi-glycosides of Tripterygium wilfordii on renal injury in diabetic kidney disease rats through NLRP3/caspase-1/GSDMD pyroptosis pathway].

This study investigated the effect of multi-glycosides of Tripterygium wilfordii(GTW) on renal injury in diabetic kidney disease(DKD) rats through Nod-like receptor protein 3(NLRP3)/cysteine-aspartic acid protease-1(caspase-1)/gsdermin D(GSDMD) pyroptosis pathway and the mechanism. To be specific, a total of 40 male SD rats were randomized into the normal group(n=8) and modeling group(n=34). In the modeling group, a high-sugar and high-fat diet and one-time intraperitoneal injection of streptozotocin(STZ) were used to induce DKD in rats. After successful modeling, they were randomly classified into model group, valsartan(Diovan) group, and GTW group. Normal group and model group were given normal saline, and the valsartan group and GTW group received(ig) valsartan and GTW, respectively, for 6 weeks. Blood urea nitrogen(BUN), serum creatinine(Scr), alanine ami-notransferase(ALT), albumin(ALB), and 24 hours urinary total protein(24 h-UTP) were determined by biochemical tests. The pathological changes of renal tissue were observed based on hematoxylin and eosin(HE) staining. Serum levels of interleukin-1ß(IL-1ß) and interleukin-18(IL-18) were detected by enzyme-linked immunosorbent assay(ELISA). Western blot was used to detect the expression of pyroptosis pathway-related proteins in renal tissue, and RT-PCR to determine the expression of pyroptosis pathway-related genes in renal tissue. Compared with the normal group, the model group showed high levels of BUN, Scr, ALT, and 24 h-UTP and serum levels of IL-1ß and IL-18(P<0.01), low level of ALB(P<0.01), severe pathological damage to kidney, and high protein and mRNA levels of NLRP3, caspase-1, and GSDMD in renal tissue(P<0.01). Compared with the model group, valsartan group and GTW group had low levels of BUN, Scr, ALT, and 24 h-UTP and serum levels of IL-1ß and IL-18(P<0.01), high level of ALB(P<0.01), alleviation of the pathological damage to the kidney, and low protein and mRNA levels of NLRP3, caspase-1, and GSDMD in renal tissue(P<0.01 or P<0.05). GTW may inhibit pyroptosis by decreasing the expression of NLRP3/caspase-1/GSDMD in renal tissue, thereby relieving the inflammatory response of DKD rats and the pathological injury of kidney.

DNA Aptamer Beacon Probe (ABP) for Monitoring of Adenosine Triphosphate Level in SW480 Cancer Cells Treated with Glycolysis Inhibitor 2-Deoxyglucose.

Early cancer screening enables timely detection of carcinogenesis, and aids in prompt clinical intervention. Herein, we report on the development of a simple, sensitive, and rapid fluorometric assay based on the aptamer probe (aptamer beacon probe, ABP) for monitoring the energy-demand biomarker adenosine triphosphate (ATP), an essential energy source that is released into the tumor microenvironment. Its level plays a significant role in risk assessment of malignancies. The operation of the ABP for ATP was examined using solutions of ATP and other nucleotides (UTP, GTP, CTP), followed by monitoring of ATP production in SW480 cancer cells. Then, the effect of a glycolysis inhibitor, 2-deoxyglucose (2-DG), on SW480 cells was investigated. The stability of predominant ABP conformations in the temperature range of 23-91 °C and the effects of temperature on ABP interactions with ATP, UTP, GTP, and CTP were evaluated based on quenching efficiencies (QE) and Stern-Volmer constants (KSV). The optimized temperature for best selectivity of ABP toward ATP was 40 °C (KSV = 1093 M-1, QE = 42%). We have found that the inhibition of glycolysis in SW480 cancer cells by 2-deoxyglucose resulted in lowering of ATP production by 31.7%. Therefore, monitoring and modulation of ATP concentration may aid in future cancer treatment.

Differential roles of CTP synthetases CTPS1 and CTPS2 in cell proliferation.

The CTP nucleotide is a key precursor of nucleic acids metabolism essential for DNA replication. De novo CTP production relies on CTP synthetases 1 and 2 (CTPS1 and CTPS2) that catalyze the conversion of UTP into CTP. CTP synthetase activity is high in proliferating cells including cancer cells; however, the respective roles of CTPS1 and CTPS2 in cell proliferation are not known. By inactivation of CTPS1 and/or CTPS2 and complementation experiments, we showed that both CTPS1 and CTPS2 are differentially required for cell proliferation. CTPS1 was more efficient in promoting proliferation than CTPS2, in association with a higher intrinsic enzymatic activity that was more resistant to inhibition by 3-deaza-uridine, an UTP analog. The contribution of CTPS2 to cell proliferation was modest when CTPS1 was expressed but essential in absence of CTPS1. Public databases analysis of more than 1,000 inactivated cancer cell lines for CTPS1 or CTPS2 confirmed that cell growth is highly dependent of CTPS1 but less or not of CTPS2. Therefore, our results demonstrate that CTPS1 is the main contributor to cell proliferation.

Discovery of Fibrinogen γ-chain as a potential urinary biomarker for renal interstitial fibrosis in IgA nephropathy.

BACKGROUND: IgA nephropathy (IgAN) is a major cause of chronic kidney disease (CKD). Renal interstitial fibrosis is a hallmark of CKD progression. Non-invasive biomarkers are needed to dynamically evaluate renal fibrosis. Data independent acquisition (DIA)-based liquid chromatography-mass spectrometry (DIA-MS) was used to identify candidate urinary biomarkers in IgAN patients with different renal interstitial fibrosis degrees. METHODS: Eighteen biopsy-proven IgAN patients and six healthy controls were recruited in a discovery cohort. Interstitial fibrosis changes were evaluated according to Oxford MEST-C scores. Urinary samples were analyzed with DIA-MS to identify hub proteins. Hub proteins were then confirmed by enzyme-linked immunosorbent assay (ELISA) in a validation cohort and the associated gene mRNA expression was analyzed using public gene expression omnibus (GEO) datasets. RESULTS: Complement and coagulation cascades pathway was the main KEGG pathway related to the over-expressed proteins. Fibrinogen γ-Chain (FGG) was selected as the potential urinary marker for further validation. Urinary FGG to creatinine ratio (uFGG/Cr) levels were higher in both disease controls and IgAN group than in healthy controls, but were not significantly different between IgAN and disease groups. uFGG/Cr was confirmed to be increased with the extent of renal fibrosis and presented moderate correlations with T score (r = 0.614, p < 0.01) and eGFR (r = -0.682, p < 0.01), and a mild correlation with UTP (r = 0.497, p < 0.01) in IgAN group. In disease control group, uFGG/Cr was higher in patients with T1 + 2 compared to those with T0. uFGG/Cr had a good discriminatory power to distinguish different fibrosis stages in IgAN: interstitial fibrosis ≤ 5% (minimal fibrosis) vs. interstitial fibrosis (mild fibrosis) > 5%, AUC 0.743; T0 vs. T1 + 2, AUC 0.839; T0 + 1 vs. T2, AUC 0.854. In disease control group, uFGG/Cr showed better performance of AUC than UTP between minimal and mild fibrosis (p = 0.038 for Delong's test). Moreover, GSE104954 dataset showed that FGG mRNA expression was up-regulated (fold change 1.20, p = 0.009) in tubulointerstitium of IgAN patients when compared to healthy living kidney donors. CONCLUSION: Urinary FGG is associated with renal interstitial fibrosis and could be used as a noninvasive biomarker for renal fibrosis in IgAN.

Effects of uridine and nucleotides on hemostasis parameters.

Several purinergic receptors have been identified on platelets which are involved in hemostatic and thrombotic processes. The aim of the present study was to investigate the effects of uridine and its nucleotides on platelet aggregation and hemostasis in platelet-rich plasma (PRP) and whole blood. The effects of uridine, UMP, UDP, and UTP at different final concentrations (1 to 1000 µM) on platelet aggregation were studied using an aggregometer. In PRP samples, platelet aggregation was induced by ADP, collagen and epinephrine 3 min after addition of uridine, UMP, UDP, UTP and saline (as a control). All thromboelastogram experiments were performed at 1000 µM final concentrations of uridine and its nucleotides in whole blood. UDP and UTP were also tested in thromboelastogram with PRP. Our results showed that UDP, and especially UTP, inhibited ADP- and collagen-induced aggregation in a concentration-dependent manner. In whole blood thromboelastogram experiments, UDP stimulated clot formation while UTP suppressed clot formation. When thromboelastogram experiments were repeated with PRP, UTP's inhibitory effect on platelets was confirmed, while UDP's stimulated clot forming effect disappeared. Collectively, our data showed that UTP inhibited platelet aggregation in a concentration-dependent manner and suppressed clot formation. On the other hand, UDP exhibited distinct effects on whole blood or PRP in thromboelastogram. These data suggest that the difference on effects of UTP and UDP might have arisen from the different receptors that they stimulate and warrant further investigation with regard to their in vivo actions on platelet aggregation and hemostasis.

Identification and biochemical characterization of a novel N-acetylglucosamine kinase in Saccharomyces cerevisiae.

N-acetylglucosamine (GlcNAc) is a key component of glycans such as glycoprotein and the cell wall. GlcNAc kinase is an enzyme that transfers a phosphate onto GlcNAc to generate GlcNAc-6-phosphate, which can be a precursor for glycan synthesis. GlcNAc kinases have been found in a broad range of organisms, including pathogenic yeast, human and bacteria. However, this enzyme has never been discovered in Saccharomyces cerevisiae, a eukaryotic model. In this study, the first GlcNAc kinase from S. cerevisiae was identified and named Ngk1. The Km values of Ngk1 for GlcNAc and glucose were 0.11 mM and 71 mM, respectively, suggesting that Ngk1 possesses a high affinity for GlcNAc, unlike hexokinases. Ngk1 showed the GlcNAc phosphorylation activity with various nucleoside triphosphates, namely ATP, CTP, GTP, ITP, and UTP, as phosphoryl donors. Ngk1 is phylogenetically distant from known enzymes, as the amino acid sequence identity with others is only about 20% or less. The physiological role of Ngk1 in S. cerevisiae is also discussed.

Indoxyl sulfate decreases uridine adenosine tetraphosphate-induced contraction in rat renal artery.

The protein-bound uremic toxin indoxyl sulfate has negative effects on a variety of physiological activities including vascular function. Uridine adenosine tetraphosphate (Up4A), a new dinucleotide molecule affects vascular function including induction of vasocontraction, and aberrant responsiveness to Up4A is evident in arteries from disorders such as hypertension and diabetes. The link between indoxyl sulfate and the Up4A-mediated response is, however, unknown. We used Wistar rat's renal arteries to see if indoxyl sulfate will affect Up4A-mediated vascular contraction. In renal arteries of indoxyl sulfate, the contractile response generated by Up4A was dramatically reduced compared to the non-treated control group. Indoxyl sulfate increased endothelin-1-induced contraction but had no effect on phenylephrine, thromboxane analog, or isotonic K+-induced renal arterial contractions. UTP, ATP, UDP, and ADP-produced contractions were reduced by indoxyl sulfate. CH223191, an aryl hydrocarbon receptor (AhR) antagonist, did not reverse Up4A, and UTP contraction decreases caused by indoxyl sulfate. The ectonucleotidase inhibitor ARL67156 prevents indoxyl sulfate from reducing Up4A- and UTP-mediated contractions. In conclusion, we discovered for the first time that indoxyl sulfate inhibits Up4A-mediated contraction in the renal artery, possibly through activating ectonucleotidase but not AhR. Indoxyl sulfate is thought to play a function in the pathophysiology of purinergic signaling.

Molecular Mechanism of Hydrotropic Properties of GTP and ATP.

Hydrotropes are small amphiphilic compounds that increase the aqueous solubility of hydrophobic molecules. Recent evidence suggests that adenosine triphosphate (ATP), which is the primary energy carrier in cells, also assumes hydrotropic properties to prevent the aggregation of hydrophobic proteins, but the mechanism of hydrotropy is unknown. Here, we compare the hydrotropic behavior of all four biological nucleoside triphosphates (NTPs) using molecular dynamics (MD) simulations. We launch all atom MD simulations of aqueous solutions of NTPs [ATP, guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP)] with pyrene, which acts both as a model hydrophobic compound and as a spectroscopic reporter for aggregation. GTP prevents pyrene aggregation effectively. Dissolution is not achieved in the presence of CTP and UTP. The higher stability of the base stacking in guanine is responsible for the higher hydrotropic efficiency of GTP. Consistent with the simulations, spectroscopic measurements also suggest that the hydrotropic activity of GTP is higher than ATP. Stacking of aromatic pyrene with the aromatic base of NTPs is a characteristic feature of this hydrotropic property. Both ATP and GTP also dissolve clusters of di- and tripeptides containing tryptophan but with equal potency. Importantly, the presence of aromatic amino acids is a necessary condition for the hydrotropic potency of ATP and GTP. Our results can have broad implications for hydrotrope design in the pharmaceutical industry, as well as the possibility of cells employing GTP as a hydrotrope to regulate the hydrophobic protein aggregation in membrane-less biological condensates.

Methylglyoxal impairs ATP- and UTP-induced relaxation in the rat carotid arteries.

Although methylglyoxal (MGO), a highly reactive dicarbonyl compound, influences the functioning of the vasculature, modulating its effects on vascular reactivity to various substances remains unclear, especially purinoceptor ligands. Therefore, we sought to investigate the direct effects of MGO on relaxation induced by adenosine 5'-triphosphate (ATP) and uridine 5'-triphosphate (UTP) in isolated rat carotid arteries. When carotid arteries were exposed to MGO (420 µM for 1 h), relaxation induced by acetylcholine or sodium nitroprusside was not affected by MGO. However, ATP- and UTP-induced relaxation was impaired by MGO compared with the control. In both ATP- and UTP-induced relaxation, endothelial denudation, incubation with the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine or the selective P2Y purinoceptor 2 (P2Y2) receptor antagonist AR-C118925XX reduced relaxation in both the control and MGO groups, while the differences between the control and MGO groups were eliminated. The cyclooxygenase (COX) inhibitor indomethacin inhibited the differences in ATP/UTP-mediated relaxations between the control and MGO groups. Moreover, N-acetyl-L-cysteine (NAC), an antioxidant, could augment carotid arterial relaxation induced by ATP/UTP in the presence of MGO. MGO increased arachidonic acid-induced contraction, which was suppressed by NAC. Following both ATP/UTP stimulation, MGO increased the release of prostanoids. These results suggest that MGO impaired ATP- and UTP-induced relaxation in carotid arteries, which was caused by suppressed P2Y2 receptor-mediated signaling and reductions in endothelial NO. Moreover, MGO partially contributed to COX-derived vasoconstrictor prostanoids through increased oxidative stress.

Polarization and ß-Glucan Reprogram Immunomodulatory Metabolism in Human Macrophages and Ex Vivo in Human Lung Cancer Tissues.

Immunomodulatory (IM) metabolic reprogramming in macrophages (Mϕs) is fundamental to immune function. However, limited information is available for human Mϕs, particularly in response plasticity, which is critical to understanding the variable efficacy of immunotherapies in cancer patients. We carried out an in-depth analysis by combining multiplex stable isotope-resolved metabolomics with reversed phase protein array to map the dynamic changes of the IM metabolic network and key protein regulators in four human donors' Mϕs in response to differential polarization and M1 repolarizer ß-glucan (whole glucan particles [WGPs]). These responses were compared with those of WGP-treated ex vivo organotypic tissue cultures (OTCs) of human non-small cell lung cancer. We found consistently enhanced tryptophan catabolism with blocked NAD+ and UTP synthesis in M1-type Mϕs (M1-Mϕs), which was associated with immune activation evidenced by increased release of IL-1ß/CXCL10/IFN-γ/TNF-α and reduced phagocytosis. In M2a-Mϕs, WGP treatment of M2a-Mϕs robustly increased glucose utilization via the glycolysis/oxidative branch of the pentose phosphate pathway while enhancing UDP-N-acetyl-glucosamine turnover and glutamine-fueled gluconeogenesis, which was accompanied by the release of proinflammatory IL-1ß/TNF-α to above M1-Mϕ's levels, anti-inflammatory IL-10 to above M2a-Mϕ's levels, and attenuated phagocytosis. These IM metabolic responses could underlie the opposing effects of WGP, i.e., reverting M2- to M1-type immune functions but also boosting anti-inflammation. Variable reprogrammed Krebs cycle and glutamine-fueled synthesis of UTP in WGP-treated OTCs of human non-small cell lung cancer were observed, reflecting variable M1 repolarization of tumor-associated Mϕs. This was supported by correlation with IL-1ß/TNF-α release and compromised tumor status, making patient-derived OTCs unique models for studying variable immunotherapeutic efficacy in cancer patients.