bluuebunny/crossref_metadata_2025_split huggingface dataset:
Silencing TRPC6 Gene Attenuates Hypertension‐induced Renal Injury in 5/6 Ablation/Infarction Model
Authors: Wang Weili , Lyu Bingqing , Li Pin‐Lan , Li Ningjun 
Published: June 2021

The transient receptor potential canonical 6 (TRPC6) channel is a member of the TRPC family. A “gain of function” mutation of TRPC6 causes familial focal segmental glomerulosclerosis (FSGS) and that overexpression of TRPC6 in podocytes causes a kidney disease consistent with FSGS in animals. Moreover, TRPC6 participates in the mechanical or pressure sensation in various cell types, including in the podocyte slit diaphragm. Therefore, TRPC6 may be the upstream mediator that transmits the pressure stress into downstream molecular pathways to cause hypertension‐induced renal injury. The present study tested the hypothesis that up‐regulated TRPC6 in the kidney mediates the hypertension‐induced renal injury in rats with 5/6 renal ablation/infarction (A/I), a model in which the renal injury is closely associated with blood pressure. Adults male Sprague Dawley rats were divided into shame group, 5/6 A/I control group and 5/6 A/I shRNA group. The left kidneys of shame and 5/6 A/I control groups were treated with intra‐renal transfection of control plasmids and the 5/6 A/I shRNA group TRPC6 shRNA plasmids. The results showed that the levels of renal TRPC6 was increased in 5/6 A/I control group compared with shame group and that the increase of TRPC6 was blocked in 5/6 A/I shRNA groups, the relative protein levels of TRPC6 were 1.0±0.26, 3.9±0.16 and 1.4±0.17 in shame, 5/6 A/I control and 5/6 A/I shRNA groups, respectively. TRPC6 shRNA had no effect on hypertension in 5/6 A/I rats (MAP: 153.9 ± 8.1 mmHg in control groups vs. 154.7 ± 8.9 mmHg in shRNA groups). However, the albuminuria was attenuated in 5/6 A/I shRNA group compared with 5/6 A/I control group. The levels of urinary albumin were 0.8±0.34 mg/Kg/24h in shame, 20.82± 3.7 mg/Kg/24h in 5/6 A/I control and 11.9±1.5 mg/Kg/24h in 5/6 A/I shRNA group, respectively (P=0.037 for control vs. shRNA groups). The regression slop between the blood pressure and the % of glomerulosclerosis was significantly steeper in 5/6 A/I control group than in 5/6 A/I shRNA group (P=0.028 by ANCOVA), indicating a significant attenuation of hypertension‐induced glomerular injuries. These data suggested that hypertension‐induced activation of TRPC6 may be the molecular sensor of pressure stress to produce kidney damage associated with hypertension. (Supported by NIH grants DK107991 to NL and DK54927 to PL).Support or Funding InformationNIH grants DK107991 to NL and DK54927 to PLThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
NFE2L2 (Nrf2) attenuates Tgf‐β1 signaling by elevation of Smad7 in mouse mesangial MES‐13 cells
Authors: Song Min‐Kyun , Ryoo In‐Geun , Ryu Dayoung , Lee Sang‐Hwan , Kwak Mi‐Kyoung 
Published: June 2021

Renal fibrosis is the principal process underlying the progression of kidney diseases. Accumulating evidence indicates that oxidative stress plays a crucial role in renal fibrotic diseases. Transforming growth factor‐β1 (Tgf‐β1) is a potent stimulator of profibrotic responses, including expression of fibrosis markers such as extracellular matrix (ECM) and α‐smooth muscle actin (α‐SMA) through a Smad‐dependent pathway. Therefore, Smad‐dependent Tgf‐β1 signaling is known to play pivotal roles in renal fibrosis. In this regard, we investigated the potential involvement of nuclear factor (erythroid‐derived 2)‐like 2 (NFE2L2; Nrf2) in renal fibrosis with Tgf‐β1–Smad signaling. We previously reported that Smad7, a negative regulator of Smad signaling, may be a linker molecule between the Nrf2 pathway and Tgf‐β1 signaling in human renal tubular epithelial HK2 cell. In this study, we confirmed that elevated protein level of Smad7 attenuates Tgf‐β1 signaling by degradation of Tgf‐β1 Receptor 1 (TβR1) in Kelch‐like ECH‐associated protein 1 (Keap1) knockdown mouse mesangial MES‐13 cell, a cellular model of Nrf2 overexpression. As one of potential underlying molecular mechanisms of Smad7 regulation by Nrf2, reduced level of Smad‐ubiquitination‐regulatory factor 1 (Smurf1), an E3 ubiquitin ligase for Smad7, was remarkable. Further, we demonstrated that a Nrf2 inducer bardoxolone methyl ester (BARD), which is on clinical trials for kidney diseases, also elevated Smad7 level and attenuated Tgf‐β1 signaling in MES‐13. Collectively, these results indicate that Nrf2 can be an effective inhibitor of Smad‐dependent Tgf‐β1 signaling, and imply the beneficial role of Nrf2 in renal fibrosis.Support or Funding InformationThis work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (NRF‐2015R1A2A1A10054384)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sex‐Unrelated Counteraction by Nicotine of the Endotoxemia‐Evoked Facilitation of Renal Vasodilator Capacity in Rats: Roles of α7/α4β2 nAChRs and HSP70
Authors: Wedn Abdalla M. , El‐Gowilly Sahar M. , El‐Mas Mahmoud M. 
Published: June 2021

Nicotine has been shown to alleviate renal inflammation and injury induced by endotoxemia. The current study investigated (i) the sex specificity of the nicotine modulation of hemodynamic and renal vasodilatory responses to endotoxemia in rats, and (ii) the roles of α7 or α4β2 nAChRs and related HSP70/TNFα/iNOS signaling in the interaction. Endotoxemia was induced by i.p. administration of lipopolysaccharide (LPS, 5 mg/kg/day.) for 2 consecutive days and changes in systolic blood pressure (SBP, tail‐cuff plethysmography) and vasodilator responsiveness of isolated perfused kidney to acetylcholine (ACh) or 5′‐N‐ethylcarboxamidoadenosine (NECA, adenosine receptor agonist) were evaluated. Compared with saline values, LPS decreased SBP and increased renal vasodilations induced by cumulative bolus injections of ACh (0.01–7.29 nmol) or NECA (1.6–100 nmol). No changes in serum creatinine or urea were observed. These effects were not sexually differentiated as they were similarly observed in the two rat genders. The LPS facilitation of renal vasodilations were attenuated after concurrent administration of aminoguanidine (iNOS inhibitor, 50 mg/kg), pentoxifylline (TNFα inhibitor, 3 mg/kg), or nicotine (0.5, 1, or 2 mg/kg). We also report that the selective blockade of α7‐nAChRs (methyllycaconitine citrate, MLA) or α4β2‐nAChRs (dihydro‐β‐erythroidine, DHβE) (i) potentiated the enhancing action of LPS on ACh or NECA vasodilations, and (ii) abolished the depressant effect of nicotine on LPS facilitation of renal vasodilatory capacity. A similar inhibition of the nicotine effect was seen after inhibition of HSP70 by quercetin. Alternatively, LPS hypotension was eliminated in rats treated with the DHβE/nicotine or quercetin/nicotine regimen in contrast to no effect for nicotine alone or combined with MLA. These findings establish that nicotine offsets the LPS facilitation of the renal vasodilator propensity, an effect that is sex independent and possibly involves a crosstalk between HSP70 and nAChRs of the α7 and α4β2 types.Support or Funding InformationSupported by the Science and Technology Development Fund, Egypt (STDF Grant No. 14895)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Exploring Structural Conformations of Human P450 1A1 with Diverse Ligands
Authors: Bart Aaron G. , Scott Emily E. 
Published: June 2021

The human P450 enzyme 1A1 (CYP1A1) is an extrahepatic P450 often expressed in tumor tissues where it may be clinically significant in procarcinogens and toxin bioactivation. Prototypical CYP1A1 ligands are planar and polycyclic aromatic compounds, such as the procarcinogen benzo[a]pyrene found in environmental combustion and tobacco smoke. The single crystal structure of CYP1A1 is with the polycyclic, planar molecule alpha‐napthoflavone (αNF) and reveals a narrow active site that is consistent with planar CYP1A1 ligands. However, a number of known CYP1A1 substrates and inhibitors do not adhere to these typical ligand features, including promising pro‐drugs in cancer therapy based on a duocarmycin scaffold and clinically used tyrosine kinase inhibitors. Such CYP1A1 ligands could not be accommodated in the active site of the CYP1A1/αNF structure. This study aims to determine how CYP1A1 binds atypical CYP1A1 substrates and inhibitors using co‐crystallization.Potential ligands for co‐crystallization trials with CYP1A1 were selected based on structural diversity, focusing on those that are not compatible with the 1A1/αNF crystal structure active site dimensions. Refinement of these ligands was performed by ranking based on their ability to inhibit CYP1A1 metabolism of a pro‐luciferin substrate. Anticancer tyrosine kinase inhibitors (erlotinib, imatinib, and ponatinib) displayed nanomolar to low micromolar IC50 values. A series of antifungal azoles, which are generally potent P450 inhibitors, resulted in a range of inhibitory responses. Compact, lower MW clotrimazole, miconazole, and tioconazole exhibited sub‐nanomolar potency, but the much larger extended azoles ketoconazole and posaconazole were also potent nanomolar inhibitors. The azoles fluconazole and letrozole were significantly less potent inhibitors with micromolar IC50 values. The pro‐duocarmycin drug ICT2700 and antitumor benzothiazole GW 610 inhibited CYP1A1 in the sub‐micromolar to nanomolar range. Compounds displaying potent inhibition are being employed in cocrystallization and structure determination. The structural results with atypical CYP1A1 ligands should provide a more robust framework for both prediction of procarcinogen and toxin activation and the development of novel CYP1A1‐activated cancer therapies.Support or Funding InformationThis work was supported by National Institutes of Health Grants F37 GM076343 (to EES).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigation of Non‐Caspase Mediated Mechanisms of Death in Response to Treatment with Novel Chalcone in Glioblastoma Cell Lines
Authors: Inbody Laura R. , Stang Christopher R.T. , Eichel Amanda M. , Orahood Cassandra B. , Dick Matt J. , Mohamed Ahmed H. , Hickey Mikaela C. , Khupse Rahul S. , Schneider Ryan A. 
Published: June 2021

Despite recent advances in cancer treatments, there is still a paucity of therapeutic options for patients with glioblastoma. We previously reported the efficacy of the synthetic chalcone, RK6, against A‐172, T98G, and U87‐MG human glioblastoma cell lines (IC50 values: 10–20 μM). Despite the moderately potent anticancer activity of RK6, the mechanism of death and putative drug target has yet to be elucidated. Previous efforts to identify potential mechanisms of death included evaluation of executioner caspase activation and production of reactive oxygen species. However, these pathways were found to be not largely involved. As such, further investigation of non‐caspase mediated forms of cell death were performed.To rule out necrosis as a mechanism of death for RK6, a trypan blue exclusion assay was conducted to examine membrane destabilization. Cellular membranes remained intact following treatment with RK6 at concentrations greater than the IC50 for six hours, suggesting necrosis is not the mechanism of death.We then sought to determine if cysteine proteases such as calpains or cathepsins were involved in the observed cell death following RK6 exposure. For this, cells were co‐treated with RK6 along with selective and non‐selective calpain and cathepsin inhibitors followed by measurement of cell viability using the XTT cell viability assay. Sublethal concentrations of the calpain and cathepsin inhibitor, N‐Acetyl‐Leu‐Leu‐Norleucinal (ALLN), selective calpain inhibitor, calpeptin, and selective cathepsin B inhibitor, CA‐074‐Me, were used to interrogate these signaling pathways. Inhibition of calpains and cathepsins concomitantly using ALLN had no effect on the efficacy of RK6 in the A‐172, T98G, and U87‐MG glioblastoma cell lines. Similarly, inhibition of cathepsin B with CA‐074‐Me did not affect the efficacy of RK6. Interestingly, inhibition of calpains with calpeptin resulted in a synergistic cytotoxic effect in all three cell lines. The results of these studies suggest that inhibition of calpains further sensitizes the cells to RK6, however concomitant inhibition of calpain and cathepsins does not provide the same effect. Further investigations are ongoing to investigate the nature of the synergy between RK6 and calpeptin and the potential role of other forms of mediated cell death, such as necroptosis, caused by this synthetic chalcone.Support or Funding InformationThis project was supported in part by an American Foundation for Pharmaceutical Education Gateway to Research Award for the second author.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Local regulation of L‐type CaV1.2 channel and vascular reactivity by adenylyl cyclase 5 during diabetic hyperglycemia
Authors: Syed Arsalan U. , Gosh Debapriya , Prada Maria Paz , Reddy Gopireddy , Nystoriak Matthew A. , Chiamvimonvat Nipavan , Santana Luis F. , Xiang Yang Kevin , Nieves‐Cintrón Madeline , Navedo Manuel F. 
Published: June 2021

Diabetes mellitus is a debilitating metabolic disorder affecting millions of people worldwide. One of the hallmark symptoms of diabetes is hyperglycemia (elevated blood glucose levels; HG). At least 60% of the diabetic patient develop hypertension and become susceptible to both macro and microvascular complications, including stroke. HG is associated with excessive vascular tone and dysfunction of the blood vessels. Recently, we demonstrated that activation of protein kinase A (PKA) leads to phosphorylation and potentiation of L‐type CaV1.2 channels in vascular smooth muscle resulting is increased vasoconstriction during HG. A fundamental gap in knowledge that remains unexplored is the upstream mechanisms underlying PKA activation. Here, we describe for the first time a critical role for local cAMP production by adenylyl cyclase (AC) isoform 5, upstream of PKA, in regulation and potentiation of CaV1.2 channels leading to vasoconstriction during HG. Consistent with this, membrane‐targeted fluorescence resonance energy transfer (FRET) biosensor for cAMP revealed a small but significant increase in cAMP production by HG, in agreement with local cAMP production. Treatment with forskolin (global cAMP activator) resulted in a significantly larger cAMP signal, aligned with cell wide global cAMP increase. In the presence of the specific AC5 and AC6 inhibitor 2,5,DDA or in vascular smooth muscle from AC5−/− mice, the HG‐induced cAMP increase was abolished. Yet, the forskolin‐induced global increase in cAMP production remained intact. Electrophysiology and pressure‐myography experiments demonstrated that the 2,5 DDA compound and genetic ablation of AC5, but not AC6, abrogated the increased in L‐type CaV1.2 channel activity and vasoconstriction in response to HG. Super‐resolution microscopy and proximity ligation assay confirmed close association between AC5 and L‐type CaV1.2 channels. Taken together, these finding demonstrate a novel role of local AC5 signaling in HG‐induced vasoconstriction due to increased CaV1.2 activity leading to vasoconstriction during diabetic hyperglycemia.Support or Funding InformationNIH R01HL098200, R01HL121059 and NIH T32‐HL086350This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Developing and characterizing human biomimetic arteriole for studying pulmonary hypertension
Authors: Bhatta Anil , Pagaduan Jayson V. , Chen Xing , West‐Foyle Hoku , Hou Annie , Liu Jiayu , Jin Qianru , Nguyen Thao D. , Kuo Scot , Berkowitz Dan , Gracias David H. , Romer Lewis H. 
Published: June 2021

Arteriolar resistance vessels control flow to vascular beds. The structure, mechanics, and function of these blood vessels are critically important to our understanding of the mechanisms of cardiovascular disease pathogenesis. Additionally, in vitro arteriolar mimetic approaches are needed to improve the study of cardiovascular disease, because models based upon rodents vascular function and cells in standard culture containers do not accurately recapitulate the native multicellular architecture of a human vessel wall. Here, we describe novel, highly parallel, mass biofabrication of biomimetic tubular vessel constructs composed of biocompatible scaffold materials, pulmonary microvascular endothelial cells (HPMEC), extracellular matrix components including fibronectin and laminin 3, and human pulmonary artery smooth muscle cells (HPASMC). These constructs had a diameter of ~250 microns, and a length of 1–3 mm. Cells were incorporated into these constructs in patterns that mimic the layering and relative alignment observed in human pulmonary arterioles. Finite Element Analysis showed that the ultra‐thin cylindrical scaffold materials used here could be displaced by the cells in these biomimetic vascular walls to an extent that was similar to displacements of thicker tube composed of softer materials. HPMEC in these biomimetic vessel constructs produced more nitric oxide, and demonstrated higher phosophorylation levels of key nitric oxide signaling proteins (eNOS and Akt) than equal numbers of cells grown on equal surface areas that were flat. Additionally, HPASMC were aligned at tunable angles that mimicked in‐vivo organization using biomolecular micropatterning. Thus, biomimetic arterioles produced in this study exhibited improved endothelial functionality together with multi‐cellular layering and anatomically accurate cellular alignment, and provide a platform for investigation of microvascular function.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Modulation of mean arterial pressure and diuresis by renomedullary infusion of selective inhibitors of fatty acid amide hydrolase
Authors: Ahmad Ashfaq , Dempsey Sara K. , Daneva Zdravka , Poklis Justin L. , Li Ningjun , Li Pin Lan , Ritter Joseph K. 
Published: June 2021

The kidneys are involved in the long term regulation of blood pressure and diuresis, which may involve various systems along with the endocannabinoid (EC) system. The EC system consists of N‐arachidonoylethanolamine (anandamide, AEA) and 2‐arachidonoylglycerol (2‐AG), cannabinoid receptors (CB‐1 and CB‐2) and hydrolyzing enzymes like fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). Previously we showed that dual inhibition of FAAH and MAGL in the renal medulla increased urinary excretion (UV) and salt excretion without affecting mean arterial pressure (MAP) and these responses are dependent on cyclooxygenase‐2 (COX‐2). The present study hypothesized that selective inhibition of FAAH in the kidney medulla of C57BL/6 mice can modulate MAP, UV, and salt excretory functions of the kidney and that these effects are COX‐2 and CB‐1 dependent. Renal medullary infusion of the selective FAAH inhibitor, PF‐3845(N‐3‐pyridinyl‐4‐[[3‐[[5‐(trifluoromethyl)‐2‐pyridinyl]oxy]phenyl]methyl]‐1‐piperidinecarboxamide), produced a significant drop in MAP only during the post treatment infusion phases but significant increases in UV and salt excretions (all P<0.05) at 15 and 30 nmol/min/kg and 30 nmol/min/kg, respectively, compared to the control pretreatment infusion phase (C1). The MAP‐lowering and UV‐stimulating effects of PF‐3845 were blocked by pretreatment of the mice with the CB‐1 antagonist, rimonabant (3 mg/kg, i.p.) which indicates the involvement of the CB‐1 receptor in both activities and these responses to PF‐3845 were not affected by pretreatment with celecoxib (15 mg/kg, i.v.). Intravenous administration of PF‐3845 significantly reduced MAP at 7.5, 15 and 30 nmol/min/kg (all P<0.05) and increased UV and salt excretion at 15 and 30 nmol/min/kg and 30 nmol/min/kg, respectively. Intramedullary administration of PF‐3845 in homozygous FAAH knock out (FAAH−/−) mice showed no change in MAP at any dose of PF‐3845 but, surprisingly, UV was increased at 7.5, 15 and 30 nmol/min/kg (P<0.05), indicating that the blood pressure‐lowering but not UV‐increasing effect of PF‐3845 was FAAH‐dependent. Analytical data showed significant increases (P<0.05) in AEA in kidneys of wild‐type mice treated with PF‐3845 versus sham‐treated controls. In summary, PF‐3845 administration into the renal medulla produces AEA which lowers MAP, increase urinary and salt excretion and these effects are dependent on CB‐1 receptors but independent of COX‐2 in C57BL/6.Support or Funding InformationThis work was supported by National Institute of Diabetes and Digestive and Kidney Disorders grant, DK102539This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
12‐LOX Oxylipins Derived from Omega‐3 Supplementation with Docosahexanoic Acid Potently Regulate Human Blood Platelets
Authors: Shami Ryan , Hawley Megan , West Joshua , Holman Ted , Holinstat Michael 
Published: June 2021

Cardiovascular disease (CVD) is the leading cause of mortality in the United States in both men and women. Thrombotic‐associated ischemic injuries due to excessive platelet activation are largely responsible for the high level of death associated with CVD. Therapies that seek to pharmacologically inhibit platelet activation, aggregation, or clot stabilization, have the potential to reduce the high mortality rates associated with CVD but are also associated with an increased risk of bleeding. The oxygenated products of polyunsaturated fatty acids—oxylipins—have been shown to be critical regulators of platelet function. Previously, it has been shown that Docosahexanoic Acid (DHA) has antiplatelet activity; therefore, we hypothesize that DHA regulates platelet function at least in part through its 12‐lipoxygenase (12‐LOX) metabolites, 11‐ and 14‐ hydroxydocosahexaenoic acid (11‐ and 14‐HDHA). Our study sought to investigate the role that 12‐LOX derived products of DHA, 11‐ and 14‐ HDHA, have on human blood platelet aggregation and activation. Platelet aggregation, granule secretion, and integrin αIIbβ3 activation were measured following agonist stimulation in the presence of DHA, 11‐ or 14‐ HDHA. Both 11‐ and 14‐ HDHA were observed to dose‐dependently attenuate aggregation in response to sub‐maximal concentrations of either thrombin or collagen. Additionally, both 11‐ and 14‐ HDHA were observed to attenuate intracellular signaling as demonstrated by a decrease in granule secretion in platelets treated with DHA compared to vehicle control treated platelets. To further the study, the effects of DHA supplementation in vivo were investigated in wild‐type mice as well as mice lacking 12‐LOX. DHA was found to play an important role in regulation of platelet function in vivo. Future studies will help to elucidate the mechanism by which DHA and its metabolites inhibit platelet function and play a role in supplementation‐dependent regulation of blood clotting. Once completed this will be the first study to clearly define a role for DHA and its 12‐LOX metabolites in the regulation of platelet aggregation and give important insight into the benefits and risks of fish oil supplementation on the cardiovascular system.Support or Funding InformationThis work was funded in part through the National Institutes of Health grants R01 GM105671 (MH), R01 HL114405 (MH), R01 MD007880 (MH)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.