bluuebunny/crossref_metadata_2025_split huggingface dataset:
The ELMO1, a Microbial Sensor Regulates Bacterial Clearance and Endo‐lysosomal Signaling
Authors: Tam Julian , Lau Mitchel , Pranadinata Rama , Ibeawuchi Stella‐Rita , Tindle Courtney , Das Soumita 
Published: June 2021

Intestinal antigen presenting cells (APCs) are a major component of the mucosal immune system and maintain the gut homeostasis and the inflammation level. Microbial sensors present on APCs recognize microbes irrespective of their pathogenicity but little is known about their mechanism that leads to host cellular responses unique to bacterial pathogenesis. Previously, we found that BAI1 (Brain Angiogenesis Inhibitor 1) recognizes bacterial LPS from pathogenic and non‐pathogenic bacteria. BAI1 binds cytosolic protein ELMO1 (Engulfment and cell motility protein 1), which facilitates the engulfment of bacteria via Rac1 activation and induces the pro‐inflammatory cytokines and regulates bacterial clearance. Interestingly, ELMO1 interacts with Salmonella effector SifA involved in bacterial survival. Therefore, we hypothesized that ELMO1 is a cytosolic sensor that interacts with bacterial effectors involved in the pathogenesis of enteric infection, controls bacterial clearance and provides differential immune responses between pathogens and commensals. In silico analysis of bacterial database revealed that Salmonella effector SifA shares a WxxxE signature motif present in several effectors from other enteric pathogens, including IpgB1 in Shigella that binds ELMO1. This signature motif is not present in commensal gut microbes. Interestingly, mutation in the WxxxE motif of SifA abolished interaction with ELMO1. SifA is involved in bacterial vacuole integrity and together ELMO1‐SifA is involved in the endosomal‐lysosomal pathway by interacting with the late endosomal protein Rab9. To further elucidate the interaction of SifA and its involvement in interrupting host defenses, we did pulldown of GST‐SifA with murine macrophage cell lysate followed by the identification of host proteins via MALDI‐TOF Spectroscopy. Three major proteins that interact with SifA are Ribophorin 1(RPN1), involved in the transport of newly synthesized proteins across the membrane of the rough endoplasmic reticulum; MOGS, a glycoside hydrolase functions within the N‐glycosylation pathway; Rab14, the endosomal trafficking protein involved in lysosomal fusion. Our functional studies showed the ELMO1‐depleted macrophages had higher acidic and proteolytic lysosomal environment and increased Cathepsin B activity at earlier time points of infection but lower Cathepsin B activity and lysosomal acidification at later time points of infection. Lastly, ELMO1 depleted cells had significant delayed clearance following enteric infection. Our results suggest that ELMO1 is a novel cytosolic sensor that determines bacterial pathogenesis and provides new insight to the degradation of bacteria via mechanisms of the endosomal‐lysosomal system.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of HIV‐1 gp120 and TAT on Endothelial Microparticle Release and Oxidative Stress
Authors: Brewster Lillian M. , Hijmans Jamie G. , Bammert Tyler D. , Stockelman Kelly A. , Levy Ma’ Ayan V. , Greiner Jared J. , DeSouza Christopher A. 
Published: June 2021

Human immunodeficiency virus (HIV)‐1 infection is associated with an increased risk and prevalence of atherosclerotic cardiovascular disease (CVD). A major factor underlying the increased CVD burden with HIV‐1‐infection is endothelial damage and dysfunction. Although the mechanisms underlying the HIV‐1‐related vasculopathy are not fully understood, HIV‐1 associated viral proteins are known to have deleterious effects on the endothelium. Endothelial cell release of microparticles and endothelial cell oxidative stress are key characteristics of a proatherogenic endothelial phenotype. The direct effect of HIV‐1 viral proteins on endothelial microparticle release and oxidative stress, however, is currently unknown. The aim of the present study was to determine the effects of X4 and R5 HIV‐1 gp120 and TAT on endothelial cell microparticle release and oxidative stress. Human umbilical vein endothelial cells (HUVECs) were cultured (3rd passage) and plated at a density of 2 × 106 cells/condition. HUVECs were treated with media alone or media containing either lavgp120 (X4; 100 ng/mL), Balgp120 (R5; 100 ng/mL) or TAT (500 ng/mL) for 24 h to generate endothelial microparticles (EMPs). Viral protein stimulated EMPs were pelleted by centrifugation and resuspended in culture media. Characterization and concentration of EMPs was determined by flow cytometry using VE‐Cadherin (CD 144‐PE). To assess oxidative stress, intracellular reactive oxygen species (ROS) production was determined. To do so, 2.5 ×104 HUVECs were plated in 96‐well tissue culture plates and allowed to adhere overnight. Cells were then incubated with 2′,7′ –dichlorofluorescin diacetate (DCFDA; 25 uM), washed and stimulated with media in the presence or absence of viral proteins. All measurements were done in triplicate and calculated as percent (%) of control by fluorescence spectroscopy. Treatment of HUVECs with X4 (117±28 MP/μL), R5 (105 ± 24 MP/μL) and TAT (101±36 MP/μL) induced markedly higher (~105%) EMP release compared with untreated (51±13 MP/μL) cells. Additionally, X4 (135 ±11%), R5 (139±13%) and TAT (142±7%) significantly increased (~40%) ROS production compared with untreated cells. Of note, the magnitude of increase in EMP release and ROS production was not significantly different between viral proteins. These results indicate that HIV‐1 gp120 and TAT induce EMP release and increase oxidative stress in endothelial cells. Viral protein augmentation of cellular ROS may be the impetus for EMP release, and requires further study. EMP generation and increased endothelial oxidative burden likely contribute to atherogenic effects of HIV‐1 viral proteins.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cardiolipin Remodeling by ALCAT1 Links Oxidative Stress and Mitochondrial Dysfunction to Cellular Senescence
Authors: Mdaki Kennedy Stanley , Shi Yuguang 
Published: June 2021

IntroductionCardiolipin is a major mitochondrial phospholipid that is required for ATP production, autophagy/mitophagy, and mitochondrial biogenesis. ALCAT1 promotes cardiolipin peroxidation by catalyzing pathological remodeling of cardiolipin with aberrant fatty acyl chains commonly found in aging, rendering cardiolipin highly sensitive to lipid oxidation by reactive oxygen species (ROS). Oxidized cardiolipin not only causes mitochondrial dysfunction, but also becomes a major source of ROS as lipid peroxides, further exacerbating oxidative stress. Recent development in the field has demonstrated a causative role of cellular senescence in aging and aging related diseases. However, the molecular mechanisms that promote cellular senescence in aging and aging‐related diseases remains elusive.ObjectiveTo determine whether oxidative stress and mitochondrial dysfunction induced by ALCAT1 link aberrant cardiolipin remodeling to cellular senescenceMethodsAdult C57BL/6 wild‐type and ALCAT1 Knockout (KO) mice were used for the studies. Primary vascular smooth muscle cells (VSMC) were isolated and cultured using standard protocols. Young cells were at passage 3 (P3) and old cells were at passage 11 (P11). Senescence associated beta galactosidase (SAβ‐Gal) and aconitase activity were used as biomarkers for cellular senescence and oxidative stress respectively. Cellular bioenergetics were assessed using a seahorse XF24 following treatment with or without xanthine/xanthine oxidase, a combination that generates superoxide. Advanced confocal live cell imaging was used to determine and monitor changes in mitochondrial dynamics, mitophagy, mtDNA, and mitochondrial membrane potential following treatment with hydrogen peroxide. TMRM, mitoTracker Green FM and mitoTracker Red, LysoTracker Green, Hoechst, and PicoGreen were used to label mitochondrial membrane potential, mitochondria, lysosome, nucleus, and mtDNA respectively. Lipid peroxidation products were quantified calorimetrically with a malondialdehyde (MDA) assay kit.ResultsWild‐type VSMC at P11 demonstrated a pronounced SAβ‐Gal staining, significant loss of aconitase activity and mitophagy function, decreased mtDNA, impaired mitochondrial function, and a significant increase in lipid peroxidation products.ConclusionALCAT1 KO protected against onset of cellular senescence by reducing sensitivity to lipid oxidation, which preserved mitochondrial function, mitophagy, and mtDNA. Taken together, our work puts forward a possible potential role of ALCAT1 in promoting cellular senescence.Support or Funding InformationNIH (2R01DK076685‐06A1), American Diabetes Association (1‐14‐BS‐185), the Barth Syndrome Foundation, and support from a NIH T32 training grant through the Barshop Institute's Biology of Aging T32 AG021890 (which has been supporting K.S.M since joining the Shi Lab in May 2016).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Low Sodium Intake Causes Kidney Cortical Hypoxia and Proteinuria in Rats
Authors: Carvalho Carla , Patinha Daniela , Fasching Angelica , Friederich‐Persson Malou , Hansell Peter , Palm Fredrik 
Published: June 2021

Reduced sodium intake induces activation of the renin‐angiotensin system to maintain sodium balance and arterial blood pressure. Renin‐angiotensin aldosterone system‐induced oxidative stress is a common pathway to intrarenal hypoxia, which could be detrimental if imposed on an already damaged kidney. We therefore investigated the effect of reduced sodium intake on kidney oxygen homeostasis and function in rats.Male Sprague Dawley rats were fed standard rat chow containing normal (0.25% sodium) or low sodium content (0.02%) for two weeks and kidney oxygen metabolism and function were studied thereafter. Acute blockade of angiotensin II AT1 receptors by candesartan (1 mg kg−1) was used to estimate the degree of renin‐angiotensin system activation in combination with measurements of intrarenal tissue angiotensin II levels.Dietary sodium restriction increased intrarenal angiotensin II tissue levels without altered arterial blood pressure. Concomitantly, a pronounced increase in kidney oxygen consumption was observed along with reduced tubular electrolyte transport efficiency and abolished cortico‐medullary oxygen gradient. Urinary protein excretion increased although renal blood flow and glomerular filtration rate were preserved.In conclusion, reduced sodium intake causing renin‐angiotensin‐aldosterone system activation significantly increases kidney oxygen consumption resulting in cortical tissue hypoxia and proteinuria. This is possibly an undesired effect, especially during pre‐existing kidney disease.Support or Funding InformationSwedish Research Council, Swedish Diabetes Foundation, Swedish Heart‐Lung Foundation and Family Ernfors Fund.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Kir4.1 is involved in Bradykinin‐induced inhibition of NCC and natriuresis
Authors: Wu Peng , Gao Zhong‐Xiuzi , Zhang Dan‐Dan , Vio Carlos P. , Gu Ruimin , Wang Wen‐Hui 
Published: June 2021

Stimulation of type II bradykinin (BK) receptor (BK2R) has been shown to increase renal Na+ excretion. However, the mechanism by which BK stimulates natriuresis is not completely understood. Immunohistochemical study demonstrates that BK2R is highly expressed in both apical and basolateral membrane of Kir4.1 positive tubules including thick ascending limb, distal convoluted tubule (DCT) and cortical collecting duct. The aim of the present study is to explore the role of BK in regulating the basolateral K+ channel and apical Na‐Cl cotransporter (NCC) in the DCT. The single channel patch‐clamp experiments show that application of BK (1–10 μM) inhibited the basolateral 40 pS K+ channel (a Kir4.1/5.1 heterotetramer) and this effect was blocked by BK2R antagonist (HOE140) but not by BK1R antagonist. Whole‐cell recordings have also shown that BK decreased the basolateral K+ conductance of the DCT and depolarized the membrane. To examine the role of Kir4.1 in mediating BK‐induced natriuresis, we performed renal clearance experiment in WT and in kidney‐specific conditional Kir4.1 knockout (Ks‐Kir4.1 KO) mice. Acute application of BK increased urinary Na+ and K+ excretion. However, the BK‐induced natriuretic effect was completely abolished in Ks‐Kir4.1 KO mice. The results strongly suggest that Kir4.1 activity is required for BK‐induced natriuresis. Our previous experiments demonstrated that Kir4.1 plays an important role in determining NCC activity such that the deletion of Kir4.1 completely inhibited NCC in the DCT. To test the hypothesis that BK‐induced inhibition of Kir4.1 may cause the inhibition of NCC thereby increasing Na+ and K+ excretion, we examine the basolateral Kir4.1 activity, the expression of NCC and thiazide‐induced natriuresis in the mice with 3 day infusion of BK by an osmotic pump. The infusion of BK for 3 days significantly decreased the basolateral Kir4.1 conductance and the negativity of the DCT membrane. Western blot showed that infusion of BK decreased the expression of total NCC and phosphorylated NCC. Renal clearance experiments demonstrated that thiazide‐induced natriuresis was blunted in the mice receiving BK infusion, suggesting that BK infusion inhibited NCC function in the DCT. Consequently, mice received BK infusion for 3 days were hypokalemic, presumably due to increasing Na and volume delivery to the collecting duct. We conclude that BK2R plays a role in tonic inhibition of the basolateral Kir4.1 channels in the DCT and that BK‐induced inhibition of NCC is, at least in part, responsible for BK‐induced stimulation of Na excretion. We also conclude that BK‐induced inhibition of Kir4.1 in the DCT is an essential step for the inhibition of NCC.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Collecting duct principal, but not intercalated, cell (pro)renin receptor modulates sodium and water transport
Authors: Ramkumar Nirupama , Stuart Deborah , Mironova Elena , Lakshmipathi Jayalakshmi , Stockand James D. , Kohan Donald E. 
Published: June 2021

The collecting duct (CD) is the predominant site for prorenin receptor (PRR) expression within the nephron. We have previously demonstrated that the CD PRR regulates epithelial Na+ channel (ENaC) activity and water transport; however, which CD cell type is involved is unclear. Hence, we examined the effects of principal cell (PC) or intercalated cell (IC) PRR deletion on renal Na+ and water handling. PC or IC PRR KO mice were obtained by crossing floxed PRR mice with mice harboring Cre recombinase under the control of the AQP2 or B‐1 subunit of the H+ ATPase promoters, respectively. In‐situ hybridization using RNAscope demonstrated abundant PRR RNA in IC with modest staining in PC in control mice with reduced cell‐specific PRR RNA in PC and IC KO mice. Compared to controls, PC KO, but not IC KO mice, had increased urinary volume (Control: 1.2 ± 0.4 vs PC KO: 2.0 ± 0.3, P < 0.05; IC KO: 1.4 ± 0.2 ml/day), reduced urine osmolality (Control: 3160 ± 279 vs PC KO: 2343 ± 247, P < 0.05; IC KO: 2748 ± 356 mosm/Kg H2O) and reduced expression of renal medullary AQP2 (50% of control). Further, PC KO mice had reduced renal medullary ENaC‐α expression with normal and low Na+ diet and lower Na+ balance (24‐hour intake – excretion) following low Na+ intake (controls: 60.5 ± 7.5 vs PC KO: 25.7 ± 10.6 μmol/day, P <0.05). In contrast, no differences in Na+ balance or ENaC expression was observed between control or IC KO mice. In acutely isolated cortical CD, under basal conditions, no significant differences in ENaC activity were observed between control, PC or IC specific PRR KO mice. Treatment with mouse prorenin (10 nM for 30 min) increased ENaC channel number and open probability by 2‐fold in control mice (basal: 0.15 ± 0.04; prorenin treatment: 0.74 ± 0.25). Addition of prorenin did not alter ENaC activity in PC specific PRR KO mice (basal: 0.28 ± 0.06; prorenin treatment: 0.33 ± 0.13) but increased ENaC activity in IC specific PRR KO mice (basal: 0.17 ± 0.08; prorenin treatment 1.29 ± 0.13). However, mean arterial pressure as measured by telemetry (Controls: 104 ± 1, PC KO: 106 ± 2, IC KO: 101 ± 4 mm Hg) was similar between controls and PC or IC KO mice on normal and low Na+ diets. Taken together, these findings indicate that under physiologic conditions, the PC, but not IC, PRR modulates ENaC expression and activity, urinary Na+ excretion and water transport. Further studies are needed to examine the role of PC and IC PRR in pathological states such as Ang‐II induced hypertension.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
SPAK (Stk39) is Involved in NKCC2 Phosphorylation and Salt‐sensitive Hypertension in Dahl Salt‐sensitive Rats
Authors: Garcia‐Pedraza Jose Angel , Ortiz Pablo A. 
Published: June 2021

In the thick ascending limb (TAL), NaCl reabsorption is mediated by the Na/K/2Cl cotransporter NKCC2, which plays a crucial role in the regulation of blood pressure. NKCC2 activity is partly controlled by phosphorylation via STE20/SPS1‐related proline‐alanine‐rich kinase (SPAK, gene name Stk39) at Thr96 and Thr101 residues in the NH2‐terminus of NKCC2. Previously, we found that NKCC2 phosphorylation was 5‐fold higher in TALs from Dahl salt‐sensitive (DSS) rats, suggesting that this pathway may be related to the salt‐sensitivity in this model. However, the kinase responsible for NKCC2 hyperphosphorylation is unknown. In addition, the role of SPAK in the development of salt‐sensitive hypertension remains unclear. We hypothesized that SPAK is involved in salt‐sensitive hypertension in part by increasing NKCC2 phosphorylation during normal or high salt diet. To test this, we generated SPAK knockout (KO) rats in a DSS genetic background via Crispr/Cas9 targeting of exon 5 in collaboration with the Gene Editing Rat Resource Center (Medical College of Wisconsin). Gene deletion was confirmed by sequencing and RT‐PCR. We used radiotelemetry to continuously measure mean blood pressure (MBP) in these rats on normal Na (0.22% Na chow) or high salt diet (4% Na chow). At baseline, SPAK KO rats had lower MBP than wild‐type DSS (WT) rats (SPAK KO: 117±1 mmHg; WT: 127±2 mmHg; p<0.05, n=7 each). After 4 weeks on high salt diet, MBP rose in all rats reaching 137±3 in SPAK KO and 154±3 mmHg in WT rats (p<0.05). The increase in MBP after switching rats from normal to high salt was lower in SPAK KO than WT rats; at 7 days (8±1 vs 13±1 mmHg, p<0.05), 14 days (12±2 vs 17±2 mmHg, p<0.05), and 28 days (20±3 vs 27±2 mmHg, p<0.05). We then explored total and phosphorylated‐NKCC2 (Thr96 and Thr101) expression in isolated TALs by Western blot. In rats fed normal salt (0.22% Na), phosphorylated‐NKCC2 (corrected over total NKCC2 expression) was lower in TALs isolated from SPAK KO (WT: 100; SPAK KO: 39±25%; p<0.05, n=4) whereas total NKCC2 expression (corrected by GAPDH) was not different between both groups (WT: 100; SPAK KO: 103±17%; n=5). After 4 weeks on high‐salt (4% Na), phosphorylated‐NKCC2 was also lower in TALs isolated from SPAK KO (WT: 100; SPAK KO: 38±13%; p<0.05, n=4) whereas total NKCC2 expression (corrected by GAPDH) was not different between both groups (WT: 100; SPAK KO: 113±22%; n=4). We conclude that SPAK KO rats exhibit a lower blood pressure at baseline and decreased salt sensitivity. This may be in part caused by lower NKCC2 phosphorylation in TALs of SPAK KO DSS rats. Our findings support an important role for SPAK in NKCC2 hyperphosphorylation and salt sensitive hypertension in DSS rats.Support or Funding InformationNIH and NIDDK R01, Grant Number: 5R01DK107263‐02.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Bile Acid Membrane Receptor TGR5 Regulates Renal AQP2 and Improves Lithiuminduced NDI
Authors: Li Suchun , Qiu Miaojuan , Kong Yonglun , Liu Qiaojuan , Hu Shan , Zhao Xiaoduo , Han Mengke , Xie Haixia , Levi Moshe , Li Chunling , Wang Weidong 
Published: June 2021

The bile acid‐activated receptors, which include the membrane G protein‐coupled receptor (TGR5) and nuclear farnesoid X receptor (FXR), are shown to play roles in kidney diseases. The purpose of current study was to investigate the role of TGR5 receptors in renal water handling and the underlying molecular mechanisms. The activation of TGR5 by lithocholic acid (LCA, a TGR5 endogenous ligand) induced aquaporin‐2 (AQP2) protein expression and intracellular trafficking in rat primary cultured inner medullary collecting duct (IMCD) cells via cAMP‐PKA signaling pathway. A synthetic specific TGR5 agonist INT‐777 has the same effects as LCA in IMCD cells. In mice with lithium‐induced nephrogenic diabetes insipidus (NDI), the activation of TGR5 by LCA markedly decreased urine output and increased urine osmolality, which was associated with significantly up‐regulated AQP2 expression in the inner medulla, whereas chenodeoxycholic acid (CDCA, a FXR endogenous ligand) had no effects. In primary cultured IMCD cells prepared from lithium‐treated rats, INT‐777 or INT‐767 (a FXR and TGR5 dual agonist), but not INT‐747 (a FXR agonist) increased AQP2 protein expression. Compared with wildtype mice, tgr5−/− mice exhibited a mild urine concentration defect after dehydration, which was associated with decreased AQP2 expression in the kidney inner medulla. In lithium‐treated tgr5−/− mice, LCA treatment failed to prevent reduction of AQP2 expression. In conclusion, TGR5 activation increases renal AQP2 expression via cAMP‐PKA signaling pathway and improves impaired urinary concentration in lithium‐induced NDI. TGR5 is thus involved in regulation of water metabolism in the kidney.Support or Funding InformationThis work was supported by the Natural Science Foundation of China (Grants 81370822, 81570635, and 81670646), by the 111 Project, China (No. B13037), and by the Natural Science Foundation of Guangdong Province, China (Grants 2014A020212623, 2014A030313168, and 2016A020215034).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.