bluuebunny/crossref_metadata_2025 huggingface dataset:
Elevated NOX1 determines oxidant load obesity and is reduced with increases in muscle mass (688.6)
Authors: Qiu Shuiqing , Mintz James , Salet Christina , Weihong Han , Chen Feng , Yu Yanfang , Su Yunchao , Fulton David , Stepp David
Published: June 2021

Increases in muscle mass, as observed with exercise, are positive influences of cardiovascular health. Recent studies from our lab have shown the increase muscle mass via deletion of myostatin improves vasodilation but the underlying mechanisms are unknown. Improving insulin resistance has been shown to improve oxidant stress, specifically via expression of NOX1. Hypothesis: Increasing muscle mass by deletion of muscle growth negative regulator myostatin, improves vascular function in mesenteric arteries from obese db/db mice via reducing NOX‐mediated oxidant stress in the endothelium. Myostatin deletion increased muscle mass in both lean (gastrocnemius 57.93%, gluteus maximus 60.95%) and obese mice (gastrocnemius 79.64%, gluteus maximus 112.32%). Fasting glucose, HbA1c and glucose tolerance are improved in obese myostatin null mice. Obese mice demonstrate superoxide‐mediated impairment of ACh‐induced vasodilation compared to lean mice. Deletion of myostatin in obese mice improved ACh‐induced vasodilation in mesenteric arteries without effects in lean mice. This improvement was blunted by L‐NAME. PGI2 and EDHF mediated vasodilation were unaffected by obesity or myostatin deletion. Treatment with sepiapterin (1×10‐6 mol/L, a precursor of the nitric oxide synthase cofactor tetrahydrobiopoterin) or GKT(1×10‐6 mol/L, NADPH oxidase inhibitor) restored impaired vasodilation in obese mice. Elevated oxidized DNA/RNA was observed in the endothelium of obese mesenteric arteries, and this elevation was blunted by myostatin deletion. NOX1 expression was increased in the endothelium of obese mice mesenteric arteries, which was reversed by myostatin deletion. Taken together, these data suggest that increasing muscle mass by deletion of myostatin improves NO‐mediated vasodilation in obese mice; this vasodilation improvement is mediated by down regulation of NOX1 in the endothelium.

Grant Funding Source: Supported by NIH R01 HL092446, R21 HL098829 and AHA‐GSA 13PRE14680055
Quercetin ameliorates hyperglycemia‐induced inflammation and apoptosis in the retina and lateral geniculate nucleus in a rat model of type 2 diabetes mellitus (688.8)
Authors: Ibarra Jessica , Bland Maia , Gonzalez Muraya , Garcia Carlos
Published: June 2021

Diabetic retinopathy (DR) is a common complication of diabetes mellitus (DM) and the leading cause of blindness worldwide. While the underlying mechanism of DR is not well understood, neuroretinal cell death due to apoptosis is implicated, with molecular abnormalities consistent with those seen in inflammation. The flavonoid quercetin has been reported to potentially have anti‐inflammatory properties in diabetic animal models. Few studies have examined the anti‐inflammatory effect of quercetin in type 2 diabetes (T2D), and none have investigated the physiological status of the lateral geniculate nucleus (LGN), which connects the retina to the visual cortex. The purpose of this study was to investigate the effects of quercetin on diabetes‐induced neuroretinal inflammation and apoptosis in a rat model of T2D. Zucker Diabetic Fatty rats (ZDF) were assigned into diabetic (ZDF) and diabetic quercetin‐fed (ZDF+Q) groups. ZDF rats were fed a high fat chow while the ZDF+Q group received a high fat chow supplemented with quercetin (0.5%). Rats on standard chow served as the control group. The levels of inflammatory and apoptotic factors in the diabetic retina and LGN were measured by ELISA and immunoblotting. The major findings of this study demonstrate that quercetin, i) significantly (p<0.05) reduced increases in the pro‐inflammatory cytokines TNF‐α and IL‐1β and increased the anti‐inflammatory IL‐10, and ii) significantly decreased the concentration of BAD and Caspase‐3 and increased Bcl‐2. These results provide evidence that quercitin mitigates inflammation, which may be a precursor to cell signaling pathways leading to retina and LGN apoptosis in diabetes.
ORAI store‐operated calcium channels are associated with proximal renal tubule dysfunction in diabetic nephropathy (689.13)
Authors: Garcia‐Vaz Eliana , Chen Gui‐Lan , Bhandari Sunil , Daskoulidou Nikoleta , Zeng Bo , Jiang Hongni , Gomez Maria F. , Atkin Stephen L. , Xu Shang‐Zhong
Published: June 2021

Proteinuria is not only an early sign of diabetic nephropathy (DN) but also contributes to the development and progression of the disease. The molecular mechanisms underlying proteinuria remain unclear. Here we explored the role of ORAI store‐operated calcium channels in DN. RT‐PCR and western blot experiments demonstrated expression of all ORAI isoforms (ORAI1‐3) in human kidney. ORAI channels were preferentially expressed in the proximal tubules and intrarenal arteries as assessed by immunohistochemistry; and ORAI channel expression was down regulated in type 1 diabetic patients with DN. Using primary human proximal tubular cells, we showed that high glucose (25 mM) significantly reduced ORAI1‐3 mRNA and protein expression, whereas insulin increased channel expression. Conversely, blockade of insulin signaling using tyrphostin A23 or wortmannin reduced channel expression. We identified a current evoked by thapsigargin and inositol 1,4,5‐trisphosphate (IP3 ) in the proximal tubular cells, sensitive to diethylstilbestrol (DES) and 2‐APB. Using ORAI1‐3/STIM1 overexpressing systems, we found that DES and BTP2 inhibited all three ORAI channels, while 2‐APB selectively blocked ORAI1 and ORAI2, but stimulated ORAI3 channels. Pharmacological inhibition of ORAI channel activity by DES, 2‐APB or BTP2, or silencing of ORAI expression by small interfering RNA significantly reduced FITC‐albumin uptake in proximal tubular cells. These results suggest that down‐regulation of ORAI channel expression or inhibition of channel activity leads to impaired protein reabsorption in the proximal tubules, representing a novel mechanism underlying diabetic proteinuria.

Grant Funding Source: British Heart Foundation, Leverhulme Trust, China Scholarship Council; Swedish Research Council
High fat diet decreases fractional sodium excretion and increases systolic blood pressure via angiotensin II‐mediated increase in epithelial sodium channel abundance (689.2)
Authors: Minas Jacqueline , Nakano Daisuke , Awayda Mouhamed , Nishiyama Akira , Ortiz Rudy
Published: June 2021

High fat diet (HFD) accelerates diabetic nephropathy via impaired sodium (Na+) regulation and improper insulin signaling. Angiotensin II (AngII) receptor (AT1) blockers (ARBs) ameliorate insulin resistance, antinatriuresis, and elevated systolic blood pressure (SBP). However, the renoprotective effects of ARB during HFD remain elusive. To assess the role of AT1 activation to improper Na+ handling in a model of metabolic syndrome (OLETF) supplemented a HFD, seven groups were studied: 1) untreated, lean LETO, 2) LETO HFD (62% fat), 3) LETO HFD + ARB (10 mg olmesartan/kg/d), 4) untreated, obese OLETF, 5) OLETF HFD, 6) OLETF ARB, and 7) OLETF HFD + ARB. ARB reduced SBP in both OLETF (151 ± 3 mmHg vs 114 ± 3) and OLETF HFD (151 ± 2 vs 101 ± 6). Creatinine clearance decreased 35% in OLETF compared to LETO and increased 2‐fold in OLETF HFD compared to OLETF. Fractional excretion of Na+ (FENa) decreased 40% in OLETF compared to LETO. HFD decreased FENa 50‐75%, whereas ARB increased FENa in all cases. HFD increased urine aldosterone excretion 35% in both LETO and OLETF. Also, HFD increased potassium FE (FEK) 2 and 4‐fold in OLETF and LETO, respectively. ARB reduced FEK levels in all groups. Also, HFD increase levels of cleaved γ‐Epithelial Na+ Channel (ENaC) content 31% in OLETFs. Collectively, these data indicate that HFD exacerbates impaired renal function by increased ENaC abundance during Ang II‐mediated hypertension.

Grant Funding Source: Supported by: NIH MHIRT and USDA Minority Research Training Fellowship
Contrasting differences of chronic endothelin A receptor blockade during the progression of renal injury in type‐1 and type‐2 diabetic nephropathy (689.3)
Authors: Spires Denisha , White Tiffani , Taylor Lateia , Williams Jan
Published: June 2021

Contrasting differences of chronic endothelin A receptor (ETA) blockade during the progression of renal injury in type‐1 and type‐2 diabetic nephropathy
Denisha Spires1, Tiffani White2, Lateia Taylor2 and Jan M. Williams2. 1Tougaloo College, Tougaloo, MS, 2Department of Pharmacology, University of Mississippi Medical Center, Jackson, MS 39216 The endothelin (ET) system has been shown to play an important role in the development and progression of diabetic nephropathy (DN) via the endothelin A (ETA) receptor. Preliminary studies from our laboratory indicate that the induction of diabetes (STZ, 50 mg/kg, i.p.) in Dahl salt‐sensitive (STZ‐SS) rats promotes the development DN that was associated with an increase in ET‐1 excretion. Therefore, the present study examined whether chronic ETA blockade with ABT‐627 prevents the progression of renal injury in STZ‐SS rats with pre‐existing renal disease. After 6 weeks of ABT‐627 (5mg/kg/day) treatment, proteinuria was markedly decreased in ABT‐627 treated STZ‐SS rats versus vehicle treated rats without any changes in arterial pressure (310±32 vs. 517±68 mg/day, respectively). The degree of glomerulosclerosis and renal interstitial fibrosis was significantly reduced in the kidneys of ABT‐627 treated STZ‐SS rats compared to vehicle STZ‐SS rats. We next determined whether treatment with ABT‐627 would be beneficial in a type‐2 diabetic model (T2DN rat) with pre‐existing renal injury. In contrast to the STZ‐SS study, ABT‐627 had no effect on the progression of renal injury in T2DN rats. In conclusion, these data indicate that treatment with an ETA receptor blocker prevents the progression of renal injury in type‐1 diabetes but not in type‐2 diabetes.

Grant Funding Source: Supported by P20GM104357
What makes SGLT2 inhibition so effective in lowering blood glucose in diabetes? (689.5)
Authors: Gerasimova Maria , Rose Michael , Koepsell Hermann , Masuda Takahiro , Mayoux Eric , Thomson Scott , Rieg Timo , Vallon Volker
Published: June 2021

Studies in knockout mice indicated that, in euglycemia, the sodium glucose cotransporters SGLT2 and SGLT1 reabsorb 97 and 3% of filtered glucose, respectively. The SGLT1 contribution is enhanced to 40‐50% during genetic or pharmacologic SGLT2 inhibition. Here we show that the SGLT2 inhibitor empagliflozin (in diet for 15 weeks; 60‐80 mg/kg/d) strongly lowered blood glucose (BG) in type 1 diabetic Akita mice (means of 187‐237 vs. 517‐535 mg/dl in vehicle group; n=10‐13/group). In steady‐state, empagliflozin did not increase urinary glucose/creatinine ratios in Akita, indicating the reduction in filtered glucose (due to lowering BG and preventing glomerular hyperfiltration [determined in awake mice by FITC‐inulin plasma kinetics; GFR means of 255 vs. 397 µl/min]) balanced the inhibition of glucose reabsorption. Akita‐diabetes, empagliflozin alone, and Akita+empagliflozin similarly increased renal membrane SGLT2 expression (by 38‐56%) but reduced the expression of SGLT1 (by 33‐37%) vs vehicle‐treated wild‐type mice (WT). Phosphoenolpyruvate carboxykinase (PEPCK) is a principal gluconeogenic enzyme and regulated by gene transcription. Renal PEPCK mRNA expression was elevated by 60% in Akita versus WT mice and normalized by empagliflozin. All mentioned differences were statistically significant. We conclude that the diabetes‐induced changes in renal SGLT2/SGLT1 expression facilitate the blood glucose lowering potential of SGLT2 inhibition by upregulating the target protein and downregulating compensating SGLT1 (the latter is also observed in mice lacking SGLT2 and prevents excessive glucose uptake in late proximal tubule). In addition, SGLT2 inhibition may attenuate diabetes‐induced renal gluconeogenesis.

Grant Funding Source: Supported by NIH grants R01DK56248, R01HL94728, P30DK079337
Elucidation of pathogenic mechanisms for Fabry disease by transcriptome analysis (690.10)
Authors: Jung Namhee , Shin Youn Jeong , Jeon Yeo‐Jin , Jung Sung‐Chul
Published: June 2021

Fabry disease is an X‐linked inborn error of glycosphingolipid metabolism associated with deficient alpha‐galactosidase A activity. The major clinical feature of Fabry disease, such as fibrosis in cardiomyopathy and renal failure has been observed with abnormal accumulation of globotriaosylceramide (Gb3) in biological fluids, vascular endothelium, heart and kidney. In addition to Gb3, increased concentration of deacylated Gb3 (lyso‐Gb3) in the plasma of symptomatic patients also has been suggested as a causative molecular event. However, the correlation between fibrogenesis and elevated levels of these sphingolipids is poorly understood. To elucidate the genetic mechanisms involved in renal fibrosis in fabry disease, we analyzed the changes of global gene expression before and after Gb3 or lyso‐Gb3 treatment on two types of kidney cell lines, human proximal renal tubular epithelial cells (HK‐2) and mouse renal glomerular mesangial cells (SV40MES13), using microarray. The results showed that Gb3 and lyso‐Gb3 regulate the expression of 199 and 328 genes in each cell type, with at least 2.0‐fold change considerations. To identify key biological functions, we classified all the genes into 4 groups based on the expression patterns (1) Gb3 up, (2) Gb3 down, (3)lyso‐Gb3 up, (4)lyso‐Gb3 down regulated genes and performed functional annotation analysis using the DAVID tool. Most biological functions were related to fibrogenesis or epithelial‐mesenchymal transition (EMT). Interestingly, the gene expression patterns were significantly different between treated with Gb3 and treated with lyso‐Gb3. The expressions of EMT related genes (F8, FOXP2, HOXA11, DLL1 and WT1) were confirmed by realtime‐PCR or Western blot. These findings suggested that Gb3 and lyso‐Gb3 lead to renal fibrosis in Fabry disease with different biochemical modulations.
Hydrodynamic delivery of mitochondrial genes in vivo protects against moderate ischemia‐reperfusion injury in the rat kidney (690.17)
Authors: Corridon Peter , Rhodes George , Zhang Shijun , Bready Devin , Xu Weimin , Witzmann Frank , Atkinson Simon , Basile David , Bacallao Robert
Published: June 2021

Ischemic preconditioning is well known to afford protection against subsequent episodes of ischemia/reperfusion (I/R) in multiple organs, including the kidney, but the mechanisms involved are not fully understood. We proposed that ischemic preconditioning results in an altered mitochondrial proteome that confers resistance to subsequent I/R injury. We subjected rats to moderate ischemic injury and used liquid chromatography – mass spectroscopy (LC/MS) to analyze the proteome of kidney cortical mitochondria isolated following 14 days of recovery from I/R. The expression of several proteins was elevated following preconditioning, and two were selected for further analysis: Isocitrate dehydrogenase 2 (IDH2) and a member of the sulfotransferase family (SULT1A1). The effect of increased expression of these proteins was evaluated by using hydrodynamic gene delivery of plasmid vectors to overexpress each protein. Strikingly, hydrodynamic delivery of IDH2 and SULT1A1 genes attenuated the peak in serum creatinine following I/R injury induced by a 40 minute bilateral clamp by 82% and 74% respectively, and by 66% and 49% respectively following I/R in uninephrectomized rats. Histological analysis showed significantly reduced proximal tubule damage. We evaluated mitochondrial respiratory chain activity in vivo using 2‐photon fluorescence microscopy and the potential‐sensitive dye TMRM, and using biochemical assays in vitro, and showed that mitochondrial respiration was maintained in animals that received hydrodynamic delivery of either gene relative to controls. In conclusion, our study supports the concept that adaptive changes in mitochondria can confer protection to ischemic damage, and the utility of hydrodynamic gene delivery as a method of manipulating kidney function.

Grant Funding Source: Supported by NIH/NIDDK DK088934
Identification of hepatocyte nuclear factor‐1β gene regulatory networks in mouse kidney cells (690.9)
Authors: Aboudehen Karam , Patel Vishal , Kim Min , Xie Yang , McDonald Jeffrey , Gordillo Ruth , Igarashi Peter
Published: June 2021

Hepatocyte nuclear factor‐1β (HNF‐1β) is a transcription factor that is expressed in renal tubules in the mammalian kidney. Mutations of HNF‐1β produce kidney cysts and electrolyte disorders. HNF‐1β has been shown to regulate the expression of cystic disease genes and solute transporters. The aim of this study was to identify the complete spectrum of genes that are regulated by HNF‐1β in the kidney. We performed genome‐wide chromatin immunoprecipitation and massively parallel sequencing (ChIP‐seq) in combination with microarray analysis to identify direct mRNA and miRNA targets in mouse mIMCD3 cells. ChIP‐seq analysis identified 4,725 mRNAs and 85 miRNAs as potential targets of HNF‐1β. Microarray analysis showed altered expression of 4,878 mRNAs and 51 miRNAs in cells expressing dominant‐negative mutant HNF‐1β. Combining the ChIP‐seq and microarray data identified 1,545 mRNAs and 22 miRNAs that were directly regulated by HNF‐1β. Pathway analysis revealed that HNF‐1β directly regulated the expression of multiple genes involved in cholesterol and sphingolipid metabolism. Liquid chromatography‐mass spectrometry showed corresponding alterations in the levels of sterols and sphingolipids in cells expressing mutant HNF‐1β. These findings provide new insights into the HNF‐1β transcriptional network in kidney cells and identify a novel role for HNF‐1β in regulating lipid metabolism in the kidney.
Loss of alpha(E)‐catenin increases cisplatin nephrotoxicity (691.2)
Authors: Wang Xinhui , Grunz‐Borgmann Elizabeth , Nichols LaNita , Parrish Alan
Published: June 2021

Cisplatin is one of the most potent and widely used antineoplastic drugs. However, the use of cisplatin is dramatically limited by its side effect, nephrotoxicity. Evidence has shown that there is an increased incidence and severity of acute kidney injury (AKI) in the elderly. We previously reported a dramatic decrease of α‐catenin expression in proximal tubular epithelium of aged kidney. We hypothesize that loss of α(E)‐catenin increases susceptibility to cisplatin. To study the effects of reduced α‐catenin, shRNA knock‐down of α(E)‐catenin (C2) was generated in NRK‐52E cells; NT3 is the non‐targeted control. Confluent cultures of cells were challenged with cisplatin (50, 100, 150 μM) for 24 hr and C2 cells exhibited a significant loss of viability at all concentrations as compared with NT3 cells. Interestingly, the expression of α(E)‐catenin was further decreased in both cell lines. Importantly, higher caspase 3/7 activity was observed in C2 cells than NT3 cells after cisplatin treatment indicating the increased susceptibility may be due to apoptosis. To elucidate the mechanism of increased apoptosis in C2 cells, we performed RNA sequencing and identified that CYFIP2, a pro‐apoptotic gene, had 6.44 fold higher expression in C2 cells than NT3 cells. This finding was confirmed by qPCR. Taken together, these results suggest that loss of α(E)‐catenin may increase cisplatin‐induced nephrotoxicity via apoptosis.

Grant Funding Source: Supported by NIH RO1AAG034154
Direct effect of glucagon‐like peptide‐1 on the autoregulatory response of the afferent arteriole (692.2)
Authors: Sorensen Charlotte , Jensen Elisa , Poulsen Steen , Holst Jens , Holstein‐Rathlou Niels‐Henrik
Published: June 2021

It has become clear that glucagon‐like peptide‐1 (GLP‐1) has many effects and the GLP‐1 receptor is expressed in many tissues outside the pancreas. GLP‐1 increases renal blood flow (RBF), glomerular filtration rate and sodium and water excretion. The aim of this study was to examine expression of the GLP‐1 receptor in the renal vasculature and assess the effects of GLP‐1 in the afferent arteriole.

The GLP‐1 receptor was localized using 125I‐GLP‐1 in kidney slices. In the isolated juxtamedullary nephron preparation, renal perfusion pressure was increased in steps and changes in afferent arteriolar diameter were measured before and after perfusion with GLP‐1. In vivo experiments were performed on rats monitoring blood pressure (BP), RBF and urine flow. GLP‐1 was infused directly into the kidney before and after infusion of the GLP‐1 receptor blocker Exendin9‐39 and indomethacin and L‐NAME.Strong binding of 125I‐GLP‐1 was found in the vascular smooth muscle cells in afferent arterioles. Perfusion with GLP‐1 in isolated kidneys completely abolished the autoregulatory response of the afferent arteriole. Intrarenal infusion of GLP‐1 increased BP, RBF and urine flow significantly in rats. Heart rate and plasma renin were unchanged. The effect on RBF was inhibited by Exendin9‐39 whereas the increase in BP and urine flow was still present. Infusion of indomethacin and L‐NAME did not change the GLP‐1 effect on BP, RBF or urine flow.
Mechanism of purinergic action on glomerular permeability for albumin (692.3)
Authors: Kasztan Małgorzata , Piwkowska Agnieszka , Kreft Ewelina , Kowalski Robert , Rogacka Dorota , Szczepańska‐Konkel Mirosława , Jankowski Maciej
Published: June 2021

Purinergic receptors are expressed on cellular components of glomerular filtration barrier and their activation may affect glomerular permeability for albumin leading to albuminuria. We investigated mechanisms underlying purinergic action on glomerular filter permeability for albumin measuring convectional albumin permeability (Palb) in single isolated glomerulus based on video‐microscopy method. Effect of nucleotides in vivo was investigated using osmotic pump implanted subcutaneously into Wistar rats. Cultured rat podocytes were used for analysis of albumin permeability, PKG dimerization and immunofluorescence of actin cytoskeleton. Various nucleotides (ATP, ADP, UTP, UDP) or non‐metabolized ATP analogues (2‐MeSATP, ATP‐γ‐S) increased Palb in time‐ and concentration‐dependent manner in vitro. Effects were dependent on P2 receptors activation, nitric oxide synthase and cytoplasmic guanylyl cyclase. ATP analogues significantly increased permeability for albumin and sub‐cortical actin reorganization in PKG‐dependent but non dimmer‐mediated route in cultured podocytes. Furthermore, ATP analogues significantly increased Palb, in vivo, but did not affect urinary albumin excretion. Our results suggest that purinergic activation involves nitric oxide synthase and cytoplasmic guanylyl cyclase which affects actin reorganization in podocytes with subsequent increase of glomerular barrier permeability for albumin.
Intravital imaging of afferent arteriole calcium dynamics (692.7)
Authors: Riquier‐Brison Anne , Burford James , Sequeira‐Lopez Maria Luisa , Gomez R. Ariel , Peti‐Peterdi Janos
Published: June 2021

The afferent arteriole (AA) of the glomerulus is the most critical resistance vessel in the autoregulation of renal blood flow and glomerular filtration rate. Calcium dynamics of the AA, including myogenic contraction and the calcium wave of tubuloglomerular feedback (TGF) have been studied in vitro, however their relevance to in vivo conditions is not known. We aimed to directly visualize the dynamics of intracellular calcium [(Ca2+)i] in cells of the AA in vivo in the intact living kidney. Multiphoton imaging of the kidney was performed <2 weeks after unilateral ureter obstruction (UUO) in mice expressing the genetically encoded calcium indicator GCaMP3 in cells of the renin lineage (Ren1dCre/GCaMP3fl). Myogenic tone of the AA was observed as regularly oscillating AA constrictions, once in every 7.1±0.2 seconds (n=19) associated with minor elevations in AA vascular smooth muscle cells (VSMC) (Ca2+)i. In contrast to the low and even GCaMP3 fluorescence along the length of the AA, high baseline and ∆(Ca2+)i were observed in 1‐2 VSMCs at the glomerular entrance which appeared to function as sphincter cells. Laser‐induced stimulation of macula densa (MD) cell permeability triggered robust, 1.9±0.2‐fold increases in AA VSMC baseline (Ca2+)i and augmented the AA myogenic responses (n=11). Focal MD stimulation triggered a propagating calcium wave along the AA towards proximal segments (with a speed of 6.8±0.9 um/s, n=5) and into the glomerular mesangium (1.2±0.1 um/s). We concluded that AA sphincter cells and MD‐derived signals have the most robust effects on AA (Ca2+)i dynamics and contractility in vivo.

Grant Funding Source: Supported by AHA
Excess oxidative stress in genetically obese rats is independent of elevated inflammatory markers (693.11)
Authors: Ernsberger Paul , Koletsky Richard
Published: June 2021

Oxidative stress is elevated in obese humans and rodents and may contribute to obesity‐associated conditions. These conditions include elevated blood pressure, lipids, glucose, and insulin resistance, all of which are components of the metabolic syndrome and are present in the spontaneously hypertensive obese (SHROB/Kol) rat model of prediabetes. In the present study, SHROB (N=27) were compared to lean SHR/Kol (N=11) littermates obtained from Charles River Labs (Wilmington,MA). Oxidative stress was measured by assay of peroxides by the ferric orange xylenol method (Sigma, St. Louis, MO) and nitrotyrosine by immunoassay. Plasma and organ lipid peroxides were increased up to 3‐fold in SHROB rats relative to lean SHR. Nitrotyrosine immunoreactivity in solublized kidney extracts was increased by genetic obesity (SHROB: 290±52 vs SHR: 4.1±1.3 pmol/g wet weight). Similar results were obtained for liver. The activity in the liver cytosol of the protective enzyme superoxide dismutase was not different (SHROB: 63±5 vs SHR: 53±7 U/mg protein). Another protective enzyme, catalase, was also unchanged in the liver cytosol (SHROB: 95±6 vs SHR: 93±13 U/mg protein). Thus, the enzymes responsible for breaking down reactive oxygen species fail to up‐regulate in response to increased oxidative stress in SHROB. We tested whether increased oxidative stress may reflect inflammation in SHROB rats. The inflammatory markers tumor necrosis factor alpha (TNF‐a) and interleukin‐6 in plasma did not differ between SHROB and SHR (P>0.3 for each). The anti‐inflammatory mediator adiponectin was actually increased in plasma from SHROB (N = 15) relative to SHR (N = 11; 12.2 ± 0.3 versus 9.27 ± 0.6 ug/ml). Thus the increased oxidative stress in genetically obese and hypertensive SHROB rats is not a consequence of a global inflammatory state.

Grant Funding Source: Supported by Ophthalmology Education Worldwide
Caveolin‐1‐dependent regulation of cellular metabolism in breast cancer: role of Nrf‐2 and MnSOD (693.19)
Authors: Hart Peter , Minshall Richard , Bonini Marcelo
Published: June 2021

Caveolin‐1 (Cav‐1) is a plasma membrane associated protein that participates in the regulation of various signaling pathways affecting key cellular functions. According to our studies, variations in the expression levels of Cav‐1 heavily impact breast cancer cell metabolic adaptation that concurs with epidemiologic observations, indicating that Cav‐1 loss contributes to the glycolytic shift in advanced breast cancer. Mechanistically, we found that Cav‐1 ablation in breast cancer epithelial cells (MCF‐7) results in a marked increase in reactive oxygen species (ROS) production (e.g., 4 fold increase in H2O2) derived from MnSOD overexpression. Because MnSOD expression is downstream of Nrf‐2 nuclear factor, that is itself repressed by the association with Cav‐1, we hypothesize that loss of Cav‐1 leads to an increase in Nrf‐2‐dependent transcription and upregulation of MnSOD, which in turn activates AMPK by enhancing H2O2 outflow from mitochondria. In fact, cells devoid of Cav‐1 were shown to turn glycolytic in an H2O2‐ and AMPK‐dependent manner. Cav‐1 reconstitution or silencing of Nrf‐2 dampened AMPK activation and restored mitochondrial polarization along with decreasing the glycolytic rate. Relevantly, cells obtained from breast cancer patients at different stages of the disease showed progressive loss of Cav‐1 parallel to the upregulation of MnSOD, Nrf2, activated AMPK and a strong shift towards a predominantly glycolytic metabolism. Additionally, immunofluorescent staining of breast cancer tissue as compared to healthy controls recapitulated these findings. In conclusion, we propose that Cav‐1 may serve a tumor suppressive role by imposing metabolic restrictions for cancer progression via repressing Nrf2, MnSOD and ROS‐induced AMPK activation.

Grant Funding Source: Supported by Dept of Defense (W911NF‐07‐R‐0003‐04), T32 Training Grant (T32HL072742‐09)
Oxidant‐antioxidant parameters in different sessions of hyperbaric oxygen therapy (693.2)
Authors: Ortiz‐Villegas Miguel , Navarro‐Barrientos Maria , Kormanovski Alexander , Castillo‐Hernandez Maria , Lara‐Padilla Eleazar , Guevara‐Balcazar Gustavo
Published: June 2021

Hyperbaric oxygen therapy (HBO) cause and increment of oxygen into the blood, through a body intermittent exposure to 100% oxygen at pressures greater than one atmosphere of pressure; it produces several therapeutic effects of which the most studied are: carbon monoxide poisoning , acute influences the vascular reactivity and reduces the heart rate ( 10 to 15% ), increases collagen production , stimulates neoangiogenesis modulates nitric oxide production , increases the uptake of reactive oxygen species and especially in the treatment of arterial gas embolism and decompression sickness.It is also well known that oxygen breathing over 1 ATA may increase reactive oxygen species (ROS). This is very important as the molecular basis for different molecular mechanisms The goal of this paper is to measure several parameters oxidant/ antioxidant in the heart of rats with HBO.

Methods: Male Wistar rats were used and were exposed to HBO at 2 atmospheres with 100% O2 for 60 minutes. TAS, TBARS , SOD , GPX , GPH , CAT and NT were measured in the left and right heart .

Results: Finding an elevation of TBARS in heart left and right since the first session until normalized in 20 session. TAS, GPX, GSH and CAT concentration were lower at the beginning than the control at the 20 session.

Grant Funding Source: COFAA / IPN
The effects of high energy radiation on the vestibular and immune function in rats (693.22)
Authors: Scott Ansley , Shenwu Ming , Tang Xuehui , Wang Jun , Yang Chunli , Zhou Wu , Zhu Hong , Mao Jinghe
Published: June 2021

The objective of the project is to study radiation effects on the vestibular and immune systems in rats. It is proposed that high energy radiation will compromise vestibular/balance system and immune function since nasopharyngeal cancer patients experience post‐irradiation vertigo and whole body radiation therapy causes immune suppression. However, the underlying mechanisms are not fully understood.

Sprague‐Dawley rats were exposed to different doses of either unilateral cranial using 6 MeV electron beam (0 to 60Gy) or whole body using 18 MeV electron beam (0 to 10 Gy). For vestibular study, seven days post irradiation, single unit recording of vestibular afferent activity was performed. A total of 309 horizontal semicircular canal afferents’ responses to sinusoidal earth‐horizontal rotations (~1Hz) were analyzed. For immune function study, total RNAs were extracted from immune organs such as thymus and spleen. The genes involved in Stress and Toxicity PathwayFinder were examined using RT2 ProfilerTM PCR Array.

Preliminary analysis shows that electron irradiation caused significant decreases in the gains of the irregular horizontal semicircular canal afferents. The gene expression profiles indicated significant changes in oxidative stress, DNA damage and repair as well as apoptosis related genes.

In conclusion, these results indicate that irradiation exposure induces specific functional deficiency of the vestibular and immune system in rats.

Grant Funding Source: Supported by NASA MSI EPSCoR (NNX12AK85A and NNX13AB31A), NIH R01DC012060 (HZ), NIH R01DC008585 (WZ)
Lipopolysaccharide‐induced histone deacetylase activity increases paracellular permeability in human lung microvascular endothelial cells (695.4)
Authors: Joshi Atul , Barabutis Nektarios , Thangjam Gagan , Birmpas Charalampos , Catravas John
Published: June 2021

Objective: Lipopolysaccharide causes disruption of the endothelial barrier and increases trans‐endothelial permeability. Inhibition of heat shock protein 90 (Hsp90) protects and restores LPS‐mediated trans‐endothelial hyper‐permeability. Hyper‐acetylation of Hsp90 suppresses its chaperone function suggesting a role of HDAC in LPS‐mediated trans‐endothelial permeability. Therefore, we investigated the role of HDAC in LPS‐mediated trans‐endothelial permeability in human lung microvascular endothelial cells (HLMVEC). Method: HDAC activity was measured using the Flour‐de‐lys assay kit and HLMVEC barrier function was measured using gold‐plated electric cell‐substrate impedance sensing (ECIS) arrays. Results: Whole cell lysates from HLMVEC exposed to LPS (0.2, 1 or 5 EU/ml) showed significant increase in HDAC activity compared to vehicle‐treated cells. Cell fractionation revealed that LPS induced HDAC activity in both the cytoplasmic and the nuclear extracts. This LPS‐induced HDAC activity was not inhibited by pre‐treatment with the PI3k/Akt inhibitor, Ly294002 (5µM) or the Hsp90 inhibitor AUY‐922 (2µM), indicating that LPS stimulates HDAC activity independently of Hsp90 or Akt. Additionally, pre‐treatment with either HDAC inhibitor (Trichostatin (TSA), 1 µM or Panobinostat (LBH), 1 µM) protected HLMVEC monolayers from LPS‐mediated hyper‐permeability and attenuated Hsp90 chaperone function by disrupting Hsp90‐CDC37 interaction. Conclusion: LPS induces HDAC activity to promote trans‐endothelial hyper‐permeability possibly via de‐acetylation and activation of the Hsp90 chaperone function. (Supported by HL093460 and HL101902)

Grant Funding Source: Supported by HL093460 and HL101902
Role of T cells in LPS‐induced lung inflammation and dysfunction (695.5)
Authors: Kvietys Peter , Nader Moni
Published: June 2021

Common pathophysiologic features of ALI/ARDS are pulmonary leukocyte infiltration, interstitial edema, and impaired gas exchange. A major component of the alveolar infiltrate is polymorphonuclear leukocytes (PMN), which are believed to be the innate immune cells responsible for the pulmonary dysfunction. In a murine LPS (i.t.) model, our experiments indicated that, at 24 hrs after LPS challenge, PMN were present in the pulmonary alveoli and there was evidence of pulmonary dysfunction, i.e., increased vascular protein leak (Evans blue) and systemic hypoxia (arterial pO2). Interestingly, the infiltrated leukocytes were not all PMN; approximately half of the leukocyte population consisted of T cells (immunohistochemistry targeting CD3+ cells). In order to determine whether CD4+ T cells were involved in the pulmonary dysfunction in our LPS model, a genetic blockade approach was used (mice deficient in CD4+ T cells). The results of these experiments indicated that the increase in vascular protein leak induced by LPS in wild type (WT) mice was completely prevented when CD4+ T cell deficient (CD4‐) mice were used. The LPS‐induced systemic hypoxemia was also substantially blunted in the CD4+ T cell deficient mice. These observations provide strong support for the hypothesis that CD4+ T cells play an important role in the pathogenesis of ALI/ARDS.

Grant Funding Source: KACST‐ MED1672‐44
In vitro (organ chambers) effects caused by respiratory acidosis on vascular reactivity of the rat aorta (695.6)
Authors: Augusto Viviane , Nadai Tales , Silveira Ana Paula , Albuquerque Agnes , Vento Daniella , Rodrigues Alfredo , Celotto Andrea , Évora Paulo
Published: June 2021

Resumo In vitro (“organ chambers”) effects caused by respiratory acidosis on vascular reactivity of the rat aorta Viviane S. Augusto, Tales R. Nadai, Ana Paula S. Cassiano, Agnes A. S. Albuquerque, Daniella A. Vento, Alfredo J. Rodrigues, Andrea C. Celotto, Paulo R. B. Evora Department of Surgery and Anatomy, Ribeirão Preto School of Medicine University of São Paulo, Brazil The mechanisms by which pH influences on vascular tone or their response to certain agonists are not fully understood, but there is evidence to suggest the involvement of nitric oxide (NO), prostacyclin (PGI2), potassiumchannels and calcium flux. The present investigation was carried out to study in vitro (“organ chambers”) effects caused by respiratory acidosis (RA) on vascular reactivity of the aorta from Wistar rats, and the endothelium‐dependent mechanisms involved. Many assays were carried out untiladoptingorgan baths bubbling a gas mixture of CO2/(40%)/02 (60%), for the construction of pH‐response curves (pH 7.4 at 6:6) registering in isometric pre‐aortic rings contracted with phenylephrine (10‐6M). As main results: 1) Relaxations were seen only in endothelial rings; 2) Endothelium‐dependent relaxations were inhibited by incubation for 30 min with indomethacin (10‐5 M), L‐NAME (10‐4 M) and tetraethylammonium (10‐3 M). These data allow concluding by the suitability of the method and the dependence of endothelial vascular response induced by extracellular RA, including the three known mechanisms of vasodilation: cAMP, cGMP and hyperpolarization.

Grant Funding Source: Supported by CNPq, FAEPA, CAPES, FAPESP
Vascular phenotype of the STE20/SPS1‐related proline/alanine‐rich kinase targeted hypotensive mouse model of Gitelman syndrome (697.6)
Authors: Siew Keith , Maguire Janet , Davenport Anthony , O'Shaughnessy Kevin
Published: June 2021

Reduced renal Na+‐Cl‐ cotransporter (NCC) activity in Gitelman syndrome leads to salt‐wasting hypotension. SPAK phosphorylates NCC and Na+‐K+‐2Cl‐ cotransporter (NKCC1) increasing activity. Our SPAK T243A loss‐of‐function knock‐in (SPAK‐KI) mice recapitulate human Gitelman syndrome by reducing phospho‐NCC. Published data on SPAK knock‐out (SPAK‐KO) mice show reduced aortic phospho‐NKCC1 and contractility, that we hypothesised would be recapitulated by SPAK‐KI. Therefore we have investigated the vascular phenotype of SPAK‐KI.

To assess the influence of altered NKCC1 activity, myograph mounted aortae (n=11 per group) were incubated with 10μM bumetanide (Bmt) to inhibit NKCC1 or 0.1%v/v ethanol (EtOH) control. Cumulative concentration‐response curves to phenylephrine were used to determine pD2 (‐log10 of the EC50) and maximum response (EMAX) values.

We found no significant difference (P>0.05) in aortic contractility between SPAK‐KI and wildtype (WT) littermates in the absence or presence of Bmt.

Group
WT (EtOH)
WT (Bmt)
SPAK‐KI (EtOH)
SPAK‐KI (Bmt)

pD2 (‐log EC50)
6.18 ± 0.10
6.17 ± 0.08
5.99 ± 0.12
6.23 ± 0.11

Emax (mN/mm)
3.3 ± 0.5
3.3 ± 0.5
4.7 ± 0.9
3.3 ± 0.7

Basal (nN/mm)
21.0 ± 1.2
20.5 ± 1.3
20.9 ± 1.2
21.0 ± 1.5

Data are mean ± SEM In conclusion, vascular contractility remains intact in the SPAK‐KI despite functional or pharmacological NKCC1 inhibition. We suggest the reported phenotypic differences of the SPAK‐KO results from their alternative model design. Recent data support physiological roles for newly discovered SPAK isoforms that are known to be absent in protein‐ablated SPAK‐KO but present in protein‐intact SPAK‐KI. These isoforms may play as yet unexplored roles in vascular contractility.

Grant Funding Source: Supported by the British Heart Foundation
Chronic treatment with antioxidant flavonoids improves the vascular reactivity in mesenteric arteries from obese rats (700.10)
Authors: Priviero Fernanda , Rojas‐Moscoso Julio , De Souza Verônica , Ravos Amanda , Claudino Mario , Rocha Thalita , Carvalho Patrícia
Published: June 2021

Impaired vascular relaxation is often observed in obese individuals, which is likely associated to the decreased NO bioavailability and increased oxidative stress. Antioxidant flavonoids are usually proposed as a therapy against the increased oxidative stress. However, glycosylated flavonoid bioavailability is limited because of its low capacity of intestinal absorption. We aimed to evaluate the effects of a chronic treatment with glycosylate hesperidin (gHD) and its enzymatically hydrolyzed compound hesperetin (HT) on the vascular relaxation of obese rats. After the 4th week of life, rats were fed with standard chow or high fat diet (59% of total fat) + fructose (1mg/ml) for 12 weeks. gHD or HT were administrated by gavage (1ml/rat/day) during the last 8 weeks. Next, we measured body weight and epididimal weight and the superior mesenteric artery was removed in order to obtain concentration response curves to acetylcholine. Endothelium‐independent relaxation was evaluated by a single concentration of sodium nitroprusside (SNP). High fat diet + fructose caused an increase in the final body weight and epididimal fat, which was not prevented by the flavonoids (Final body weight: Control: 414 ± 15g, High Fat Diet: 504 ± 17g, gHD: 497 ± 39g, HT: 536 ± 22g; Epididmal fat: Control: 7 ± 1g, High fat diet: 19 ± 2g, gHD: 15 ± 1g, HT: 21 ±2g) Further, obese rats showed decreased vascular relaxation to acetylcholine (pEC50: 7.35 ± 0.35 vs 5.78 ± 0.42, for control and high fat diet, respectively). However, chronic treatment with antioxidant flavonoids prevented this decrease (pEC50: 6.97 ± 0.11 and 7.53 ± 0.05, for gHD and HT, respectively), suggesting that the impaired relaxation is because of the increased pro oxidant agents. Maximal responses to acetylcholine were similar among the groups. No differences were seen in the relaxant response to 1uM of SNP. In conclusion, either gHD or HT prevented the impaired vascular relaxation observed in obesity with no changes in the body weight and epididimal fat accumulation.

Grant Funding Source: Financial Support: FAPESP and CNPq
Low‐level lead exposure increased bioavailability of nitric oxide mediated by angiotensin II and PI3K/AKT pathway in rat aorta (700.2)
Authors: Fiorim Jonaina , Ribeiro Rogério , Azevedo Bruna , Simões Maylla , dos Santos Leonardo , Padilha Alessandra , Alonso Maria , Salaices Mercedes , Stefanon Ivanita , Vassallo Dalton
Published: June 2021

We previously demonstrated that 7‐day exposure with a low concentration of lead acetate increases blood pressure (BP) and nitric oxide (NO) bioavailability, which could be a compensatory mechanism in the initial stages of lead exposure.We investigated the involvement angiotensin II and PI3K/AKT pathway in this increased NO bioavailability. Wistar rats were treated with lead (1st dose 4 μg/100g and, subsequent doses 0.05 μg/100g, i.m cover daily loss) or vehicle. After lead treatment, blood levels were of 9.98μg/dL. In aortic rings from lead‐treated rats, phenylephrine (PHE 10‐10‐10‐4M) contractile responses were reduced; L‐NAME (LN‐10µM) increased PHE responses more in lead‐treated rats than untreated rats. Wortmannin (WT‐0,1µM) and PD123319 (PD‐1µM) normalized PHE responses in treated rats, and in presence of either WT, PD or losartan (LOS) that effect of LN on reactivity from lead treated group was blunted. Lead also increased release of NO, which was blocked by LOS, WT and PD. Our results suggest the involvement of angiotensin II and PI3K/AKT pathway in increased NO bioavailability in lead rats. This might be an alternative way to increase the NO bioavailability, as a compensatory mechanism against the increased SBP in the early stages of lead exposure.

Grant Funding Source: CAPES, CNPq and FAPES.
Pendrin localizes to the adrenal medulla where it modulates catecholamine release (701.1)
Authors: Lazo‐Fernandez Yoskaly , Aguilera Greti , Beierwaltes William , Verlander Jill , Kim Young , Pacak Karel , Plotsky Paul , Wall Susan
Published: June 2021

Pendrin is an aldosterone‐sensitive, Na+‐independent Cl‐/HCO3‐ exchanger expressed in intercalated cells within the renal cortex where it modulates blood pressure. Since pendrin gene ablation stimulates the release of renin, but not aldosterone, we hypothesized that pendrin is expressed in the adrenal gland and modulates adrenal function. Thus, we examined rat and mouse adrenal pendrin mRNA and protein abundance by PCR, immunoblot and immunohistochemistry and examined adrenal responses in wild type and pendrin null mice. Pendrin protein was detected in adrenal lysates from wild type, but not pendrin null mice. In both mice and rats, we observed pendrin mRNA and protein in the adrenal medulla but not in the adrenal cortex. Since the adrenal medulla produces catecholamines, further experiments examined plasma epinephrine responses to 5 and 20 min of immobilization stress in wild type and pendrin null mice. Basal epinephrine concentration was similar in wild type and pendrin null mice. Immobilization stress increased epinephrine in both groups, but responses were ~50% higher in pendrin null vs wild type after either 5 or 20 min of immobilization stress (20 min: KO, 1562 ± 263, n=4, vs WT, 1041 ± 129 pg/ml, n=6, P < 0.05). We conclude that pendrin is expressed in the adrenal medulla where it may blunt stress‐induced epinephrine release.

Grant Funding Source: Supported by NIH
Angiotensin II modulates sex steroid receptors and their metabolizing enzymes in rat cardiac fibroblast (701.10)
Authors: Madhavpeddi Lakshmi , Gonzales Rayna , Hale Taben
Published: June 2021

Sex steroids influence angiotensin II (AngII)‐induced cardiac remodeling; however, the importance of local sex steroid metabolism in this process is not known. We previously demonstrated that AngII increased aromatase levels, the enzyme that converts testosterone (T) to 17β‐estradiol, in cultured coronary vascular smooth muscle cells. In the present study we investigated the impact of AngII on sex steroid receptor and enzyme expression in primary rat cardiac fibroblasts. Fibroblasts were isolated from adult male rats and treated at P1 in 2% charcoal‐stripped FBS with AngII or vehicle (18h), followed by T (6h) in the presence or absence of anastrozole (aromatase inhibitor; 6.5h). Cardiac fibroblasts expressed receptors ERα, ERβ, and AR, as well as the enzymes aromatase and 5α‐reductase. AngII significantly reduced mRNA expression of ERβ. 5α‐reductase, AR, and ERβ levels increased when anastrazole was administered with T. This is the first demonstration that cardiac fibroblasts express the enzymes and receptors necessary for local sex steroid metabolism and action. Enzymes and receptor levels are differentially impacted by AngII. Additionally, aromatase inhibition in the presence of T alters steroid receptor and 5α‐reductase (testosterone metabolizing enzyme) levels. Given the major role of the fibroblast in pathogenic cardiac remodeling, the relative balance of T to estrogen in these cells may play an important role in the development of AngII‐mediated fibrosis.

Grant Funding Source: Supported by American Heart Association