bluuebunny/crossref_metadata_2025 huggingface dataset:
Upregulation of L‐type calcium channel function in the colon of P/Q‐type calcium channel deficient mice (1110.9)
Authors: Rodriguez‐Tapia Eileen , Fried David , Galligan James
Published: June 2021

The objective of this study was to examine the role of P/Q‐type voltage‐gated calcium channels (VGCCs) in enteric neuromuscular transmission in mice with a spontaneous mutation in P/Q channels (tottering, tg mice). Methods: Intracellular recordings of inhibitory junction potentials (IJPs) were obtained from circular muscle cells in segments of distal colon maintained in vitro. IJPs were evoked by a brief train of electrical stimulation (1s, 10 Hz, 10‐90 volts). Results: Electrical stimulation produced a voltage dependent increase in IJP amplitude and duration. IJPs were significantly larger in tissues from tg mice. Nifedipine (L‐type calcium channel blocker 1 μM) did not change IJPs in tissues from WT mice but it reduced IJPs in tissues from tg mice by 40 and 32% (P<0.05) at 60 and 70 V stimulation respectively. Conclusions: These results suggest that absence of functional P/Q‐type VGCCs causes an upregulation of L‐type calcium channel in the colon of tg mice. This could occur in inhibitory motorneurons, smooth muscle cells or both.

Grant Funding Source: Supported by R01DK094932
Ligand‐directed activation of extracellular signal‐regulated kinases (ERK1/2) in enteric neurons (1114.1)
Authors: Duraffourd Celine , Kumala Erica , Anselmi Laura , Brecha Nick , Sternini Catia
Published: June 2021

Opioids, acting at µ opioid receptors (µORs) are commonly used for chronic pain treatment. Opioids differ in their efficiency to induce µOR internalization, a key event in receptor signaling and function. Here, we tested the effect of opioids with different internalizing efficiency on the activation of the ERK family of Mitogen‐Activated Protein Kinases (MAPKs), a µOR downstream signaling, in enteric neurons, which mediate constipation, a major side effect of chronic opioids. Cultured rat myenteric neurons were stimulated with μOR‐internalizing agonists (the endogenous opioid analogue DAMGO or fentanyl at 1 µM), or morphine (10 µM) a poor internalizing agonist, for 0‐30 min at 37oC. ERK1/2 phosphorylation was determined by Western Blot with phospho‐p44/p42 and total ERK1/2 antibodies. DAMGO and fentanyl, but not morphine induced a rapid and transient ERK1/2 activation, which was blocked by naloxone, MAPK inhibitor, hypertonic sucrose, dynamin inhibition or mutation. To determine the effects of chronic µOR activation on ERK1/2 phosphorylation, cells were treated with 10 µM morphine or 1 µM fentanyl for 4 days before exposure to DAMGO, fentanyl or morphine. In enteric neurons chronically treated with morphine, DAMGO, fentanyl and morphine induced ERK1/2 activation, whereas in enteric neurons chronically treated with fentanyl only DAMGO and fentanyl induced ERK1/2 activation. Chronic morphine induced activation of the transcription factor, cAMP response element‐binding protein (CREB), which was abolished by MAPK pathway blockade. Chronic µOR activation induces ligand‐dependent changes of µOR downstream signaling, which are likely to contribute to the development of opioid‐induced constipation.

Grant Funding Source: NIDDK54155 and 41301
Sirtuin 1 induces autophagy to suppress liver ischemia/reperfusion injury in mice (1115.1)
Authors: Biel Thomas , Flores‐Toro Joseph , Dean Joseph , Law Brain , Zendejas Ivan , Behrns Kevin , Kim Jae‐Sung
Published: June 2021

Autophagy clears dysfunctional proteins and organelles in a timely manner. Defective autophagy and subsequent mitochondrial dysfunction is a key mechanism of I/R injury to the liver. Sirtuin 1 (SIRT1) is an NAD+‐dependent deacetylase that induces longevity and stress resistance, but the role of SIRT1 in hepatic I/R is unknown. Immunoblotting analysis after in vivo I/R of human and mouse livers and simulated in vitro I/R of mouse hepatocytes revealed that ischemia decreases SIRT1 to less than 20% of normal values, which is not recovered by reperfusion. I/R, however, did not decrease the expression of mitochondrial sirtuins such as SIRT3 and 5. Biochemical analysis showed that SIRT1 depletion during I/R is mediated by multiple proteases, including calpains. Specific overexpression of SIRT1 mitigated defective mitochondrial autophagy, onset of the mitochondrial permeability transition, and subsequent hepatocyte death after both in vitro and in vivo I/R. In striking contrast, hepatocytes from SIRT1 knockout mice were significantly susceptible to I/R injury. Thus, our results suggest that SIRT1 suppresses I/R injury in the liver by enhancing mitochondrial autophagy.

Grant Funding Source: NIH DK079879, DK090115, EY019688, HL064024 and AG028740
The impact of P‐selectin deficiency on obesity‐associated non‐alcoholic fatty liver disease (1116.8)
Authors: Kuo Chao Yu , Day Yuan Ji , Hung Li Man
Published: June 2021

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease in the worldwide, and occurs in patients who do not abuse alcohol. Metabolic syndrome is a major risk in the progression of NAFLD, steatosis and NASH (nonalcoholic steatohepatitis). In addition, obesity induced chronic low‐grade inflammation also play a crucial role in NAFLD formation. P‐selectin, an adhesion molecule, regulates leukocyte aggregation to vascular endothelium cell, induces the initial inflammatory response. In the study, we aimed to investigate the impact of P‐selectin deficiency on high fat diet (45% caloric from fat, HFD) induced NAFLD. C57BL6 wild‐type mice and P‐selectin KO mice were randomly divided into four groups, and fed with normal chow diet or HFD : wild‐type fed with chow diet (C) or high‐fat diet (H), P‐selectin KO mice fed with chow‐diet (PC) or high fat diet (PH). After 8 weeks HFD feeding, significantly increase body weight in wild‐type mice. The mice also exhibited higher liver weight, hyperglycemia, hyperinsulinemia and hyperlipidemia. Compared with the chow diet fed animal, the high fat diet impaired glucose tolerance and insulin sensitivity, enhanced glucose‐stimulated insulin secretion. High fat diet also induced plasma aspartate aminotransferase (AST) and alanine aminotranferease (ALT) elevation with imply hepatocyte damage. HFD promoted NF‐κB related inflammatory signaling in liver. However, P‐selectin KO mice reversed high‐fat diet induced AST, ALT elevation and NF‐κB inflammatory pathway in liver.

P‐selectin KO mice also exhibited lower body weight, improved glucose intolerance and insulin resistance. Our studies indicate that P‐selectin is a crucial metabolic inducer. Deletion of P‐selectin correct high fat diet induced metabolic disturbance, liver injury and slow down the progression of NAFLD.
Age and SPARC dependent cardiac collagen changes (1120.7)
Authors: Toba Hiroe , de Castro Brás Lisandra , Weintraub Susan , Jin Yu‐Fang , Bradshaw Amy , Lindsey Merry
Published: June 2021

Secreted protein acidic and rich in cysteine (SPARC), a collagen‐binding matricellular protein, has been implicated in procollagen processing. The aim of this study was to investigate age and SPARC dependent changes in cardiac extracellular matrix (ECM). We studied 6 groups of mice (n=4/group): young (4‐5 month old), middle‐aged (11‐12 m.o.), and old (18‐23 m.o.) C57BL/6J wild type and SPARC‐null mice. The left ventricle was decellularized in 1% SDS with protease inhibitors in PBS to enrich for ECM proteins. Protein extracts (10 μg) were separated by SDS‐PAGE, digested in‐gel and analyzed by HPLC‐ESI‐MS/MS. Relative quantification was by spectral counting (Scaffold). We identified 321 proteins, of which 27 proteins were ECM or ECM‐related proteins. Of these proteins, 16 showed age‐dependent changes. Collagens I, III, IV and VI, dystrophin, and laminin b2 increased with age, which may explain previously reported age‐associated increases in cardiac stiffness. SPARC deletion blunted the age changes observed in collagens I and III and laminin b2. Of interest, fibrillin and elastin showed a significant increase with age in the Null mice, suggestive of increased microfibril deposition in the absence of SPARC. In summary, our data suggest SPARC is a possible therapeutic target for aging induced cardiac dysfunction.

Grant Funding Source: Supported by HHSN 268201000036C (N01‐HV‐00244), R01 HL075360, HL051971, 1SC2 HL101430, 5I01BX000505
Disrupted gene expression in human cerebral cavernous malformations (1123.3)
Authors: Shishoian Taline , Stockton Rebecca
Published: June 2021

CCM is a serious human disease affecting 35 million people worldwide, characterized by grossly dilated brain capillaries forming clustered caverns. CCM is caused by familial (inheritable) mutations in KRIT1, CCM2 or PDCD10 genes, or sporadic occurrence of unknown origin. Hemorrhagic stroke may occur and symptomatic lesions are removed surgically since no drug therapy exists. All three CCM genes are expressed in all tissues, so inherited mutations might be expected to produce vascular lesions everywhere. Instead CCMs are confined to the brain, suggesting there are brain capillary‐specific additional genetic disruptions contributing to CCM pathology. My project focuses on assessing expression of target genes in CCM endothelial cells to identify commonly deregulated genes in both familial and sporadic CCMs. I purified RNA from resected human tissue, then performed Reverse‐Transcription PCR to synthesize a cDNA library representing differential expression between sample and control normal endothelial cells. We conducted gene expression studies first by microarray to identify target genes, then validation using quantitative PCR (qPCR) to compare normal and diseased tissue, to determine statistically significant changes in CCM samples. We find disruptions in functional GO cluster groups including apoptosis and matrix synthesis.

Grant Funding Source: NIH
Cortical voluntary activation adjustments induced by non‐exhausting eccentric exercise (1125.2)
Authors: Girard Olivier , Racinais Sébastien , Banzet Sébastien , Koulmann Nathalie , Chennaoui Mounir , Drogou Catherine , Chalabi Hakim
Published: June 2021

We tested the hypothesis that an eccentric exercise would exacerbate fatigue‐induced impairments in cortical voluntary activation and delay the recovery process, compared with a concentric‐matched trial. Fourteen males randomly performed two 30‐min walking exercises (velocity: 1 m.s‐1; grade: 25%; load: 12% of body weight), one downhill (DW) and one uphill (UP). Neuromuscular test sessions including tibial nerve and motor cortex stimulations during maximal isometric voluntary contractions of the plantar flexors were performed before and 3 min, 24 h, 48 h and 72 h post‐exercise. DW immediately reduced voluntary torque by 14.1% (P<0.001), while smaller strength losses occurred after UP (‐6.0%; P=0.15). Torque remained significantly depressed at least 3 days into recovery post‐DW (‐14.1%, ‐11.5% and ‐7.2% after 24 h, 48 h and 72 h, respectively; P<0.05). After exercise, cortical voluntary activation (‐15.9% vs. ‐8.5%; P<0.05) and resting twitch (‐42.6% vs. ‐30.2%; P=0.06) values were reduced below baseline with larger reductions after DW compared to UP, and were restored near baseline within 24 h. The capacity of the motor cortex to optimally drive the plantar flexors that accompanies peripheral fatigue is acutely impaired by non‐exhaustive eccentric exercise, but recovers within 24 h, and is therefore unlikely to explain the persisting reductions in voluntary strength capacity with the development of muscle soreness.

Grant Funding Source: Supported by Aspetar and the French Délégation Générale pour l’Armement under contracts 07ca704
Systemic inhibition of AT1 receptors does not alter respiratory‐sympathetic synchronization in anesthetized rats (1130.13)
Authors: Favero Michele , Menezes Miguel , Yamamoto Kenta , de Paula Patricia , Menani Jose , Mifflin Steve
Published: June 2021

Sympathetic nerve discharge contains a respiratory‐related rhythm.

Arterial chemoreceptors possess angiotensin AT1 receptors which have been reported to increase chemoreceptor afferent discharge. To determine if angiotensin might modulate respiratory‐sympathetic interactions, we tested if synchronization between phrenic nerve activity (PNA) and renal sympathetic nerve activity (RSNA) would be altered by systemic blockade of AT1 receptors. Spectral analysis and cross correlation were used to examine the synchronization in anesthetized, vagotomized and mechanically ventilated Sprague‐Dawley rats (300‐400g, n=5). Intravenous injections of losartan (a selective angiotensin II type‐1 receptor (AT1)antagonist, 1 mg/ml/kg body weight) inhibited the increase in blood pressure following iv. angiotensin II injection (50 ng/0.1 ml) from 40±7 mmHg to ‐6±8 mmHg (n=5, P<0.05). Following losartan, we estimated coherence between spectral power of PNA and RSNA at central respiratory drive frequency, defined as frequency of the main peak in PNA before and after losartan injection. Losartan did not alter the spectral power of either PNA or RSNA (P>.05). Coherence did not differ before (0.78 ± 0.21) compared to after (0.72 ± 0.34) losartan. Phase difference also did not change before (0.21 ± 0.25) and after (0.20 ± 0.24) losartan. These results suggest that systemic inhibition of AT1 receptors does not alter respiratory‐sympathetic synchronization.

Grant Funding Source: HL088052; CAPES (18890‐12‐1); FAPESP; CNPq
Reduced GABAergic synaptic transmission in the nucleus tractus solitarius in Mecp2‐null mice (1130.20)
Authors: Chen Chao‐Yin , Lin Yen‐Chu , Rogawski Michael , Lien Cheng‐Chang , Maezawa Izumi , Jin Lee‐Way
Published: June 2021

Rett syndrome (RTT), a devastating neurodevelopmental disorder, is caused by loss‐of‐function mutations in the X‐linked methyl‐CpG binding protein 2 (Mecp2) gene. Respiratory dysfunction is one of the major clinical features, presumably due to defective GABAergic function because enhancing GABAergic transmission improved the respiratory phenotype and prolonged survival in RTT mice. We test the hypothesis that deficiency in Mecp2 gene reduces GABAergic signal transmission in the NTS where respiratory information is first received and integrated. Spontaneous inhibitory postsynaptic currents (sIPSCs) and miniature IPSCs (mIPSCs) were recorded from NTS neurons in brainstem slices obtained from age‐matched male Mecp2‐null mice and wild‐type littermates. Compared to those from wild‐type mice (n = 11 neurons), NTS neurons from Mecp2‐null mice (n = 12 neurons) had a left‐ward shift in cumulative probability of sIPSC amplitudes (p < 0.05) without significant changes in inter‐event intervals (p > 0.05). Similarly, there was a significant left‐ward shift in mIPSC cumulative probability of amplitudes (p < 0.05) but not inter‐event intervals (p > 0.05). The data are consistent with the hypothesis and suggest that reduced GABAergic signaling via post‐synaptic mechanism(s) in the NTS contributes to respiratory dysfunction in RTT.

Grant Funding Source: supported by: 1R01 HD064817, International Rett Syndrome Foundation
Mechanisms of chronic intermittent hypoxia‐induced enhancement of paraventricular nucleus glutamatergic tone (1130.21)
Authors: Bardgett Megan , Guo Qing , Toney Glenn
Published: June 2021

Sleep apnea increases risk for cardiovascular diseases in part by increasing sympathetic nerve activity (SNA). Chronic intermittent hypoxia (CIH), a rodent model that mimics repetitive hypoxemias that occur in patients with sleep apnea, increases mean arterial pressure (MAP) through a hypothalamic PVN dependent mechanism. The goal of this study was to determine effects of CIH on glutamatergic NMDA receptor tone and investigate the underlying mechanisms. After 7 days of CIH, male Sprague‐Dawley rats were anesthetized and PVN NMDA receptors were blocked bilaterally with AP5. Compared to normoxic controls, CIH exposed rats had greater reductions of renal SNA (CIH: ‐17±7 vs control: 2±2%) and MAP (CIH: ‐8±3 mmHg vs control: ‐3±1 mmHg). CIH decreased PVN expression of NMDA receptor subunits NR1 (72% of control, n=5, P<0.05), NR2A (33% of control, n=2), and NR2B (49% of control, n=6, P<0.05), but increased expression of the excitatory amino acid transporter 2 (158% of control, n=2), suggesting a possible compensatory mechanism to maintain excitatory neurotransmission. CIH reduced expression of postsynaptic density protein 95 (PSD‐95, 73% of control, n=5, P<0.05), a critical adaptor protein that links NMDA receptors to neuronal nitric oxide synthase (nNOS) activation. Interestingly, CIH did not change PVN expression of nNOS (105% vs control, n=3). We conclude that support of SNA and MAP after 7 days of CIH involves increased NMDA receptor‐dependent glutamatergic tone in PVN, which involves reduced expression of NMDA receptors offset by reduced glutamate uptake. A further contribution could arise from reduced NO‐mediated inhibition caused by reduced NMDA receptor‐nNOS coupling by PSD‐95. HL 102310 & 088052 (GMT)
Effect of an emetic gastrointestinal input on the processing of labyrinthine inputs by cerebellar rostral fastigial nucleus neurons (1130.7)
Authors: Catanzaro Michael , Miller Daniel , Cotter Lucy , McCall Andrew , Yates Bill
Published: June 2021

Multiple inputs can elicit nausea and vomiting, including ingested toxins and vestibular signals. Amongst the nervous system regions believed to participate in generating motion sickness is the cerebellar rostral fastigial nucleus (rFN). In addition, there is evidence that gastrointestinal inputs are transmitted to rFN through a variety of pathways, including projections from the dorsal motor nucleus of the vagus and area postrema. These observations raise the hypothesis that GI inputs could change motion sickness susceptibility by altering the processing of labyrinthine inputs by rFN neurons. To test this hypothesis, we recorded activity from rFN neurons in decerebrate cats, and compared their responses to whole‐body rotations before and after the intragastric infusion of the emetic compound copper sulfate (CuSO4). Some (18%) rFN neurons had complex responses to vestibular stimulation, particularly responses to only one direction of rotation (known as STC behavior). Such complex responses would be expected in a brain area that participates in generating motion sickness. The spatial and temporal characteristics of these responses changed little after injection of CuSO4. Although infusion of CuSO4 affected gains of responses to rotations of ~25% rFN neurons, the effects were inconsistent (CuSO4 augmented responses in some cells and decremented responses in others). CuSO4 resulted in pronounced changes in responses to rotations of a significantly larger fraction (Chi‐squared test, p<0.05) of neurons in other brain regions that mediate nausea and vomiting, including the parabrachial nucleus and the “vomiting center” (caudal medullary lateral tegmental field). These data suggest that integration of vestibular and GI inputs by rFN neurons plays a limited role in affecting motion sickness susceptibility.

Grant Funding Source: NIH grant R01‐DC03732
Femoral artery occlusion amplifies TRPA1 function in skeletal muscle sensory neurons (1132.5)
Authors: Xing Jihong , Lu Jian , Li Jianhua
Published: June 2021

A prior study showed that transient receptor potential channel A1 (TRPA1) in thin‐fiber muscle afferents contributes to the amplified reflex autonomic responses observed in rats with femoral artery ligation. Thus, in this study we characterized current responses induced by activation of TRPA1 in skeletal muscle dorsal root ganglion (DRG) neurons of control limbs and limbs with 24 hrs of femoral ligation using whole‐cell patch clamp methods. Our results show that AITC (a TRPA1 agonist, 50‐200 µM) produced a dose‐dependent increase of amplitudes of inward current responses. Also, the peak current amplitude induced by AITC is significantly larger in DRG neuron of ligated limbs. There is no difference in size distribution of DRG neurons with AITC‐induced current responses between two groups. However, arterial occlusion increases the percentage of the AITC‐sensitive DRG neurons compared with control. AITC‐induced currents in DRG neurons are significantly attenuated by exposure to 10 µM of HC‐030031, a selective inhibitor of TRPA1. In addition, capsaicin (a TRPV1 agonist) evokes a greater increase in the amplitude of AITC‐currents in DRG neurons of ligated limbs. Overall, our data suggest that a greater TRPA1 function is developed in muscle afferent nerves with the hindlimb ischemia and TRPV1 is partly responsible for augmented TRPA1 responses induced by arterial occlusion.

Grant Funding Source: NIH P01 HL096570 and AHA EIA 0840130N
Functional recovery of AQP2 recessive mutations through hetero‐oligomerization with wild‐type counterpart (1133.2)
Authors: El Tarazi Abdulah , Lussier Yoann , Da Cal Sandra , Bissonnette Pierre , Bichet Daniel
Published: June 2021

Mutations in Aquaporin‐2 (AQP2) induce nephrogenic diabetes insipidus (NDI), a water reabsorption defect of the kidney challenging water homeostasis. While the accepted NDI model claims that recessive (rec) mutations are misfolded and monomeric by nature, the partial activity (Pf= 15‐25% of wt‐AQP2) found with some mutations (D150E, V24A) challenges this notion, and even suggest the presence for mixed wt/rec heteromers in heterozygotes. We have tested this hypothesis in Xenopus oocytes and showed that not only do wt/rec associate (co‐IP assays), but activity of the rec‐AQP2 mutant is shown to be mostly restored (63‐100% of wt‐AQP2) in the process. Also, using bidirectional vectors for dual transfection in cell lines, we find a recovery of targeting for rec‐AQP2 when in presence of wt‐AQP2. Finally, the negative results found when analyzing the strictly monomeric R187C mutant support the notion that the functional recovery of (at least some) rec‐AQP2 mutants is secondary to wt/rec heteromerization. In conclusion, we show that recessive mutations may be functionally recovered through wt/rec oligomerization, contributing to the overall activity in heterozygotes, a notion which could be relevant to all homomeric proteins.
Prenatal steroid exposure promotes expression of renal injury indices in African American females (1134.11)
Authors: Gwathmey TanYa , Chappell Mark , Nixon Patricia , Washburn Lisa
Published: June 2021

Administration of steroids to expectant mothers at risk for pre‐term delivery is standard practice, but may have detrimental effects on kidney development and function in the offspring. African‐Americans (AA) experience higher rates of both pre‐term birth and kidney disease than other racial and ethnic groups, and the onset of kidney disease occurs much earlier in life for AA. This study examined the expression of specific markers of renal injury in a cohort of AA females (age 14) either born prematurely (<32weeks gestation) and exposed in utero to betamethasone (BMX; N=12), born prematurely but not exposed (PRE; N=18) or born at term (>37 weeks; N=12). In comparison to term AA, the oxidative stress marker 8‐Isoprostane tended to be higher in PRE [10.9 + 1.0 vs. 8.9 + 1.9 pg/g creatinine; P = 0.06], but was higher in the BMX cohort [13.1+ 1.6 pg/g creatinine; P < 0.05]. Angiotensinogen excretion, a biomarker for glomerular damage and precursor to the fibrotic and inflammatory peptide angiotensin II, did not differ between BMX and PRE [0.14+0.04 vs. 0.12+0.04 ng/g creatinine], but was higher than in term subjects [0.06+0.01 ng/g creatinine; P<0.05]. While urinary angiotensin II tended to be higher in BMX [0.20+0.09 pg/g creatinine], no significant differences were observed between groups [PRE: 0.07+0.01 vs. Term: 0.11+0.06 pg/ g creatinine]. The microalbumin/creatinine ratio was higher in PRE [0.25+0.18 mg/g] and BMX [0.25+0.13 mg/g] as compared to term [0.04+0.01 mg/g]. In summary, we show that pre‐term birth is associated with increased expression of urinary 8‐Isoprostane, angiotensinogen and microalbumin, and is exacerbated by prenatal exposure to betamethasone. These findings suggest preterm birth and the accompanying prenatal steroid exposure may result in an increased susceptibility to renal injury and that indices of damage can be detected early during adolescence.

Grant Funding Source: Supported by AHA 12CRP9390000 and HD047584
Time‐dependent serum, urinary and tissue biomarker responses to renal ischemia/reperfusion injury in rats (1134.13)
Authors: Richards Toni , Glascock Christopher , Horn Todd , McFarland Kelli , Plato Craig
Published: June 2021

Acute kidney injury (AKI) secondary to acute renal ischemia is associated with high mortality and morbidity with few effective treatments. To elucidate potential novel therapeutic targets, the current study sought to characterize the acute serum, urinary and tissue biomarker response profiles to discrete durations of renal ischemia/reperfusion (I/R)‐induced AKI in rats. At d0, rats underwent sham (n=4) or warm, bilateral renal I/R (n=8/group; 30’ or 40’ ischemia) surgery using our proprietary vascular clamp. Rats were single‐housed in metabolic cages for balance of study (24 or 48 hrs post‐reperfusion). In 24 hr intervals post‐reperfusion, urine volumes were determined and sampled, and serum was collected. Clinical chemistry and ELISAs were applied to urine and serum analyses. At endpoint, renal tissue was collected for analysis of mRNA expression (quantitated via Luminex) and ED1+ immunohistochemistry. I/R injury elicited perturbations in multiple indices of renal function compared to sham rats including increased renal mass, serum BUN and creatinine (30’: 4.1±1.2 fold; 40’: 10.8±1.5 fold at 48 hr), urinary protein:, albumin:, Kim‐1: (30’: 131.4 fold; 40’: 60.4 fold at 48 hr), and MCP‐1:creatinine (30’: 10.4 fold; 40’: 86.6 fold at 24 hr), and decreased creatinine clearance (30’: ‐76%; 40’: ‐95% at 48 hr),. I/R injury altered tissue mRNA expression of several biomarkers (pro‐inflammatory, pro‐fibrotic, pro‐ and anti‐oxidative stress, tissue remodeling, apoptosis, angiotensin II receptor, endothelin receptor) and increased renal macrophage infiltration. The biomarker expression profile was dependent on both ischemia and reperfusion durations. The distinct biomarker expression profiles of varying durations of renal I/R in rats can subsequently be utilized to define potential therapeutic targets and possible new AKI diagnostic markers.
Podocyte NLRP3 inflammasome activation and glomerular injury by adipokine visfatin: in vitro and in vivo evidence (1134.7)
Authors: Boini Krishna , Xia Min , Abais Justine , Li Pin‐Lan
Published: June 2021

NLRP3 inflammasome formation and activation have recently been reported to be an important early mechanism responsible for glomerular inflammation and injury in obese mice. However, the precise mechanism of obesity‐induced NLRP3 inflammasome activation remains unknown. The present study explored whether adipokine visfatin mediates obesity‐induced podocyte NLRP3 inflammasome activation and consequent glomerular injury in in vitro and in vivo models. Confocal microscopic analysis showed that visfatin treatment increased the colocalization of NLRP3 with Asc or caspase‐1 in podocytes indicating the formation of NLRP3 inflammasomes. This visfatin‐induced inflammasome formation was abolished by pretreatment of podocytes with Asc siRNA. Correspondingly, visfatin treatment significantly increased the caspase‐1 activity and IL‐1β production in podocytes, representing activation of NRLP3 inflammasomes, which was significantly attenuated by Asc siRNA. Further RT‐PCR and confocal microscopic analysis demonstrated that visfatin treatment significantly decreased the podocin expression (podocyte damage), and Asc siRNA transfection attenuated this visfatin‐induced podocin reduction. Similar findings were observed in mouse model in vivo. It was shown that visfatin infusion induced NLRP3 inflammasome formation and activation in glomeruli of Asc+/+ mice, but not in Asc‐/‐ mice. Further functional studies found that visfatin infusion significantly increased the urinary albumin and protein excretion in Asc+/+ mice compared to Asc‐/‐ mice. Fluorescent immunohistochemical examinations using podocin and desmin as podocyte injury markers showed that the inflammasome formation induced by the visfatin were mostly located in podocytes as demonstrated by co‐localization of NLRP3 with podocin or desmin. Based on these results, it is concluded that visfatin induces NLRP3 inflammasome formation and activation in podocytes and thereby results in glomerular injury and sclerosis.

Grant Funding Source: supported by NIH grants DK54927 and HL‐75316
The endoplasmic reticulum stress inhibitor, 4‐phenylbutyrate, attenuates chronic kidney disease induced by a model of cardiorenal syndrome (1136.12)
Authors: Carlisle Rachel , Ask Kjetil , Dickhout Jeffrey
Published: June 2021

Endoplasmic reticulum stress has been identified as a feature of chronic kidney disease and appears to be associated with proteinuria. We have found the endoplasmic reticulum stress inhibitor, 4‐phenylbutyrate (4‐PBA), reduced renal damage in a mouse model of acute kidney injury. Therefore, we hypothesized that inhibition of endoplasmic reticulum stress through augmenting protein folding with a low molecular weight chemical chaperone, 4‐PBA, would attenuate chronic kidney disease. Chronic kidney disease was induced in uninephrectomized wild type mice. After recovery from uninephrectomy, an angiotensin II infusion pump (1.5 ng/min/g) and a deoxycorticosterone (DOCA) pellet were subcutaneously implanted and mice were provided with 1% NaCl in the drinking water for three weeks. Blood pressure was measured and animals were placed in metabolic cages for 24 h urine collection bi‐weekly, starting one week prior to uninephrectomy. Co‐treatment with 4‐PBA (1 g/kg/day) prevented angiotensin II/DOCA/salt‐induced hypertension, cardiac hypertrophy, proteinuria, and kidney injury, as demonstrated by reduced α‐smooth muscle actin expression, protein cast formation and damaged glomeruli. However, 4‐PBA‐treated mice suffered from significantly higher mortality rates. Inhibition of cardiac hypertrophy by 4‐PBA may have been the cause of the increased mortality through congestive heart failure. Despite the higher mortality rate in 4‐PBA‐treated mice, we conclude that 4‐PBA prevents hypertension, proteinuria, cardiac hypertrophy, and chronic kidney disease in this angiotensin II/DOCA/salt model of cardiorenal syndrome.

Grant Funding Source: Supported by CIHR OSO‐115895.
Interaction of PPARα, CD36 and Th17 in angiotensin II‐induced hypertension (1136.9)
Authors: Alatawi Abdullah , Oyekan Adebayo
Published: June 2021

Peroxisome Proliferator‐Activated Receptor alpha (PPARα) is known to exert many cardiorenal protective effects. In contrast, T‐ helper 17 (Th17) and CD36 have been implicated in hypertension (HTN) and Inflammation. PPARα has been found to promote CD36 function. We hypothesized that PPARα exerts anti‐inflammatory effect by inhibiting CD36‐induced Th17 activation in the kidney in Angiotensin II (AngII)–induced renal injury. The aim of this study is to evaluate the role of PPARα in amelioration of hypertensive renal injury due to CD36‐mediated Th17 activation in HTN. In animals treated with WY14643, a PPARα agonist, or GW6471, a PPARα antagonist, no significant difference in blood pressure (BP) was found between control and Ang II animals. However, GW6471 increased BP (188 ± 23 Vs154 ± 9 mmHg; P<0.01). Ang II increased proteinuria (P<0.001). WY14643 decreased proteinuria caused by Ang II at 14 days (36 ± 5 Vs 64 ± 19 mg/day; P<0.05), while GW6471 was without effect. Ang II did not affect PPARα or CD36 protein expression and WY14643 and GW6471 were also without effect. However, urine IL‐17 was higher in animals treated with Ang II and GW6471 (P<0.05). On the other hand, WY14643 caused a reduction in urine IL‐17 (P<0.01). We conclude that PPARα has renoprotective and anti‐inflammatory effects through inhibition of Th17 but not CD36. This mechanism may be important in the treatment of renal injury after Ang II treatment.
Minimal piggyBac vectors for chromatin integration (1138.12)
Authors: Solodushko Victor , Troyanovsky Boris , Bitko Vira , Fouty Brian
Published: June 2021

We describe novel transposon piggyBac vectors engineered to deliver transgenes as efficiently as currently available piggyBac systems, but with significantly less helper DNA co‐delivered into the host genome. To generate these plasmids, we identified a previously unreported aspect of transposon biology, that the full‐length terminal domains required for successful plasmid‐to‐chromatin transgene delivery can be repositioned from the transgene delivery cassette to the other (helper) parts of the plasmid. This design decreased the size of the required terminal domains within the delivered gene cassette of the piggyBac vector from about 1,500 to just 98 base pairs. Herein we tested the transposition efficiency of these minimal piggyBac transposons on different cell types: HEK293, HeLa, L929, and primary pulmonary artery smooth muscle cells. Transposition efficiency was defined as transgene (RFP) positivity at 28 days. The specificity of integration was determined by PCR and qPCR. Conclusion: Relocating most of the long terminal‐repeat sequences from the delivered gene cassette of minimal piggyBac vectors into the helper region of the same plasmid decreased the amount of non‐essential (helper) DNA incorporated into the host cell genome without a significant decrease in transposition efficiency. This may reduce the risk of target cell transformation.

Grant Funding Source: Supported by the American Heart Association to V.S. (12GRNT12070291)
Levels of antioxidant activity and oxidative stress in metabolic syndrome Puerto Rican participants (1138.9)
Authors: Collazo‐Roman Marielys , Muñoz‐Forti Kevin , Gonzalez Adrian , Jimenez Giselle , Mangual Roberto , Perez Yamilette , Quintana Xiomara , Rodriguez Juan , Pérez‐Cardona Cynthia , Ferder Leon , Suarez‐Martinez Edu B.
Published: June 2021

Metabolic syndrome (MetS) is defined as a of cluster factors that increase the risk of cardiovascular diseases. The criteria for diagnosis for MetS include increased triglycerides, reduced HDL cholesterol levels, raised plasma glucose concentration, and body mass index > 30 kg/m2. In the Puerto Rican population the prevalence of MetS is estimated in approximately 43.3%. Some studies suggested that MetS is linked to higher levels of oxidative stress (OxS). However, there is no data available using OxS markers at Puerto Rican community. Our objective was to determine levels of oxidized lipids and the activity of antioxidant enzymes Glutathione Peroxidase (GPx) and lipid peroxidation. We further expect to evaluate if there is there exist an association between the obtained levels and the presence of MetS in a Puerto Rican population. We hypothesized GPx activity will be significantly lower in participants with MetS and that lipid peroxidation will be higher in these persons. Data was analyzed using Prism Graph Pad and SPSSv12.0 Softwares. Our preliminary results showed no significant differences in plasma lipid peroxidation (n=224; p=0.229) and GPx (n=70; p=0.163). This study is important because excessive accumulation of oxidative molecules may be prevented by ingesting antioxidant supplements and this may create awareness of the consequences of the MetS and the potential benefit of intaking antioxidants.

Grant Funding Source: Support by R25GM096955 (NIH RISE Program)
Peculiarities of trichophytia epidemiology in Astana (1139.4)
Authors: Batpenova Gulnar , Kazieva Aliya , Dzhetpisbaeva Zulfiya , Pshembaeva Saule
Published: June 2021

To study the epidemiological aspects of the trichophytia in Astana Materials: The analysis of reported cases of Trichophytia in Astana from 2007 to 2011.

It should be noted that since 2007 (23 cases) to 2010 (267 cases) there was an increasing of more than 10 times, and in 2011 the number of patients has decreased to 151. Analysis of gender showed an absolute predominance among male patients, almost 2 times. For example, in 2007, men were infected trichophytia ‐ 15 and women 8. In 2010: men ‐ 172, women ‐ 95, in 2011 there were 100 male patients, 51 woman. The average age of the patients was ‐ 20 years. 10%(62) of the patients were married. It is should be noted prevalence of sexual infection in 39% (243), 14%(87) were infected when caring for pets and cattle. In 31% (193) the source of infection is not known. 6%(37) were infected through household items from patients trichophytia.

Social status of the patients was as follows : 174 ( 28 %) ‐ the unemployed, 124 (20%) employees, about the same workers, and 15% (93)‐ pupils and students, reported cases of Trichophytia among the private entrepreneurs and military ‐ 2 (0.3 %) and 1 (0.16%), respectively.

Thus, the current trend towards an increased incidence of Trichophytia preserved. This pathology is most often affects children and young adults . Among the modes of transmission, in recent years, often indicates a sexual way, which requires strengthening health education among young people.
Aliskiren effects upon prorrenin/renin receptor expression on heart and kidney in 5/6 nephrectomy‐induced hypertension in rats (1140.12)
Authors: Lopez Pedro , Anguiano‐Robledo Liliana , Avila‐Ramirez Massiel Alfonsina
Published: June 2021

Prorrenin / renin receptor (PRR), a new element of the renin‐angiotensin system has been implicated in development and progression of cardiovascular and renal diseases. Recently, Aliskiren, a direct renin inhibitor, has been used to treat these diseases. The aim of this work was to study Aliskiren effects upon PRR expression in cardiac and renal tissue during 5/6 nephrectomy‐induced hypertension. Male Wistar rats underwent 5/6 nephrectomy (5/6 Nx) or sham operation (S). Both groups received Aliskiren, 10 mg/kg/day, po, throughout the experiment. Control groups received vehicle. Seven days later, 24‐hour urine was collected and blood pressure was measured. PRR expression was determined by immunoblot. Our results showed that, compared to sham animals, 5/6 Nx rats treated with Aliskiren had lowered blood pressure and improved creatinine seric values, proteinuria and creatinine clearance. We found in nephrectomized rats that PRR increased seven days after surgery in heart and kidney, and aliskiren reduced PRR expression in both tissues. These results confirm the antihypertensive and renoprotective effects of aliskiren and interestingly, showed that aliskiren modifies PRR expression

Grant Funding Source: Supported by SIP20130546
α1D‐Adrenoceptors blockade revert angiotensin II‐dependent vascular remodeling in rats (1140.7)
Authors: Gallardo‐Ortíz Itzell , Rodríguez‐Hernández Sandra , López‐Guerrero Juan , Villalobos‐Molina Rafael
Published: June 2021

We explored the cardiovascular effects of angiotensin II (Ang II) continuous infusion focusing on α1A/1D‐adrenoceptors (α1‐ARs) functional expression, and hypertrophy in aorta and tail arteries of Wistar rats. Minipumps filled with Ang II (200 ng/kg/min), were implanted in male Wistar rats during 4 weeks. Some rats were co‐treated Ang II+losartan, AT1R blocker, or Ang II+BMY7378, α1D‐AR antagonist starting after 2 weeks maintaining the Ang II infusion for 2 more weeks. The systolic blood pressure (SBP) was measured by tail‐cuff; at 4 weeks aorta (α1D‐AR vessel) and tail (α1A‐AR vessel) were isolated for functional and structural analyses. Ang II increased SBP (209±8 vs 115±2 mmHg). Phenylephrine‐induced contraction in aorta was ≍45% higher in Ang II‐treated rats; while in tail responses were similar among all groups. Ang II hypertrophied aorta, whereas no change occurred in tail artery. Losartan reversed hypertension and hypertrophy in aorta; while BMY reversed hypertrophy without modifying hypertension. Data indicate that Ang II‐induced hypertrophy in aorta involves AT1R and α1D‐AR, but not α1A‐AR. Ang II‐induced α1D‐AR‐mediated vascular remodeling occurs independently of hypertension. These findings identify a α1D‐AR‐sensitive mechanism in the hypertrophy induced by Ang II; however, this effect is independent of blood pressure elevation.

Grant Funding Source: Grant IN214812 PAPIIT, DGAPA, U.N.A.M.
Apocynin increases the eNOS expression in aortas, increases [NO] and decreases [ROS] in endothelial cells of SHR (1146.4)
Authors: Graton Murilo , Perassa Ligia , Potje Simone , Gomes Ana Carolina , Antoniali Cristina
Published: June 2021

NOX enzymes are major source of reactive oxygen species (ROS) in many cellular types. Studies suggest the participation of NOX in hypertension. Previous experiments showed that chronic treatment of SHR with apocynin, a NOX inhibitor, prevents the development of hypertension. The aim of this study was evaluate possible alterations in eNOS (endothelial nitric oxide synthase) expression and ROS and nitric oxide (NO) concentrations in aortas and endothelial cells (EC) of SHR treated with apocynin. Wistar and SHR male rats were treated with apocynin (30mg/Kg/day, p.o.) from 4th to 10th week of life. The animals were sacrificed, the thoracic aorta and endothelial cells were removed. The basal expression of eNOS was lower in SHR aorta than in Wistar aorta. eNOS expression was not altered in aortas of Wistar treated with apocynin, but the treatment increased eNOS expression in SHR aortas when compared to untreated SHR. Aortas from treated or untreated SHR showed increased DHE fluorescence than Wistar aortas. EC of treated SHR showed reduced basal [ROS] than EC of untreated SHR. ACh doesn’t increased [ROS] in EC of SHR, Wistar or treated SHR. Basal [NO] in EC of treated SHR was higher than in EC of untreated SHR and similar to Wistar EC. ACh increased [NO] in EC of treated SHR when compared to Wistar and untreated SHR EC. Apocynin prevents hypertension in SHR and increases the eNOS expression in aortas of SHR. Apocynin increases [NO] and decreases [ROS] in endothelial cells of SHR.
Mitochondrial processing peptidases as a target therapy for vascular endothelial dysfunction in atherosclerosis (1146.8)
Authors: Pandey Deepesh , Bhunia Anil , Chang Fumin , Bergman Yehudis , Kuo Maggie , Serbo Janna , Berkowitz Dan , Romer Lewis
Published: June 2021

Oxidized Low density lipoprotein (OxLDL) acts via its receptor LOX‐1 and elicits endothelial dysfunction that is considered a hallmark for initiation and progression of atherosclerosis. However, the precise mechanism by which this occurs is still unclear and is the main focus of current study. Our time course, subcellular fractionation, and immunofluorescence data support our hypothesis that Arg2 is a dual‐targeted single gene and single mRNA product found in both mitochondrial and cytosolic fractions of HAEC ‐ a novel pattern of effector system activation in mammalian vascular injury. These data indicate that OxLDL‐mediated activation of Arg2 in HAEC and in mouse aortic intima occurs via post‐translational modification and subcellular translocation of pre‐existing pools of Arg2 from mitochondria to the cytosol. Accumulated Arginase 2 in cytosol diminishes the concentration of cytosolic L‐arginine and leads to eNOS uncoupling. OxLDL‐evoked changes in cytosolic Arg2 activity are muted by Rho‐kinase inhibition, and do not occur at all in LOX‐1 null endothelial cells. In addition, inhibition or siRNA knockdown of the mitochondrial processing peptidase (MPPα) reduces the cytosolic abundance and activity of Arg2 following OxLDL stimulation. Blocking MPP with the biochemical inhibitor O‐phenanthroline also attenuates OxLDL‐mediated changes in HAEC production of ROS and NO. Finally, knockdown of MPPα by adenovirally delivered MPP shRNA interference in isolated mice aortas was associated with improved endothelium‐dependent vascular relaxation as assessed by wire myography following OxLDL exposure, when compared with aortas treated with scrambled shRNA. Hence, inhibition of Arg2 translocation to cytosol by targeting MPP could provide novel therapeutic avenues for the treatment of atherosclerosis and other cardiovascular diseases.

Grant Funding Source: NIH, AHA
Fetal bovine serum influences the effect of a ligand on the activity of the SV23 splice variant of human constitutive androstane receptor (1148.1)
Authors: Lau Aik Jiang , Chang Thomas
Published: June 2021

The naturally occurring SV23 splice variant of human constitutive androstane receptor (hCAR‐SV23) is activated by di‐(2‐ethylhexyl)phthalate (DEHP), which is detected as a contaminant in fetal bovine serum (FBS). We determined the effect of charcoal‐stripped, dextran‐treated FBS (CS‐FBS), dialyzed FBS, and regular FBS on hCAR‐SV23 activity in a cell‐based reporter gene assay. In transfected HepG2 cells cultured in Minimum Essential Medium (MEM) supplemented with 10% FBS, basal hCAR‐SV23 activity varied with the type of FBS (regular > dialyzed > CS). DEHP increased hCAR‐SV23 activity when 10% CS‐FBS, but not regular or dialyzed FBS, was used to supplement MEM. With increasing concentrations (1‐10%) of regular or CS‐FBS, hCAR‐SV23 basal activity increased, whereas the DEHP‐mediated hCAR‐SV23 activity remained constant (regular FBS) or slightly increased (CS‐FBS). Subsequent experiments identified the following optimal conditions to conduct the hCAR‐SV23 activity assay: 1% CS‐FBS in Minimum Essential Medium‐Reduced Serum (MEM‐RS) for plating of cells and for the first 24 h after plating; and then in serum‐free MEM‐RS. Under these conditions, artemisinin, artemether, and arteether increased hCAR‐SV23 activity. In contrast, they decreased it in cells cultured in MEM supplemented with 10% regular FBS. In conclusion, the type and concentration of FBS influence the effect of a ligand on hCAR‐SV23 activity.

Grant Funding Source: Canadian Institutes of Health Research and Michael Smith Fdn. for Health Res.
Alterations of epigenetic signatures in hepatocyte nuclear factor 4α deficient mouse liver determined by improved ChIP‐qPCR and (h)MeDIP‐qPCR assays (1148.4)
Authors: Zhang Qinghao , Lei Xiaohong , Lu Hong
Published: June 2021

Hepatocyte nuclear factor 4α (HNF4α) is a liver‐enriched transcription factor essential for liver development and function. In hepatocytes, HNF4α regulates a large number of genes important for nutrient/xenobiotic metabolism and cell differentiation and proliferation. Currently, little is known about the epigenetic mechanism of gene regulation by HNF4α. In this study, the global and specific alterations at the selected gene loci of representative histone modifications and DNA methylations were investigated in Hnf4a‐deficient female mouse livers using the improved MeDIP‐, hMeDIP‐ and ChIP‐qPCR assay. Hnf4a deficiency significantly increased hepatic total IPed DNA fragments for histone H3 lysine‐4 dimethylation (H3K4me2), H3K4me3, H3K9me2, H3K27me3 and H3K4 acetylation, but not for H3K9me3, 5‐methylcytosine,or 5‐hydroxymethylcytosine. At specific gene loci, the relative enrichments of histone and DNA modifications were changed to different degree in Hnf4a‐deficient mouse liver. Among the epigenetic signatures investigated, changes in H3K4me3 correlated the best with mRNA expression. Additionally, Hnf4a‐deficient livers had increased mRNA expression of histone H1.2 and H3.3 as well as epigenetic modifiers Dnmt1, Tet3, Setd7, Mllt3, Ehmt2, and Ezh2. In conclusion, the present study provides convenient improved (h)MeDIP‐ and ChIP‐qPCR assays for epigenetic study. Hnf4a deficiency in young‐adult mouse liver markedly alters histone methylation and acetylation, with fewer effects on DNA methylation and 5‐hydroxymethylation. The underlying mechanism may be the induction of epigenetic enzymes responsible for the addition/removal of the epigenetic signatures, and/or the loss of HNF4α per se as a key coordinator for epigenetic modifiers.

Grant Funding Source: Supported by NIH grants ES019487, CA143656, and CA159350