bluuebunny/crossref_metadata_2025 huggingface dataset:
Occludin redistribution induced by platelet activating factor in rat mesentery microvessels is blocked by increased cAMP
Authors: Adamson R H , Sarai R K , Lenz J F , Altangerel A , Curry F E
Published: June 2021

Increased intracellular cAMP blocks the acute hyperpermeability response to inflammatory mediators such as platelet activating factor (PAF) or bradykinin in many models of microvascular permeability. Increased cAMP stabilizes the distribution of several endothelial adhesion molecules (e.g., occludin and VE‐cadherin) against inflammatory mediators in cell culture, but cAMP effects on adhesion molecule distribution has not been extensively investigated in intact microvessels. We tested the hypothesis that pretreatment (30 min) with rolipram (10μM) and forskolin (5μM) (R/F) to elevate intracellular cAMP would block redistribution of occludin that is induced by PAF in single perfused vessels of rat mesentery. In control vessels occludin revealed by immunofluorescence was distributed peripherally, was generally very thin and nearly continuous and co‐localized with VE‐cadherin (VE). Perfusion with PAF (10 nM) induced large acute Lp increases within 5–10 min. In vessels fixed near the peak of PAF Lp response occludin was discontinuous and closely co‐localized with VE. Pretreatment with R/F blocked Lp increases and blocked the discontinuities in the occludin. The results are consistent with the model that increased cAMP acts to stabilize peripheral endothelial adhesion structures, including the tight junction, and the stabilization contributes to blocking acute increases in permeability. NIH 28607.
ICAM‐1 mediates constitutive, leukocyte independent and inducible, leukocyte dependent permeability pathways in microvessels
Authors: Sumagin Ronen , Sarelius Ingrid H.
Published: June 2021

Using confocal microscopy and fluorescence based solute permeability (Ps) measurements, we identified two independent, ICAM‐1 mediated Ps pathways in arterioles and venules in cremasters of anesthetized (65 mg/kg Nembutal, ip) mice. We showed that under control conditions in wild type (WT) mice there is a constitutive PKC‐dependent Ps pathway [1.0±0.10 (arterioles) and 2.2±0.46 (venules) ×10‐6 cm/sec], which was significantly decreased in ICAM‐1 knockouts [(KO) 0.54±0.07 and 0.77±0.11 ×10‐6 cm/sec]. PKC inhibitor (Bisindolylmaleimid l, 1μM in 0.01% DMSO) decreased Ps in WT mice to similar levels measured in ICAM‐1KO mice. Likewise, a PKC activator (PMA, 1μM in 0.01% DMSO), successfully restored Ps values in ICAM‐1KO vessels to levels measured in WT controls. On the other hand, during TNFα induced inflammation, a significant (p<0.05), 2‐fold increase in Ps in venules and 2.5‐fold increase in Ps in arterioles occurred in a Src‐dependent and PKC independent manner. Blockade of Src (PP2, 2μM in 0.01% DMSO) but not PKC significantly decreased the TNFα dependant increase in Ps in both arterioles and venules. We conclude that both constitutive and inducible Ps in the microvasculature are regulated by ICAM‐1 mediated, outside‐in signaling but the regulatory pathways diverge on the downstream signals. Supported by: NIH HL 18208, HL 75186 and AHA 0615677T
HIV‐1 transgenic expression in rats alters tight junction protein expression and increases permeability in the lung epithelium
Authors: Fan Xian , Lassiter Coy , Joshi Pratibha C , Koval Mike , Guidot David M
Published: June 2021

Despite improvements in therapy, HIV‐1 infection still causes serious lung infections and respiratory failure. Many of the manifestations of HIV‐1 infection are mediated by viral‐related proteins, such as gp120 and Tat, and not by direct infection of cells. Tight junction (TJ) protein structure and function is associated with HIV‐1‐related breakdown of the blood‐brain barrier, but to date this has not been examined in the lung. We used the hemizygous NL4‐3Δgag/pol transgenic rat to test the hypothesis that HIV‐1‐related proteins interfere with alveolar epithelial TJ assembly and barrier formation. We assessed alveolar epithelial barrier function both in vivo and in primary alveolar epithelial monolayers in vitro. Transgenic HIV‐1 expression significantly decreased lung liquid clearance in vivo and decreased alveolar epithelial monolayer integrity in vitro. In parallel, HIV‐1 transgenic expression significantly decreased the TJ proteins claudin‐4, claudin‐18, occludin and ZO‐1 by western blot analysis, and fluorescence staining showed that ZO‐1 and occludin protein localization to alveolar epithelial cell junctions was abnormal. Our findings suggest that HIV‐1‐related proteins interfere with tight junction assembly and function within the alveolar epithelium, and this could explain in part why HIV‐1‐infected individuals are at higher risk for respiratory failure.
ATP released from pulmonary neuroepithelial bodies activates Clara‐like cells in the NEB microenvironment via P2Y2 receptors
Authors: De Proost Ian , Pintelon Isabel , Wilkinson William J , Goethals Sofie , Brouns Inge , Van Nassauw Luc , Riccardi Daniela , Timmermans Jean‐Pierre , Kemp Paul J , Adriaensen Dirk
Published: June 2021

Pulmonary neuroepithelial bodies (NEBs) are densely innervated groups of neuroendocrine cells, shielded from the airway lumen by Clara‐like cells (CLCs). In ex vivo mouse lung slices, NEB cells respond with a [Ca2+]i rise to stimulation with high K+, followed by a delayed [Ca2+]i increase in CLCs, indicative of an indirect activation. Aim was to unravel the mechanism of this potential interaction between NEBs and CLCs. Because accumulation of ATP is known for NEB cells, we focused on a possible purinergic signaling pathway. Using ‘sniffer patching’ to detect ATP release, stimulation with high K+ was shown to elicit quantal ATP release from NEBs. Use of enhanced ATP hydrolysis and the P2 receptor blocker suramin confirmed that ATP was responsible for activating CLCs following application of high K+. A combined pharmacological and Ca2+ imaging approach revealed the involvement of functional P2Y2 receptors, and immunohistochemistry verified the expression of P2Y2 receptors on CLCs. It is concluded that stimulation of NEBs in ex vivo lung slices evokes exocytosis of ATP and subsequent selective purinergic activation of surrounding CLCs in the NEB microenvironment, which recently has been assigned a potential pulmonary stem cell niche.

Support: FWO grants G.0085.04 and G.0081.08 (DA); UA grants NOI BOF 2003, GOA BOF 2007 (DA) and KP BOF 2006 (IB); BBSRC grant BB/D01591X/1 (PJK, DR)
Genome‐wide RNAi screen identifies degradation of protein damage as a novel and essential hypertonic stress response
Authors: Choe Keith Patrick , Strange Kevin
Published: June 2021

Hypertonic stress causes cellular and molecular damage in animal cells. Little is known about the mechanisms that detect and repair this damage. To begin addressing this problem, we used the genetic model organism C. elegans. These free‐living nematodes experience periods of desiccation in their native environments. Under laboratory conditions, C. elegans survives extreme hypertonic stress induced by raising environmental NaCl levels. To begin identifying genetic pathways required for survival under these conditions, we screened ~19,000 C. elegans genes by RNAi feeding. This screen identified 55 genes that are essential for survival during high NaCl hypertonic stress. Interestingly, 40% of these genes (22/55) encode proteins that function to transport and degrade damaged proteins, including components of the ubiquitin‐proteasome system and lysosomes. Western analysis demonstrated that hypertonic stress increases high molecular weight ubiquitin conjugates, suggesting increased ubiquitination of damaged proteins. In addition, protein aggregation detected with a fluorescent reporter dramatically increased under hypertonic conditions. Our data suggest that hypertonicity causes protein damage that is repaired by ubiquitin‐directed degradation and provide the foundation for a comprehensive genetic, biochemical, and molecular characterization of this novel osmotic stress response.
Influence of salinity on the localization and expression of the CFTR chloride channel in the ionocytes of the European sea‐bass Dicentrarchus labrax during ontogeny.
Authors: Bodinier Charlotte , Boulo Viviane , Lorin‐Nebel Catherine , Charmantier Guy
Published: June 2021

The European sea‐bass is a euryhaline teleost whose high osmoregulatory abilities allow sea‐lagoon migrations. Cellular and molecular approaches were combined to determine the osmoregulatory function of CFTR (“Cystic Fibrosis Transmembrane Conductance Regulator”) in different organs during ontogeny. At hatch, immunolocalization shows an apical localization of CFTR in the digestive tract and tegumental ionocytes, suggesting ion excretion. During ontogeny, the secretion site shifts from the tegument to the gills. In the kidney, the CFTR expression is transitory before the larva‐juvenile transition. Juvenile‐like physiological abilities are acquired just before metamorphosis. Sea water (SW) induces dehydration. In the posterior intestine, the basal CFTR may facilitate ionic absorption, perhaps in relation to water uptake through aquaporin. Excess ions are excreted through the gills and rectum, as shown by the apical CFTR. In fresh water (FW), CFTR is apical, suggesting chloride secretion. Expressions of protein and transcript of CFTR were followed after a transfer from SW to FW. In gills and intestine, after the transfer, the transcript expression and the protein quantity decrease, to lower the chloride eflux. These changes in CFTR expression according to salinity constitute one of the adaptive mechanisms for sea‐lagoon migrations.
Histone H2AX becomes phosphorylated in response to high NaCl in apoptotic cells, but not in viable ones
Authors: Dmitrieva Natalia I. , Burg Maurice B.
Published: June 2021

High NaCl induces DNA breaks. The breaks persist as long as NaCl remains elevated, but are rapidly repaired when NaCl is lowered. H2AX is phosphorylated in response to DNA damage from many causes, such as ionizing radiation, but whether this occurs in response to high NaCl has been controversial. In mIMCD3 cells H2AX does not become phosphorylated when osmolality is elevated to 500–600 mosmol/kg by adding NaCl (Am J Physiol Renal 285:F266,2003). In contrast, in HeLa cells elevation of osmolality to 500–600 mosmol/kg causes H2AX phosphorylation (Am J Physiol Renal 291:F1014,2006). The purpose of the present studies was to understand the difference. We find that 100% of mIMCD3 cells survive 24 hours after osmolality is increased to 500 mosmol/kg, but only 60% of Hela cells survive. Knowing that H2AX becomes phosphorylated during apoptosis, we identified apoptotic cells by fluorochrome labeling of activated caspases, and H2AX phosphorylation by immunostaining. Elevating NaCl to 500 mosmol/kg causes apoptosis in the HeLa cells and H2AX phosphorylation that is limited to the apoptotic cells. Exposure of HeLa cells to 400 mosmol/kg induces DNA breaks (Comet assay) without apoptosis or H2AX phosphorylation. We conclude that high NaCl‐induced DNA breaks do not cause H2AX phosphorylation in cells that survive, but that H2AX phosphorylation occurs in the cells that die by apoptosis. Supported by the Intramural Program of NHLBI
Disruption of the Actin Assembly and Microtubules Induces Export Trafficking and Activation of Human Short ClC‐3 in Cos‐7 Cells
Authors: McCloskey Diana T. , Dai Yan‐Ping , Lynch Aisling , Hume Joseph R. , Yamboliev Ilia A.
Published: June 2021

Human short ClC‐3 (sClC‐3) is associated primarily with lysosomal vesicles with only a fraction residing at the surface membrane of resting cells. Hypotonic swelling induces export trafficking to the cell surface and activates sClC‐3. To determine if the actin cytoskeleton and microtubules participate in sClC‐3 export trafficking, we disrupted these structures using Cytochalasin D (1 μM, 2h), Nocodazole (0.3 μM, 2h) or the combination of both drugs, prior to hypotonic cell exposure (230 mOsm, 15 min). Biotinylation of surface membrane protein, followed by immunoprecipitation and Western immunoblot analysis demonstrated that either drug induced export trafficking of both native ClC‐3 and expressed Flag‐sClC‐3 in COS‐7 cells in isotonic solution. This was associated with over a 2‐fold increase in the isotonic volume‐sensitive outwardly rectifying current (VSOAC). The concomitant incubation with both drugs caused little additional export of sClC‐3, but stimulated the isotonic VSOAC 3‐fold. Hypotonic exposure induced further export of sClC‐3 and activated VSOAC in cells incubated with both inhibitors, suggesting that both actin and tubulin networks contribute to normal cell volume regulation. As disruption of the actin and microtubular assembly is a cell stress factor, potentiated VSOAC could act as a survival response. This study was supported by NCRR grant P20RR 15581.
SNARE Proteins And SNARE Regulators Mediate Aquaporin2 Exocytosis In Renal Cells
Authors: Procino Giuseppe , Barbieri Claudia , Tamma Grazia , De Benedictis Leonarda , Pessin Jeffrey , Svelto Maria , Valent Giovanna
Published: June 2021

Aim: The expression and the functional involvement of SNARE proteins and SNARE‐regulating proteins (Munc18b and Munc18c) in catalyzing aquaporin 2 vesicles fusion at the apical membrane is investigated in a mouse cortical collecting duct cell line.

Methods: Localization of SNARE and SNARE‐regulating proteins was performed using a combination of confocal microscopy and cell fractionation. SNARE complex formation was demonstrated by co‐immunoprecipitation. Functional involvement of each protein was demonstrated coupling RNA interference to apical surface biotinylation in order to quantify the effect of protein knockdown on AQP2 exocytosis in response to FK stimulation.

Results: Both VAMP2 and VAMP3 were found associated with AQP2 immunoisolated vesicle. Syntaxin 3 was found associated with the apical plasma membrane along with its functional partner Munc18b. Upon FK stimulation, Synataxin 3 is able to dissociate from Munc18b and to form a stable complex with VAMP2, VAMP3 and SNAP23. siRNA experiments indicate that Syntaxin 3, VAMP2, VAMP3 and SNAP23 are required for AQP2 vesicle fusion at the apical membrane. Moreover, Munc18 knockdown induced accumulation of AQP2 at the apical plasma membrane.

Conclusion: These findings, taken together, propose a complementary set of SNARE proteins and SNARE‐regulators responsible for AQP2 storage vesicle fusion into the apical membrane in the renal collecting duct.
Identification of a regulatory phosphorylation site in a cell cycle and swelling regulated ClC Cl‐ channel
Authors: Falin Rebecca , Morrison Rebecca , Ham Amy‐Joan L , Strange Kevin
Published: June 2021

clh‐3 encodes two splice variants, CLH‐3a and CLH‐3b, of a C. elegans voltage‐gated ClC Cl‐ channel. CLH‐3b is expressed in the worm oocyte and is activated during meiotic cell cycle progression or in response to cell swelling. Channel activation is brought about by serine/threonine dephosphorylation mediated by the type 1 phosphatases GLC‐7α and GLC‐7β. GCK‐3 is a Ste20 kinase and homolog of mammalian PASK/SPAK, which regulates cation‐coupled Cl‐ cotransporters. GCK‐3 binds to a 101 amino acid C‐terminal splice insert unique to CLH‐3b. When co‐expressed with CLH‐3b, GCK‐3 dramatically inhibits channel activity and alters gating kinetics and voltage sensitivity. Knockdown of GCK‐3 in worm oocytes constitutively activates CLH‐3b. Using mass spectrometry, we identified a serine residue (S747) located within the 101 amino acid splice insert that is phosphorylated in the presence of GCK‐3. S747 and surrounding amino acids conform to the Ste20 kinase phosphorylation motif. Mutation of S747 to alanine prevents GCK‐3 mediated channel inhibition and alterations of channel voltage‐dependent properties. We conclude that phosphorylation of S747 is critical for regulation of CLH‐3b activity. S747 may be phosphorylated directly by GCK‐3 or other downstream kinases.

Supported by NIH R01 grant DK51610
Calcium‐dependent phosphorylation of TPD52 is mediated by a novel 46 kDa CAMKII isoform
Authors: Chew Catherine S. , Chen Xunsheng , Zhang Hanfang , Zhang Han
Published: June 2021

In the GI tract, tumor protein D52 is highly expressed in cells that undergo classical exocytosis and has been implicated as a regulator of this process. We have shown that the cholinergic agonist, carbachol, and the calcium ionophore, ionomycin, increase the phosphorylation of S136 in the D52 protein in gastric mucosal cells. The goal of this study was to identify the protein kinase(s) involved. A phosphospecific antibody was produced and used to identify kinase(s) that specifically phosphorylate S136. D52 kinase activity in extracts from mouse and rabbit gastric glands and HEK293 cells was resolved on ion exchange and gel filtration columns. A single protein kinase with an apparent Mr of 46 kDa was identified using “in gel” assays with D52 as a substrate. The 46 kDa protein kinase bound to calmodulin Sepharose 4B, cross‐reacted with several pan‐specific CAMKII antibodies and, upon activation with Ca2+/calmodulin, also cross‐reacted with anti‐active CAMKII (pT286/287) antibody. Carbachol‐stimulated phosphorylation of S136 was inhibited by the CAMKII inhibitor, KN93, with an IC50 of ∼20 μM and by the calmodulin antagonist, W7, with an IC50 of ∼30 μM. We conclude that a novel, low Mr CAMKII isoform is expressed in gastric mucosa and in kidney and that this protein kinase regulates calcium‐dependent D52 function(s) in these tissues. (Supported by NIH R01 DK31900).
Caveolar endocytosis is essential for tumor necrosis factor (TNF) ‐induced occludin internalization in vivo
Authors: Marchiando Amanda M , Shen Le , Schwarz Brad T. , Guan Yanfang , Watson Alastair JM , Montrose Marshall H. , Turner Jerrold R.
Published: June 2021

TNF causes epithelial barrier dysfunction in vitro and in vivo and is accompanied by redistribution of the tight junction protein occludin. Mechanistic characterization of this vesicular trafficking process and associated tight junction regulation has been limited by the inability to image occludin internalization in real time. Our aim was to characterize the route and mechanisms of TNF‐induced occludin endocytosis in vivo.

We developed mice expressing EGFP‐occludin and mRFP1‐ZO‐1 in intestinal epithelium, and both fusion proteins colocalized with their endogenous counterparts. Jejunal mucosa was imaged by confocal microscopy of live anaesthetized mice.

TNF (5μg i.p.) triggered focal intra‐junctional occludin aggregation followed by occludin endocytosis. The small endocytic vesicles contained caveolin‐1 but not clathrin heavy chain. Occludin endocytosis was prevented by dynasore, a dynamin II inhibitor; L‐t‐LacCer, a synthetic lipid inhibitor of caveolar endocytosis; and cholesterol chelation with MβCD; while neither macropinocytosis nor clathrin‐mediated endocytosis inhibitors blocked internalization. Occludin endocytosis was also defective in caveolin‐1 knockout mice, though TNF activated myosin normally.

These data are the first to image occludin protein trafficking in vivo and demonstrate the pivotal role of caveolin‐1‐dependent caveolar endocytosis in this process.

Supported by NIH.
Oxidative stress does not mediate hypertension and renal injury in ovariectomized DS rats
Authors: Lopez‐Ruiz Arnaldo , Zhang Huimin , Reckelhoff Jane
Published: June 2021

Blood pressure (BP) increases in postmenopausal women. BP is higher in ovariectomized (OVX) Dahl salt sensitive rats (DS) compared to intact females (INT). Estrogen is an antioxidant. We hypothesized that OVX leads to increased oxidative stress (ROS), increased BP and renal injury. INT and OVX, 13 weeks of age (n=4–6/grp), were given low salt (LS) diet 0.3%, and supplemented with either tempol (T) (1mmol/L, p.o.) or water for 2 weeks. BP was measured via femoral catheter. ROS was measured by lucigenin in kidney. Urinary albumin (UAlb) and nitrate/nitrite excretion (UNOx) were measured at the end. OVX on LS had higher MAP than INT, and T had no effect in either group. ROS was higher in OVX than INT, and was reduced with T in OVX. UAlb was higher in OVX, and was not affected by T in either group. UNOx was higher in INT than OVX, and was increased with T only in OVX. Despite the fact that ROS are higher and UNOx increases with T in OVX, ROS play no role in mediating HT or renal injury in OVX DS rats on LS. These data suggest that antioxidants may not reduce BP or renal injury in postmenopausal women. (Supported by NIH HL66072)
ANGIOTENSION‐INDUCED HYPERTENSION AND CHANGES IN RENAL FUNCTIONS IN MALE AND FEMALE sEH KNOCKOUT MICE
Authors: Yan Qingshang , Raksaseri Promporn , Weldon Steven M. , Kabcenell Alisa K. , Hebert Steven , Wang Tong
Published: June 2021

We have previously demonstrated that knockout of sEH exhibits lower blood pressure and GFR and reduced Na+ and K+ excretions in males but not in females. In this study we examined the effects of Ang II on systemic blood pressure and renal functions in male and female wild‐type (WT) and sEH knockout (KO) mice by both acute and chronic infusion of Ang II. Acute iv infusion of Ang II, for 2 hours (10 ng/kg BW/min), increased MBP significantly in both genders of KO and male WT mice but not in female WT mice. The MBP increased (mmHg) by 15.5 in male WT, 18.4 in male KO, 20.3 in female KO and 3.5 in female WT. Acute infusion of Ang II also slightly reduced GFR and increased both absolute (ENa) and fractional (FENa) Na+ excretions in all groups of mice, including female WT mice. Chronic infusion of Ang II, by osmotic minipump for 2 weeks (1mg/ Kg BW/ day), did not change MBP significantly in WT mice; MBP increased by 10.1 and 0.4 mmHg in male and female WT mice (P >0.05). In contrast, AngII increased MBP in both genders of KO mice. MBP increased by 27.9 mm Hg in male KO and 31.4 mm Hg in female KO mice. Chronic infusion of Ang II reduced ENa and FENa in female WT mice but had no effects on urine volume, GFR, and Na+ and K+ excretions in sEH KO mice and in male WT mice. These results indicate that female KO mice are more sensitive than female WT mice to Ang II‐induced hypertension. Male WT and sEH KO mice show similar blood pressure and renal function responses to Ang II infusion.
Rosiglitazone Decreases Blood Pressure in Female Dahl Rats: Role of Nitric Oxide and Oxidative Stress
Authors: Sartori‐Valinotti Julio C. , Ryan Michael J. , Zhang Huimin , Reckelhoff Jane F.
Published: June 2021

In women the prevalence of hypertension (HT) and salt‐sensitivity of blood pressure (BP) increases after menopause (PM). The peroxisome proliferator activator receptor gamma agonist, rosiglitazone (RO), reduces BP in humans and animals, including male Dahl salt‐sensitive rats (DS). We tested the hypothesis that RO decreases BP in intact (Int) and ovariectomized (Ovx) female DS by increasing nitric oxide (NO) and decreasing oxidative stress (OS). Int and Ovx DS (10 weeks; n=15/group) received low salt diet (LS: 0.3% NaCl) or LS+RO (100mg/kg diet) for 1 week and then challenged for 1 week to high salt (HS: 8% NaCl) or HS+RO. Urinary Nitrate/nitrite (N/N) and F2‐isoprostanes (F2Ip) were assessed on HS. On LS, telemetric BP was similar in Int and Ovx, and RO decreased BP to similar levels. On HS, BP increased to higher levels in Ovx than Int and RO decreased BP to similar levels in Int and Ovx. RO increased N/N only in Ovx. F2Ip were lower in Ovx than Int and not affected by RO. Our data suggest that in Ovx on HS, RO protects against the greater increase in BP, possibly by increasing NO bioavailability, and that OS plays no role in salt‐sensitivity of BP in female DS. RO may be a new therapeutic approach for treating HT in PM women (supported by NIH HL 66072, AHA 0725561B)
Estrogen is nephroprotective in the rat remnant kidney in a rat model of intrauterine growth restriction
Authors: Grigore Daniela , Ojeda Norma B. , Alexander Barbara T.
Published: June 2021

Recent epidemiological studies suggest that an enhanced susceptibility to renal injury may originate in fetal life. Our laboratory utilizes a model of placental insufficiency in the rat to induce intrauterine growth restriction (IUGR) and hypertension in IUGR offspring. Pre‐pubertal female IUGR are hypertensive; however, blood pressure (BP) returns to normotensive values after puberty. Ovariectomy (OVX) leads to a significant increase in BP (plus 17 mmHg) in IUGR with no effect in control suggesting estradiol may contribute to BP regulation in female IUGR. We hypothesized that susceptibility to renal injury may be enhanced by IUGR, an effect exacerbated by OVX. Despite the increase in BP by ovariectomy, IUGR did not manifest renal injury as determined by expression of TGFβ1, a cytokine key to the progression of renal disease; mRNA expression measured by Real‐Time PCR. Exposure to a secondary insult, reduction of renal mass (RRM), also did not lead to renal injury in intact control or IUGR. However, RRM did elicit a marked 4‐fold increase in remnant kidney TGFβ1 mRNA expression in OVX IUGR with no effect in OVX control. At the protein level, TGFβ1 was also significantly increased by RRM in OVX IUGR compared to OVX control (507 pgTGFβ1/ml vs. 344 pgTGFβ1/ml). Thus, renal TGFβ1 was enhanced in response to RRM in OVX IUGR, suggesting that estradiol plays a protective role against the development of renal injury in a model of IUGR.
Intracerebroventricular (icv) injection of replication‐deficient adenovirus encoding Cre‐recombinase (Ad‐Cre) reduces estrogen receptor alpha (ERα) staining in the subfornical organ of ERαflox female mice
Authors: Xue Baojian , Badaue‐Passos Daniel , Beltz Terry G , Johnson Ralph F , Johnson Alan Kim , Hay Meredith
Published: June 2021

The estrogen receptor knock out mouse (ERKO) has been used in our early studies that implicate ER involvement in protecting females from angiotensin II (ANG II)‐dependent hypertension. However, the relative role of peripheral vs central ER in this effect remains uncertain. This is mainly because of the difficulties in experimentally dissecting this complex system where different forms of ER (i.e., ERα and ERβ) are expressed in numerous sites and cell types both in the brain and in the periphery. In the present study, a ¡°floxed¡± ERα transgenic mouse line harboring a loxP‐flanked (exon II) for the ERα (ERαflox ) was used to create an animal model in which it is possible to knock down one specific ER subtype (i.e., ERα) in a targeted body structure/region. Ad‐Cre or a control empty vector (Ad‐Con) was injected into the lateral ventricle (2 μl) in ER αflox female mice. Ten days later, coronal brain sections were double immunostained for ERα and Cre‐recombinase. Cre‐treated ER αflox mice showed reduced ERα staining in the subfornical organ (SFO) along with intense Cre staining at this site. These results indicate that using the Cre recombinase/loxP system in combination with viral gene transfer of Cre, conditional gene deletion can be achieved in the SFO and other cardiovascular regulatory sites in the CNS. This will allow us to assess the role of central ER subtypes in ANG II‐induced hypertension. (Support: NIH HL‐59676 and HL‐62261)
Changes in cell function and protein expression of mouse renal principal cells, mpkCCD, after carbon nanoparticle (CNP) exposure
Authors: Amos Adam David , Witzmann Frank A. , Chernoff Ellen A. , Hong David , Lai Xianyn , Ringham Heather N. , Blazer‐Yost Bonnie L.
Published: June 2021

To assess effects of CNP exposure on renal epithelial cells, 3 types of CNP, single‐wall carbon nanotubes (SWCNT), multi‐wall carbon nanotubes (MWCNT), and fullerenes (4–200 μg/cm2, 48 hrs) were applied to mpkCCD cells, a renal barrier epithelial line, and functional changes determined via electrophysiology. CNP‐treated cells showed significant decreases in transepithelial resistance compared to control. To determine CNP‐induced structural changes, confluent cells were incubated with CNP (21.1 μg/cm2, 48 hrs). Actin filaments and nuclei were visualized in fixed cells. Cultures treated with SWCNT and MWCNT showed an increase in large, multinucleated cells. CNP agglomerates were surrounded by actin filaments. Differential protein expression analysis by LC‐MS/MS revealed changes in several proteins, including intra‐ and extra‐cellular membrane‐associated proteins stathmin‐like 2 and enolase (increased) and myotubularin related protein 9, gap junction alpha‐8 protein coiled‐coil domain‐containing protein 44, zona pellucida glycoprotein 4, olfactory receptor 586, phosphomannomutase 2, RAN binding protein 1, and actin (decreased). These results indicate a profound impact of CNPs on renal epithelial cell structure and function and merit further study of effects of CNPs on barrier epithelial cells. Funding: IUPUI Undergraduate Research Opportunities Program & AFOSR Grant FA9550‐06‐1‐0083.
Role of Age‐Dependent Changes in MMP Expression in Acute Kidney Injury
Authors: Akintola Adebayo D. , Chen Gang , Catania Jeffrey M. , Burghardt Robert C. , Parrish Alan R.
Published: June 2021

Aging is associated with an increased incidence and severity of acute renal failure. However, the molecular mechanism(s) underlying the increased susceptibility to injury remain undefined. Given the emerging role of MMPs in ischemic renal injury, the renal mRNA expression of MMPs and their endogenous inhibitors, TIMPs (tissue inhibitor of metalloproteinases), were also examined in young, aged‐AL and aged‐CR rats. Interestingly, age‐dependent increases in the expression of MMP‐7, ‐9, ‐13, and ‐14 were seen, and these changes were attenuated by caloric restriction. The expression of TIMP‐1, but not TIMP‐2 or ‐3, was increased by age and attenuated by caloric restriction. Changes in MMP expression gene expression were paralleled by an age‐dependent increase in collagenase, gelatinase, and membrane type‐MMP activity. Specific increases in MMP‐7, ‐9, and ‐13 activity was seen in urine and kidney tissue, respectively. These results suggest an association between the age‐related increase in susceptibility to injury and overexpression of select MMPs (‐7, ‐9, ‐13, and ‐14) in the kidney, implying a therapeutic potential for inhibition of these MMPs in acute kidney injury.
ACE‐independent ANG II generating pathway in type II diabetic renal vascular disease
Authors: Park Sungmi , Bivona B J , Feng Y , Lazartigues E , Harrison‐Bernard L M
Published: June 2021

The db/db mouse exhibits progressive type II diabetic renal disease. Studies were performed to test the hypothesis that ACE‐independent ANGII generation in the diabetic kidney contributes to afferent arteriole (AA) reactivity. Conscious non‐fasted blood glucose levels and body weight were elevated (523 ± 22 mg/dl, 54 ± 1g vs 161 ± 15 mg/dl, 30 ± 1g), while 24hr blood pressure measured by radiotelemetry was not different (108 ± 3 vs 107 ± 3 mmHg; n=5, 6) in db/db compared to db/m mice, respectively. Circadian rhythm for blood pressure was absent and heart rate was decreased in db/db mice (484 ± 16, 537 ± 12 bpm; P<0.05). AA were studied using the mouse in vitro blood perfused juxtamedullary nephron technique utilizing euglycemic (5mM) and hyperglycemic (30mM) solutions for db/m and db/db mice, respectively. AA responses to ANGI (1–1000nM) ± ACE inhibitor (1mM Captopril) ± Serine proteinase inhibitor (1mM PMSF) were measured. Baseline AA diameters of db/db (14.9 ± 0.5μm; n=22) were significantly larger than db/m (13.3 ± 0.6μm; n=19, P<0.05) mice. Significant vasoconstriction to 10, 100, 1000nM ANGI was observed in both groups (−10±2, −31±4, −30± 5% db/db, n=10; −11 ± 3, −30 ± 5, −30 ± 5 db/m, n=8) due to conversion of ANGI→ANGII. There were similar responses to ANGI in db/m and db/db mice. Vasoconstriction produced by ANGI→ANGII was significantly attenuated by Captopril and Captopril + PMSF in db/m mice (−30 ± 5, −10 ± 3, −2 ± 7%; n=8, 3, 3 at 1μM ANGI); however, there was no significant difference between ANGI and ANGI + Captopril in db/db mice. Interestingly, ANGI responses in db/db were blocked by ACE + chymase inhibition (−30 ± 5, −30 ± 7, −7 ± 5 %; n=10, 3, 3 at 1μM ANGI). Vasoconstictor responses to 100nM ANGII were intact in the presence of inhibitors (−17 ± 7, −15 ± 3%). These data suggest that intrarenal ANGII may be formed from ANGI by increased chymase activity in diabetes. NIDDK‐62003 & P20RR018766
Early diabetic kidney damage in the mouse VCD model of menopause
Authors: Keck Maggie , Romero‐Aleshire Melissa Jill , Cai Qi , Hoyer Patricia B , Brooks Heddwen L
Published: June 2021

The extent to which ovarian hormones impact the development of diabetic nephropathy is not well known. To characterize how changes in hormones across the menopausal transition impact the development of early diabetic kidney damage we used the 4‐vinylcyclohexene diepoxide (VCD) model of menopause, which induces gradual ovarian failure (OF) in B6C3F1 mice. 2 weeks after mice entered OF, diabetes was induced with streptozotocin. Follicle numbers and gross ovarian histology were not different between cycling diabetic and non‐diabetic mice or between OF‐diabetic and OF‐non‐diabetic mice. After 6 weeks of diabetes blood glucose was elevated in OF‐diabetic mice compared to cycling diabetic mice (336 ± 31 vs 218 ± 37 mg/dL, P<0.05). Glomerular area was not different between control mice and cycling diabetic mice, however glomerular area was significantly increased in OF‐diabetic mice compared to cycling diabetic mice (118 vs. 100%, P<0.05). There were no differences between any groups in the degree of glomerulosclerosis. Using microarray analysis and real‐time PCR we found that mRNA expression of midkine was increased 1.74 fold (P<0.05) and immediate early response gene 1 was increased 1.85 fold (P<0.05) in kidneys from OF‐diabetic mice compared to cycling diabetic mice. These results suggest that the changes in ovarian hormone production that occur at menopause exacerbate the development of diabetic kidney disease.
Mechanisms underlying altered P2X3 receptor‐mediated responses in the ovariectomized rats
Authors: Ma Bei , Fan Juan , Yu Lihua , Zhang Yanmin , Ni Xin
Published: June 2021

P2X3 and P2X2/3 receptors appear to be expressed in peripheral tissues and dorsal root ganglia (DRG) and participate in producing peripheral pain. However, the mechanisms underlying the P2X receptors mediated nociception in the different ovarial hormone levels remain unclear. We found that after ovariectomy, the peripheral mechanical allodynic and nocifensive behavioral responses were developed, which could be reversed by P2X receptor antagonists, while the visceromotor reflex to colorectal distention was decreased, with the longer latency and higher threshold. Immunohistochemial studies revealed that P2X3 expression was significantly higher in small and medium dorsal root ganglion neurons. Real time PCR results showed that the P2X3 mRNA was in a higher level in both dorsal root ganglion neurons and rectocolon. Patch‐clamp recording results showed an increased number of cells responded to ATP with a transient current which was mediated by P2X3 receptors, while no significant change in the amplitude of current. These results indicate P2X3 is likely an important mechanism contributing to the altered receptor functions in DRG and rectocolon after ovariectomy. (Supported by NSFC No: 30570597 and program of Changjiang scholar and innovative team)
Glucose flux is elevated in men, but not women, during the post‐exercise recovery period
Authors: Brooks George Austin , Henderson Gregory C. , Fattor Jill A. , Horning Michael A. , Faghihnia Nastaran , Johnson Mathew L. , Luke‐Zeitoun Mona
Published: June 2021

We tested whether glucose rate of appearance (Ra), disappearance (Rd), and metabolic clearance rate (MCR) would remain elevated following exercise. 10 men and 8 women received a primed‐continuous infusion of [6,6‐2H2]glucose tracer to measure glucose kinetics under 3 different conditions: 1) before, during and 3 h after 90 min of exercise at 45% VO2peak (E45), 2) before, during, and 3 h after 60 min of exercise at 65% VO2peak (E65), and 3) in a time‐matched sedentary control trial (C). Glucose Ra, Rd, MCR, and carbohydrate oxidation (CHOox) declined in C from the pre‐exercise time points to those corresponding to post‐exercise recovery in both men and women (P < 0.05). In men and women, Ra, Rd, and MCR increased above C during exercise and were higher in E65 than E45 (p < 0.05). Average Ra, Rd, and MCR remained elevated above C over 3 h of post‐exercise recovery in men following exercise in E45 and E65 (P < 0.05). In contrast, average Ra and Rd did not remain significantly elevated during post‐exercise recovery in women, although MCR did remain significantly elevated in E65 in women during post‐exercise recovery (P < 0.05). During post‐exercise recovery, CHOox was lower than in C in both sexes (P < 0.05). Depressed CHOox with elevated glucose disposal (men) or maintained glucose disposal (women), characterizes post‐exercise recovery as a time of CHO mobilization and partitioning toward non‐oxidative disposal. NIH grant AR 42906.
Effects of central and peripheral angiotensin AT1 receptor antagonism on cardiac sympathetic nerve activity in conscious sheep
Authors: May Clive , Hood Sally , Ramchandra Rohit , Watson Anna
Published: June 2021

Possible causes for the increased cardiac sympathetic nerve activity (CSNA) in heart failure (HF) are elevated circulating levels of angiotensin II (Ang II) and increased activity of central angiotensinergic mechanisms. We have, therefore, investigated the effects of blockade of the peripheral and central angiotensin systems on directly recorded CSNA in conscious normal sheep and sheep with pacing induced HF. Arterial pressure, CSNA and arterial baroreflex curves were measured prior to intravenous irbesartan (1 mg/kg/h) and after 90 minutes infusion. Similar measurements were made before and following central angiotensinergic blockade with intracerebroventricular (ICV) infusion of losartan (1 mg/h for 5 h). In sheep in HF, after 90 minutes of irbesartan, there were no significant changes in CSNA or heart rate (HR) with a tendency for mean arterial pressure (MAP) to decrease. These changes resulted in a leftward shift in the baroreflex relation of CSNA to diastolic blood pressure. After 5 hours ICV infusion of losartan in HF animals CSNA had decreased by 50% (18 ± 4 to 9 ± 3 spikes/sec) while MAP was unchanged. Our results indicate that the large increase in CSNA in HF is dependent to only a minor extent on circulating angiotensin, whereas central angiotensinergic mechanisms are a major factor causing the increased sympathetic drive to the heart. Funded by NHMRC, Australia and NIH.
Reduction of superoxide in the nucleus tractus solitarius improves cardiovascular dysfunction in chronic heart failure
Authors: Wang Wei‐Zhong , Wang Han‐Jun , Gao Lie , Zucker Irving H. , Wang Wei
Published: June 2021

Overproduction of the central superoxide has been suggested to contribute to increased sympathetic outflow and blunted baroreflex in chronic heart failure (CHF). The nucleus tractus solitarius (NTS) is a crucial region involved in cardiovascular regulation. This work was designed to determine the level of superoxide production in the NTS, and further investigate the effects of superoxide inhibition in the NTS on cardiovascular function in CHF. CHF was produced by coronary artery ligation. Protein expression of the NADPH oxidase subunits gp91 and p47 in the NTS were higher (53 % and 48%) in CHF than in sham rats. The superoxide production derived from NADPH oxidase in the NTS was greater (84 %) in CHF than in sham rats. Bilateral injection of the NADPH oxidase inhibitor apocynin or the superoxide dismutase mimetic tempol into the NTS produced a greater effect on lowering sympathetic activity and increasing baroreflex sensitivity in CHF than in sham rats. At the level of NTS barosensitive neurons (n=35), picoinjection of tempol (100 pmol/5 nl) decreased the neuronal discharge (−37%) and enhanced the neuronal barosensitivity (52 %), and the degree of tempol's effect on the NTS neuron was greater in CHF than in sham rats. These data demonstrate that superoxide in the NTS is increased in CHF, and a reduction in the NTS superoxide presents a beneficial effect on the sympathetic hyperactivity and the baroreflex impairment in CHF.
The Role of Ionotropic Glutamate Receptors in the Nucleus Raphe Magnus on Hypercapnia‐induced Hyperpnea and Regulated Hypothermia
Authors: Nucci Tatiane Borodinas , Gargaglioni Luciane Helena , Branco Luiz Guilherme
Published: June 2021

Introduction: Nucleus Raphe Magnus (NRM) is one of the cellular groups of the brainstem that is involved in the physiological responses to hypercapnia. We assessed the role of ionotropic glutamate receptors in the NRM in the ventilatory responses to hypercapnia. Methods: Pulmonary ventilation (VE) and body temperature (Tb) of male Wistar rats were measured before and after microinjection of Kinurenic Acid (KY, an ionotropic glutamate receptors antagonist, 10ηmol/0,1μL) into the NRM, followed by 60 min of hypercapnia exposure (7% CO2). Results: Intra‐NRM microinjection of vehicle or KY did not affect VE or Tb under normoxic conditions. Hypercapnia caused an increase of pulmonary ventilation in all groups, which resulted from increases in respiratory frequency and tidal volume (VT). The hypercapnic ventilatory response was significantly increased in Kinurenic Acid microinjected group compared with vehicle. The hypercapnia‐induced regulated hypothermia was not affected by Kinurenic Acid treatment. Conclusion: This data suggest that the glutamate acting on ionotropic receptors in the NRM exerts an inhibitory modulation on the hypercapnia‐induced hyperpnea. Supported by:CNPq and FAPESP.
Interactions between gonadal steroids and neonatal caffeine exposure on HVR in adult male rats
Authors: Bairam Aida , kinkead Richard , Lajeunesse Yves , Fournier Stéphanie , Montandon Gaspard , Joseph Vincent
Published: June 2021

Caffeine is commonly used to treat apnea in newborn infants, but the long‐term consequences of this treatment are poorly understood. We previously showed that neonatal caffeine treatment induces long lasting changes on respiratory control in adult male rats but not in females. To test the interaction of neonatal caffeine treatment and sexual hormone on respiratory pattern, adult male and female rats, that received by gavage either caffeine (15mg/kg/day – postnatal days 3–12) or water (control) were sham‐operated or gonadectomized. After 14 days of recovery, ventilatory parameters were measured under normoxia and in response to moderate hypoxia (12% O2 – 20 min). In sham males, a significant interaction (treatment x hypoxia) was obtained for the increase in respiratory frequency (p<0.001) and the decrease in inspiratory time (p<0.01) in hypoxia. No interaction was obtained in gonadectomized rats. In females, no interaction between hypoxia and nicotine was observed in sham or ovarectomized rats. This suggests that endogenous circulating testosterone in adult males is necessary to reveal the alterations induced by caffeine on respiratory control. Supported by CIHR.
Effect of acetic acid feeding during exercise on appetite and glycogen repletion after exercise in rats
Authors: Yaji Keiko , Okada Sachiko , Kondo Emi , Tai Shinji , Okamura Koji
Published: June 2021

We examined the effect of acetic acid feeding during exercise and ambient temperature after exercise on appetite and glycogen repletion in rats. Five‐week‐old male rats were provided meal twice daily and trained by treadmill run for 7d. On the 8th day, rats divided into 6 groups (n = 5) ran on treadmill at 30 m/min for 30min×4 sets. The rats were given oral gavage of 1.5 ml water (W) or a solution containing 300 mg/100 mL acetic acid (A) during a 10 min rest period allocated between 4 exercise sessions. After exercise, 5 rats given W or A during the exercise session were killed immediately, and the remaining groups were given a standard laboratory diet and water ad libitum for 1h under room temperature (R, 26°C) or cooling temperature (C, 20°C), respectively. These rats were killed at 4h after the start of feeding. The food intake was not statistically different among the groups, whereas AC was greater than the other groups (WR, 4.9 g (SD 2.8); WC, 4.7 (2.9); AR, 4.9 (2.0); AC, 7.0 (2.1)). The glycogen content in the liver and soleus muscle was greater in AC than the other groups (liver, WR, 23.6 mg/g tissue (9.2); WC, 25.0 (9.7); AR, 25.4 (5.9); AC, 35.0 (5.6); soleus, WR, 1.84 (1.1); WC, 1.95 (1.4); AR, 2.36 (1.6); AC, 3.08 (1.0)). These results suggest that combination of acetic acid feeding during exercise and feeding under cool temperature after exercise might enhance glycogen repletion in liver and skeletal muscle due to greater appetite after exercise.
Temporal sequencing of brain activations in menopausal hot flashes
Authors: Freedman Robert R , Murphy Eric , Diwadkar Vaibhav A
Published: June 2021

The insula, associated with interoception and representation of internal physiological states is a terminus in the thermoreceptive spinothalamocortical pathway within which the dorsal raphe (DR), is an early signaling region. Menopausal hot flashes (HF) are internal thermal events that activate the insula but the precedents of this activity are unknown. We assessed the time course of activity in the DR and insula in the context of HFs using fMRI conducted on 20 postmenopausal women with HFs (mean age 52 yrs). Subjects were heated with ventral and dorsal heating pads (42°C) throughout. HF onset was signaled with an increase of 2μmho/30 s in sternally recorded SCL's acquired synchronously with fMRI. Data were analyzed using SPM2 in a 30 image (1 minute) tri‐partitioned (Baseline, Pre‐Flash, post‐Flash: 10 images/partition) window around HF onset. Extracted time series from the raphe and insula were normalized to the mean activation value in Baseline. A significant interaction between partition and region, F(2,2388)=3.39, p<.034, indicated a rise in DR but not insula activity in the pre‐Flash partition. These results document a temporally ordered pattern of detectable physiological events associated with endogenously generated thermal events, and suggest that activity in the human medulla, central to homeostatis, precedes the detectable onset of menopausal flashes and the response of regions such as the insula.
Evaporative Heat Loss and Changes in Core Temperature Set‐Point during Different Absolute Workloads
Authors: Jay Ollie , Kenny Glen
Published: June 2021

Previous work suggests changes in core temperature set‐point during exercise are determined by relative workload (i.e. % of VO2max). Concurrent rates of metabolic heat production (M‐W), whole‐body evaporative (EHL) and dry heat loss (DHL) were measured by whole‐body direct calorimetry in 5 males throughout 90‐min of cycling at 3 absolute oxygen uptakes of 1.82±0.05 (hi), 1.26±0.05 (med) and 0.65±0.03 (low) L‐min’ at 30°C and 30% RH. Due to the variation in VO2max between participants (3.24 to 4.44 L·min−1) these workloads corresponded to a range of relative workloads within each condition. Rectal temperature (Tre) was also measured throughout. Sustained levels of M‐W obtained throughout exercise were 500±17 (hi), 348±10 (med) and 203±10 W (low). After 90‐min, Tre was steady at 0.96±0.13, 0.70±0.10 and 0.23±0.03°C above rest for hi, med and low respectively. EHL was 423±18, 290±10 and 150±16 W respectively and DHL was 41±10, 45±15 and 58±14 W. Within condition, EHL varied little between participants despite relative workloads differing by 15–20%. End‐exercise EHL appears to be determined by absolute metabolic workload rather than relative workload. Variations in core temperature set‐point within condition are attributed to individual differences in sweating sensitivity during the early stages of exercise; and effects of body mass and composition upon the ‘passive’ system.

Supported by NSERC Grant.