bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple cell‐death mechanisms are triggered in hepatotoxicity induced by acrolein, an environmental pollutant and lipid peroxidation product
Authors: Mohammad Mohammad , Avila Diana , Zhang Jingwen , Jokinen Jenny , Arteel Gavin , McClain Craig , Barve Shirish , Joshi-Barve Swati
Published: June 2021

Acrolein is a common environmental, food and water pollutant. It is produced via lipid peroxidation and metabolism of certain amino acids and drugs. Acrolein is a product and an initiator of lipid peroxidation and oxidative stress. Acrolein is cytotoxic to hepatocytes. Primary hepatocytes and human hepatoma cells were used to study acrolein‐induced hepatotoxicity, ER stress, mitochondrial death pathways and the induction of apoptosis. Acrolein was cytotoxic and caused a dose‐dependent loss of survival of hepatic cells. Acrolein exposure caused a rapid and dramatic decrease in the intracellular antioxidant glutathione and overall antioxidant capacity; activated the signaling kinases JNK and p38 MAPK; induced ER stress and activated ER stress proteins PERK, eIF2α, Gadd153/CHOP and ATF‐3, ‐4, and ‐6; and disrupted mitochondrial integrity/function by fluorescence microscopy, High‐Content Cellomics analysis, and depletion of cellular ATP. We postulate that acrolein triggers multiple cell death mechanisms which together contribute to its hepatotoxicity. Our study defines basic mechanisms contributing to liver injury caused by oxidative stress and reactive aldehydes such as acrolein.
Potassium channel openers elicit endothelium‐dependent enhancement of nerve‐mediated vasoconstriction
Authors: Kerr Paul M , Naomi Fariha , Chen Ting-Yeuh , Mittal Rohan D , Plane Frances , Narang Deepak
Published: June 2021

The vascular endothelium modulates arterial diameter via the release of diffusible relaxing and contracting factors and by direct electrical coupling with smooth muscle cells (endothelium‐dependent hyperpolarization; EDH). Hyperpolarization of the endothelial cell membrane potential facilitates Ca2+ influx through non‐selective cation channels and spreads electrotonically to the smooth muscle cells via gap junctions. We hypothesized that pharmacological activators of Ca2+‐activated potassium (KCa) would cause endothelium‐dependent attenuation of nerve‐mediated vasoconstriction in the rat perfused mesenteric bed. The presence of CYPPA, an activator of small conductance KCa channels, nerve‐mediated vasoconstriction was significantly attenuated in the endothelium‐intact perfused mesenteric bed. However, on washout, a rebound effect was observed with nerve‐mediated constriction significantly enhanced. Addition of indomethacin enhanced CYPPA‐induced depression of nerve‐evoked constriction but prevented the rebound increase in vasoconstriction. In endothelium‐denuded preparations, CYPPA caused a smaller depression of vasoconstriction also with no rebound. These data indicate that activators of KCa channels may activate both endothelium‐dependent dilator and contractile pathways. Supported by HSFC.
Role of TRPC1 and TRPC3 channels in constriction and relaxation of mouse thoracic aorta
Authors: Kochukov Mikhail Y , Balasubramanian Adithya , Abramowitz Joel , Birnbaumer Lutz , Marrelli Sean P
Published: June 2021

Canonical transient receptor potential (TRPC) channels expressed in smooth muscle (SMC) and endothelial cells (EC) are candidates for regulating intracellular Ca2+ and membrane potential in the vasculature. We examined the role of TRPC1 and TRPC3 channels in the mechanisms of vasocontraction and relaxation in mouse aorta.

Experiments were performed with wild type (WT), TRPC1, and TRPC3 knockout (KO) mice. Contraction of aorta to phenylephrine (Phe) and relaxation to carbachol (CCh) were measured using an isometric tension bath. Intracellular Ca2+ was measured in cultured aorta EC by Fura 2. Whole cell K+ current was measured in freshly isolated SMC.

Aorta from TRPC1 but not TRPC3 KO mice showed augmented contractility to Phe compared to WT. Contractions to Phe were significantly potentiated in WT (but not TRPC1) aorta in the presence of a BKCa blocker Penitrem A. In SMC, Phe‐stimulated BKCa current was reduced in TRPC1 KO compared with WT. Relaxations to CCh were significantly reduced in aorta from TRPC1 and TRPC3 KO mice. EC from TRPC1 and TRPC3 KO mice demonstrated a reduced CCh‐mediated Ca2+ response. Additionally, Ca2+ response of WT EC was attenuated by a TRPC3 blocker Pyr3.

Data suggest that TRPC1 facilitates BKCa channel activation in SMC to promote attenuation of agonist‐mediated constriction. Both TRPC1 and TRPC3 contribute to endothelium‐dependent relaxation of aorta and agonist‐mediated Ca2+ influx in EC.

Supported by NIH R01 HL088435 and the Intramural Research Program of the NIH, project Z01‐ES‐101684.
Key role of Pannexin‐based hemichannels in flow‐induced nitric oxide production in resistance arteries
Authors: Perez Francisco R , Gaete Pablo , Boric Mauricio , Figueroa Xavier
Published: June 2021

Nitric oxide (NO) production and connexin‐based channels play a key role in the control and coordination of vascular function. Blood flow induces NO production by the activation of endothelial NO synthase phosphorylation at serine 1177 (P‐eNOSS1177) through the PI3K/Akt pathway. The participation of connexin‐ or pannexin‐formed hemichannels in the response to flow has not been evaluated. We used the rat arterial mesenteric bed to study the involvement of hemichannels in flow‐induced NO production. Mesenteric vessels were stimulated with flow by increasing the perfusion rate from 2 to 10 mL/min and hemichannel activity was assessed by ethidium or Lucifer Yellow uptake. Flow increased NO production, Akt‐dependent P‐eNOSS1177, ATP release and endothelial hemichannel activity. Treatment with 18‐beta‐glycyrrhetinic acid or probenecid, but not La3+, blocked the hemichannel activation and Akt dependent‐P‐eNOSS1177 induced by flow. The increase in P‐eNOSS1177 was prevented by the specific inhibition of P2Y1 receptors with MRS2179 or global inhibition of purinergic receptors with PPADS, but not by the P2X receptor blocker iso‐PPADS. These results suggest that pannexin hemichannel‐mediated ATP release is involved in the activation of NO production in response to flow, which may be involved in the development of cardiovascular diseases.

FONDECYT 1100850, 1090757, 1111033, Anillos ACT‐71.
Re‐evaluating the interstitial fluid pressure‐volume (compliance) relationship
Authors: Dongaonkar Ranjeet M. , Quick Christopher M. , Stewart Randolph H. , Laine Glen A.
Published: June 2021

Recently we formulated an analytical framework to characterize interstitial fluid balance by integrating microvascular, interstitial, and lymphatic function. Eight standard equations characterizing fluid and protein fluxes were solved simultaneously to yield analytical solutions for interstitial fluid pressure (Pi) and protein concentration. The resulting simple and transparent balance point approach reveals that Pi results from a ratio of the microvascular and the lymphatic parameters, and is independent of interstitial compliance (C). Furthermore, steady‐state interstitial fluid volume (V) is a function of Pi and is determined by C. However, previous attempts to characterize C have been constrained by the practice of isolating tissue samples to measure V. This practice made it impractical to characterize the entire Pi ‐V relationship. To overcome these limitations we developed a Pallid bat wing model allowing continuous simultaneous measurements of Pi and resulting changes in V. Manipulation of microvascular pressure to induce changes in Pi allows characterization of the Pi ‐V curve under negative as well as positive Pi ranges in a single experiment. This approach not only allows characterization of the entire Pi ‐V relationship in a single animal but also makes characterization of biomechanical properties such as stress relaxation of the interstitium possible. (Supported by the AHA and NIH)
Pericytes: not only an anatomical “internet” coordinating coronary vessels, but also key functional players?
Authors: Nees Stephan , Weiss Dominik R , Juchem Gerd
Published: June 2021

Aims

determination of the distribution of pericytes (P) in the human coronary system and their functional characterization. We found that interconnected P not only engird the endothelial cell tube (E) of all coronary microvessels, but also form a continuous intimal tissue net in the subendothelium of all coronary arteries and veins. Regardless of vascular origin P are typically connected to the E via abundant gap junctions. Smooth muscle cells extend down the arterial tree but stop abruptly at the junction between muscular arterioles (diam. >20 μm) and endarterioles (diam. 10–20 μm). The latter, the most important vessel segments for blood flow regulation, thus consist only of an inner E and outer tube of P. Pure cultures of P can be established in presence of human placental serum and passaged whilst retaining their typical histological and functional features. P reveal a high procoagulatory potency (activation of X, formation of the prothrombin complex). In co‐culture with endothelial cells of human coronary venular origin spontaneous formation of microvessels can be observed that resembles closely natural angiogenesis. The tip cells in capillary sprouts, though to direct this process, appear to be P and not endothelial cells. Thus, the interconnected P in the heart probably play a central role in the control of myocardial blood flow, hemostasis and angiogenesis. Supported by the Friedrich‐Baur‐Stiftung Munich.
Human erythrocyte shape creates a capillary oxygen delivery advantage
Authors: Blinman Thane
Published: June 2021

Textbooks still describe the “purpose” of the biconcave disk of the human erythrocyte (RBC) is “to maximize surface area for gas exchange.” This appears incorrect: 1) RBC surface area does not govern diffusion radius or limit the oxygen diffusion coefficient, and 2) quasi fractal mechanisms are almost universally employed for surface area advantages. Here, a mathematical model is used to explore oxygen delivery (DO2) advantage of the RBC shape, especially at high oxygen consumption (VO2) states (e.g. fight or flight, sepsis, etc). An axisymmetric model of a capillary and its service volume of target cells is simulated containing stacked RBCs, “parachute” RBCs, spherocytes, or bare Hb. Flow velocity, Hb saturation, and local VO2 are varied according to a Monte Carlo algrorithm. A no‐slip condition is approximated for laminar flow within the capillary. The model shows that the flow boundary layer of the capillary would tend to distribute bare hemoglobin to the axial center of the capillary. In contrast, RBC shape (normal and parachute, but not spherocyte) concentrates the Hb within a cylindrical manifold close to the capillary wall. Since target cell oxygen delivery depends on the square of the diffusion radius, even small reductions in radius convey a DO2 advantage in physiological stress. This model reveals constraints on design of artificial blood substitutes.
Sphingolipids signal rapid loss of P‐glycoprotein transport activity at the blood‐brain barrier
Authors: Miller David S. , Campos Christopher R. , Hawkins Brian T. , Cannon Ronald
Published: June 2021

Blood‐brain barrier P‐glycoprotein is a major obstacle to CNS pharmacotherapy. We previously showed that TNF‐α signals through TNFR1, ET‐BR, iNOS and PKCβ1 to rapidly reduce P‐glycoprotein transport activity in rat brain capillaries (Hartz et al., Mol Pharmacol 69, 462, 2006; Rigor et al., J Cereb Blood Flow Metab 30, 1373, 2010). Here we investigate sphingolipid signaling downstream of PKCβ1. Exposing capillaries to sphingosine or sphingosine‐1‐phosphate (S1P) rapidly (minutes) and reversibly reduced P‐glycoprotein transport activity by a S1PR1‐dependent mechanism. Blocking sphingosine kinase activity or S1PR1 attenuated the ability of TNF‐α exposure or PKCβ1 activation to reduce P‐glycoprotein activity. Using in situ brain perfusion in rats, we found that infusing S1P into the cerebral vasculature increased brain accumulation of 14C‐verapamil, indicating loss of P‐glycoprotein activity; this effect was blocked by S1PR1 antagonists. S1P did not alter 3H‐sucrose accumulation, indicating no change in paracelllular permeability. We are currently testing S1PR1 agonists for possible use in improving delivery to the brain of CNS‐acting drugs that are P‐glycoprotein substrates.
Protective role of Ac‐SDKP and thymosin β4 on cardiac rupture after myocardial infarction
Authors: Peng Hongmei , Carretero Oscar A. , Yang Xiao-Ping , Xu Jiang , Peterson Edward , Rhaleb Nour-Eddine
Published: June 2021

N‐acetyl‐seryl‐aspartyl‐lysyl‐proline (Ac‐SDKP), released from its precursor thymosin β4 (Tβ4), has anti‐inflammatory, pro‐angiogenic and anti‐fibrotic effects. We hypothesized that Tβ4 and Ac‐SDKP reduce cardiac rupture after myocardial infarction (MI). C57BL/6J mice were subjected to MI and divided into groups: 1) sham MI, 2) MI + vehicle (VEH), 3) MI + Ac‐SDKP or 4) MI + Tβ4. Ac‐SDKP and Tβ4 (1.6 mg/kg/day) were given i.p. via osmotic pump for 7 days before MI and 7 more days after MI. Ejection fraction (EF) was assessed by echocardiography. Infarct size was similar in all MI groups. Interstitial collagen fraction (ICF) and capillary density were measured and ICAM‐1 was detected by Western blot. Ac‐SDKP and Tβ4 decreased cardiac rupture rate, both tended to increase EF but the difference did not reach statistical significance (Table). Decline in cardiac rupture by Ac‐SDKP or Tβ4 may be due to angiogenesis, and decreased inflammation and metalloproteinase activity. The use of Ac‐SDKP and Tβ4 may reduce cardiac rupture after MI.

The NIH (R01HL071806).
Incorporating Human Ventricular Fiber Architecture in Patient‐Specific Computational Models
Authors: Villongco Christopher T , Frank Lawrence R , Krummen David E , Nigam Vishal , Kerckhoffs Roy CP , Omens Jeffrey H , McCulloch Andrew D
Published: June 2021

Patient‐specific models of the cardiac ventricles require a realistic representation of the myofiber architecture to accurately simulate mechanical and electrical properties of healthy and failing hearts. Diffusion tensor magnetic resonance imaging (DT‐MRI) techniques have made it possible to reconstruct the fiber anatomy of the ventricles. However, in vivo DT‐MRI scans are still impractical to obtain for individual patients. The aim of this study develops a method to register DT‐MRI measurements from an explanted human heart into an anatomical model and warping the result to patient‐specific anatomies using large deformation diffeomorphic mapping.

An explanted human organ donor heart was subjected to a DT‐MRI scan. The raw tensor data was registered into an anatomical model of the ventricles by fitting the measured diffusion tensor components in log‐Euclidean space. A material coordinate transformation model defined a local orthogonal coordinate system representing the fiber, sheet, and sheet‐normal directions of the myocardium from the eigenvectors of a locally interpolated diffusion tensor. Diffeomorphic maps of the deformation F between the donor and patient models were computed and used to warp the donor fiber architecture into the patient models. The registration error between the eigenvectors of the measured tensor and its interpolated tensor was quantified by a scalar metric of axes overlap. It found 74% of the registered DT‐MRI data (112,419 of 152,651 voxels) had mean angular errors of 12.5°±7° (SD) in the fiber, 20°±11.5° (SD) in the sheet, and 18°±11.6° (SD) in the sheet normal axes.
Identification of critical residues within the extracellular loop of the Epithelial Sodium Channel (ENaC)
Authors: Swann Samantha R , Whisenant Ty , Booth Rachell E
Published: June 2021

The epithelial Na+ channel (ENaC) plays a pivotal role in fluid homeostasis through its function in sodium reabsorption. Malfunction of this channel can lead to salt sensitive hypertension or hypotension. Although passage specific and constitutively active for the transport of Na+ ions, ENaC has exhibited self‐inhibition in the presence of excess Na+ as well as altered regulation from other ions such as Cl−. ENaC's structure is likely heterotrimeric and composed of α, β, and γ‐subunits, all contributing to proper function. In an effort to elucidate structural features of mouse αENaC, critical residues, E361 and K487, were identified using a functional yeast screen. Also, the effects of specific mutations within the extracellular region (K189 to A491) of mENaC were studied under various pH levels and in the presence of excess Cl−, K+ or Na+. Variations in the growth of yeast cells expressing wild‐type and mutant ENaC were correlated to changes in extracellular ion concentration. Residues involved in potential interactions with ions that contribute to ENaC regulation in yeast will be further characterized in mammalian cells. (Supported by NIH‐NIGMS R15GM086798)
Purinergic Signaling Reciprocally Contributes to the TRPV4‐Mediated Mechano‐Sensitive Response in the Aldosterone‐Sensitive Distal Nephron
Authors: Mamenko Mykola , Zaika Oleg L , OˈNeil Roger G , Pochynyuk Oleh
Published: June 2021

Aldosterone‐sensitive distal nephron (ASDN), which includes the connecting tubule (CNT) and cortical collecting duct (CCD), is essential for regulation of electrolyte balance and circulating volume. Mechanical stimuli affect epithelial transport in ASDN. The ASDN cells respond to mechanical stress by increasing [Ca2+]i. Mechanical stimulation is also known to enhance ATP release from the epithelial cells. P2Y2 receptors play the pivotal role in purinergic signaling in ASDN. Here we combined ratiometric Ca2+ imaging in freshly isolated split‐opened ASDNs with targeted deletion of P2Y2 receptors and TRPV4 channels to test if purinergic signaling contributes to mechano‐sensitive response in the distal nephron. We found that ATP, via stimulation of P2Y2 receptors, increases [Ca2+]i in a biphasic manner: transient Ca2+ peak is followed by a sustained plateau. ATP‐induced [Ca2+]i responses are identical in principal and intercalated cells in CNT and CCD. Both intracellular and extracellular Ca2+ sources account for [Ca2+]i elevation in the ASDN cells. Mechano‐sensitive responses are significantly attenuated in P2Y2 −/− mice. Furthermore, TRPV4 channels are essential for sustained elevation of [Ca2+]i in response to purinergic stimuli. We concluded that reciprocal activation of TRPV4 channels by purinergic cascade is essential for proper mechano‐sensitive response in ASDN.
TRPV4 is a mechanotransducer of fluid flow in principal cells (PC) and intercalated cells (IC) of the renal collecting duct system
Authors: Berrout Jonathan , Jin Min , Mamenko Mykola , Zaika Oleg L , Pochynyuk Oleh , O'Neil Roger G
Published: June 2021

The sites of expression and function of TRPV4, a Ca2+‐permeable channel, were evaluated in the mouse kidney. Immunhistochemical localization studies in kidney slices and isolated split‐opened connecting tubule (CNT) and cortical collecting duct (CCD) demonstrated strong expression of TRPV4 along the entire collecting duct system with no detectable expression in upstream segments. Expression was highest in PC, the aquaporin‐2 (AQP‐2) positive cells, with a high level of colocalization with AQP‐2 near the apical/subapical region, and low in IC (AQP‐2 negative). Separate Ca2+ imaging studies (fura 2 imaging) of split‐opened CNT/CDD on coverslips, demonstrate that GSK1016790A (50 nM), a selective TRPV4 agonist, activates TRPV4 channels in both PC and IC, although less robustly in IC. Further, a step increase in fluid flow over the luminal surface rapidly stimulated a sustained Ca2+ influx in PC and IC of both CNT and CCD. This response was abolished following treatment with ruthenium red, a TRPV channel blocker, or in CNT/CCD isolated from TRPV4 knockout animals. Inhibition of the phospholipase C (PLC) signaling cascade with the PLC inhibitor, U73122 (10 uM), largely abolished the flow‐dependent response, implicating TRPV4 as a downstream transducer of flow. It is concluded that TRPV4 is widely expressed in the collecting duct system and that it functions as a mechanotransducer of fluid flow in both PC and IC.
Bone loss and renal Ca2+ wasting in experimental colitis is accompanied by downregulation of TRPV5 in renal distal convoluted tubules
Authors: Kiela Pawel R , Radhakrishnan Vijayababu M , Thurston Robert D , Larmonier Claire B , Ramalingam Rajalakshmy , Laubitz Daniel , Ghishan Fayez K
Published: June 2021

Dysregulated Ca2+homeostasis likely contributes to the etiology of IBD‐associated bone loss. Experimental colitis leads to decreased expression of Klotho, a protein which supports renal Ca2+ reabsorption by stabilizing TRPV5 channel on the apical membrane of distal tubule epithelial cells. We investigated whether colitis leads to changes in renal Ca2+ handling and expression of TRPV5 utilizing TNBS, and two adoptive T‐cell transfer models (CD4+CD45RBHi or CD4+IL10−/−). Colitic mice had normal serum levels of Ca2+ and PTH. μCT analysis demonstrated changes in bone structure along with changes in serum sRANKL, IL‐6, osteocalcin, TNF, and total serum tDPD crosslinks. Increased fractional Ca2+excretion was accompanied by reduction of TRPV5 protein expression, with concomitant increase in its sialylation status. In mIMCD3 cells transduced with Ad‐TRPV5, proinflammatory cytokines reduced TRPV5 surface expression followed by its degradation. TNF induced interaction of TRPV5 with UBR4 ubiquitin ligase and siRNA‐mediated UBR4 knockdown prevented TNF induced TRPV5 degradation. The cytokine effect on TRPV5 was not observed in cells stably transfected with membrane‐bound Klotho. We propose that TNF and IFNγ act to decrease Klotho expression and activity, increased sialylation of TRPV5, impaired interaction of TRPV5 and galectin‐1, and ultimately to UBR4‐dependent endocytosis and degradation of TRPV5.
Loss of Wnk3 is Compensated for by ks‐Wnk1 in the Kidney of the Mouse
Authors: Mederle Katharina , Oppermann Mona , Paliege Alexander , Castrop Hayo
Published: June 2021

Wnk3 kinase is expressed throughout the nephron and acts as a positive regulator of NKCC2 and NCC in vitro. Here we addressed the in vivo relevance of Wnk3 by the use of Wnk3‐deficient mice. Wnk3−/− mice were viable and showed no gross abnormalities. Net tubular function was similar in WT and Wnk3−/− mice as assessed by determination of 24‐hr urine output (1.7±.2 in WT and 1.6±.2 mL in Wnk3−/−, p=.52) and ambient urine osmolarity (1680±170 vs. 1780±170 mosmol/kg; p=.11, n=10). Water restriction (48 hrs) increased urine osmolarity to 3440±220 and 3200±180 mosmol/kg in WT and Wnk3−/−, respectively (n=10, p=.41). During a salt‐restricted diet (7d, 0.02% NaCl) urine osmolarity tended to decrease in WT (1584±200 mosmol/kg; p=.34 vs. standard diet) whereas it was reduced significantly in Wnk3−/− mice (1183±130 mosmol/kg; p=.025 vs. standard diet). Glomerular filtration rate (343±22 vs. 315±13 mL/min), renal blood flow (1.35±.1 vs. 1.42±.04 mL), and plasma renin concentration (160±38 vs. 135±19 ng AngI/mL/hr) were similar in WT and Wnk3−/− (p>.05). The abundance of the kidney‐specific Wnk1 isoform (ks‐Wnk1) was markedly enhanced in Wnk3−/− compared to WT mice, whereas expression of full‐length Wnk1 and Wnk4 was unchanged. In summary, the function of Wnk3 can largely be compensated for by other kinases, most likely by ks‐Wnk1, and this may cause compensatory activation of downstream targets like eNaC.
WNK4 phosphorylates mouse thiazide‐sensitive Na‐Cl cotransporter NCC at threonine 48
Authors: Na Tao , Wu Guojin , Peng Ji-Bin
Published: June 2021

With‐No‐lysine (K) 4 (WNK4) is a serine/threonine kinase and mutations in WNK4 are associated with a genetic form of hypertension. WNK4 was shown to regulate the thiazide sensitive sodium chloride cotransporter NCC via SPAK and OSR1 kinases, which phosphorylate NCC and increase its transport activity. It is unclear whether WNK4 phosphorylates NCC directly. In this study, we purified human WNK4 kinase domain (amino‐acids 160–440) produced in E. coli. The WNK4 kinase domain was capable of phosphorylating GST‐fusion OSR1 D164A mutant. We then tested the ability of the WNK4 kinase domain in phosphorylating GST‐fusion mouse NCC cytoplasmic terminal regions using in vitro kinase assay. The NCC N‐terminal region (amino‐acids 1–135) was significantly phosphorylated by the WNK4 kinase domain whereas no strong phosphorylation was detected in the C‐terminal region (amino‐acids 604–1002). By truncation and site directed mutagenesis in the NCC N‐terminal region, threonine 48 was identified to be the main site of WNK4 phosphorylation in mouse NCC. Phospho‐mimicking mutation at this site (T48D) increased NCC‐mediated sodium uptake and NCC protein abundance in Xenopus oocytes by 84.2% and 106.5%, respectively. Furthermore, the phosphorylation of NCC 1–135 by OSR1 T185E was enhanced by the phospho‐mimicking mutation. In conclusion, direct phosphorylation of NCC by WNK4 may be involved in WNK4‐mediated regulation of NCC.
Characterization of a Druggable Binding site in the Renal Outer Medullary Potassium Channel
Authors: Nguyen Thuy Tuong , Sheehan Jonathan H. , Meiler Jens , Denton Jerod S.
Published: June 2021

The Renal Outer Medullary potassium (K+) channel, ROMK, has been postulated as a target for a novel class of loop diuretic. We recently used high‐throughput screening and medicinal chemistry to develop a highly selective small‐molecule inhibitor, termed VU591, with which to explore ROMK's physiology and therapeutic potential. Electrophysiological experiments suggested that VU591 blocks the intracellular pore of the channel. In an effort to characterize the VU591 binding site, a comparative homology model of ROMK based on the X‐ray structure of Kir2.2 was used to identify the pore‐lining residues to target for alanine‐scanning mutagenesis. Two‐electrode voltage clamp analysis of mutagenized channels expressed in Xenopus oocytes identified several pore‐lining residues that appear to be important for VU591‐dependent inhibition of ROMK. We are currently refining the mutagenesis and employing ligand‐docking simulations to help define molecular structure of the VU591 binding site.

NIH R01 award to Jerod S. Denton DK082884.
Remission of nephrotic syndrome resolves plasminogen/plasminuria and reverses the ability of urine to activate ENaC
Authors: Friis Ulla Glenert , Andersen Rene F , Rittig Søren , Buhl Kristian B , Jensen Boye L
Published: June 2021

Recently it was reported that urinary plasmin activates the epithelial sodium channel (ENaC) and may mediate sodium retention. The present study investigates if remission of nephrotic syndrome (NS) affects glomerular filtration of plasminogen, its activation to plasmin, and the ability of the patients urine to activate ENaC.

A paired design study was conducted collecting samples during active nephrotic syndrome and at stable remission. Single cell patch clamp experiments were performed on single collecting duct cells (CDC) with urine samples from six patients investigating the potential to generate an ENaC mediated current.

Urine samples from active NS significantly increased the inward current in CDC (201 ± 31%, n=6), whereas at remission, the observed current was significantly lower (29 ± 10%, n=6). The ability of nephrotic urine to enhance inward current was abolished by amiloride (2 μM) (19 ± 10%, n=6).

Our data demonstrate significantly increased inward (ENaC) current induced by urine collected during active NS compared to remission, suggesting ENaC inhibition by amiloride to be a new therapeutic target for oedema control.
The good, the bad and the ugly? The role of exercise on obesity‐induced chemerin expression
Authors: Lloyd Jesse William , Owusu Priscilla , Sieburg Michelle , Heckstall Ebony Monique , Kim Yuho , Zerfass Kristy , Keslacy Stefan
Published: June 2021

Exercise has been shown to alleviate insulin resistance in obesity‐induced diabetes. Although chemerin, a recently discovered adipokine, has been involved in chemotaxis and adipocyte cell‐differentiation in vitro, its role in vivo is still unclear.

Purpose

To determine the effect of exercise on obesity‐induced chemerin expression.

Methods

Forty‐four mice (DIO C57Bl/6J) were separated into 2 cohorts: high fat diet (HFD) n=22 and normal diet (ND) n=22. Following a 12 week‐feeding period, glucose tolerance test was performed with blood samples taken at 0, 30, 60, 90, and 120 min. Chemerin plasma concentration and protein expression were assessed using ELISA and western blot respectively.

Results

HFD induced diabetes. Diabetes resulted in enhanced circulating chemerin levels (ND: 134.63ng/ml vs. HFD: 193.38ng/ml p=0.0009). At 30 min following glucose gavage, circulating chemerin level was significantly higher in HFD compared to ND group (179.6 vs. 112.8 ng/ml respectively P=0.0036). Our preliminary data suggested that exercise upregulated chemerin expression in adipose tissue.

Conclusion

Our data suggest that exercise regulates diabetes‐increased chemerin expression. A better understanding of chemerin regulation by exercise need to be further studied as it may represent a novel therapeutic target.

Study supported by Syracuse University and the School of Education creative writing grant.
Intermittent hypoxia conditioning improves basal and stimulated nitric oxide production in spontaneously hypertensive rats
Authors: Manukhina Eugenia B. , Jasti Dinesh , Downey H. Fred
Published: June 2021

Earlier we reported that intermittent hypoxia conditioning (IHC) slows development of hypertension in young spontaneously hypertensive rats (SHR) and stimulates nitric oxide (NO) synthesis. Now we have compared basal and stimulated NO production in IHC and sham conditioned, 4–5 week old SHR. IHC protocol: 20 days, 5 to 8 cycles of 9.5–10% O2, each 5 to 10 min, separated by 4 min normoxia. Sham protocol: same regimen but no change in inspired O2. The 20 day rise of systolic BP of IHC rats was less than in sham rats (from 131±2 mmHg to 162±2 mmHg vs 202±3 mmHg in sham rats, P<0.001). Basal NO production was evaluated by the contractile response to norepinephrine (NE, 10− 8M) of isolated aortic rings in the presence and absence of L‐NAME (10−3 M), an NO synthase inhibitor. After L‐NAME, NE‐induced contraction increased more (P<0.05) in IHC rats (66±16%) than in sham rats (35±3%). Endothelium‐dependent relaxation (EDR) was evaluated by the response to acetylcholine of NE‐precontracted rings. In 8‐week old IHC rats, EDR was not impaired and did not differ from that of 4–5 week old SHR (60±6%). EDR was impaired in 8‐week old sham‐IHC rats compared to 4–5 week old rats (28±6 vs 55±5%, P<0.002). Therefore, the antihypertensive effect of IHC is associated with improvement of both basal and stimulated NO production. These effects may indicate an important role of NO in the antihypertensive action of IHC. (Support: UNTHSC Cardiovasc. Res. Inst.)
Cyclooxygenase‐2‐dependent neuroinflammation and oxidative stress in rostral ventrolateral medulla contribute to neurogenic hypertension following chronic systemic inflammation
Authors: Wu Kay Li-Hui , Chan Julie Yu-Hwa , Chan Samuel H.H.
Published: June 2021

Inflammation plays a significant role in pathogenesis of hypertension. This study aimed to investigate whether chronic systemic inflammation (CSI) induces hypertension via triggering neuroinflammation and oxidative stress in the rostral ventrolateral medulla (RVLM) where the sympathetic premotor neurons are located. CSI was induced via a continuous intraperitoneal infusion of E coli. Lipopolysaccharide (LPS) to normotensive Sprague‐Dawley rats. CSI was confirmed by a sustained increase in plasma level of C‐reactive protein. CSI resulted in the activation of perivascular microglia, increases in proinflammatory cytokine and intercellular adhesion molecule‐1 expression, and decrease of endothelial nitric oxide synthase expression in RVLM. CSI also promoted a long‐term pressor response accompanied by an increase in tissue level of superoxide and a reduction in expression of a voltage‐gated K+ channel, Kv4.3, in the RVLM. CSI‐induced hypertension and cellular events were significantly prevented by intracisternal (IC) infusion of cytokine or cyclooxygenase‐2 (COX‐2) inhibitors. CSI‐induced hypertension was also attenuated by IC infusion of tempol. These data suggest that CSI activates the perivascular microglia to evoke the COX‐2‐dependent neuroinflammation and oxidative stress in the RVLM, leading to oxidative stress‐associated neurogenic hypertension.
Effect of Differentially Selective S1P Receptor Agonists on Cardiovascular Function after Acute and Long‐Term Administration in Rat Suggestive of Subtype‐Specific Bradycardia (S1P1) and Hypertension (S1P3)
Authors: Fryer Ryan M , Muthukumarana Akalushi , Harrison Paul C , Mazurek Suzanne G Nodop , Reinhart Glenn A
Published: June 2021

Sphingosine‐1‐Phospate (S1P) and S1P receptor agonists elicit mechanism‐based effects on cardiovascular function in vivo. Indeed, FTY‐720 (subtype non‐selective S1PX agonist) produces mild hypertension in patients (2–3 mmHg in a 1‐yr trial) as well as acute bradycardia independent of changes in blood pressure. However, the precise receptor subtypes responsible is controversial, possibly dependent upon the CV effect in question (e.g. bradycardia/hypertension), and perhaps even species‐dependent since functional differences in rodent, rabbit, and human have been suggested. Thus, the cardiovascular effects of FTY‐720 after acute i.v. infusion in anesthetized rats, and after oral administration for up to 15‐d in telemetry‐instrumented conscious rats, was tested. The more selective S1P1,5 agonist, BAF‐312, was tested in the same models. We demonstrate that acute i.v. infusion (0.1, 0.3, 1.0 mg/kg/20min) of both FTY‐720 and BAF‐312 elicits bradycardia in rat, results suggestive of an S1P1 mediated mechanism. However, while FTY‐720 (0.5, 1.5, 5.0 mg/kg/d) elicited dose‐dependent hypertension after multiple days of oral administration in rat (mean MAP=102, 107, 109 mmHg vs. 97 mmHg in vehicle controls), BAF‐312 (0.3, 3.0, 30.0 mg/kg/d) had no effect on blood pressure suggesting that hypertension produced by FTY‐720 is mediated by a non‐S1P1 subtype, likely S1P3. Moreover, hypertension in the presence of FTY‐720 may imply that FTY‐720 affects renal sodium handling in rats. In summary, results in anesthetized and conscious rats administered FTY‐720 or BAF‐312 suggest that S1P1 receptors mediate bradycardia while hypertension is mediated by S1P3 receptor activation.
Morphological characterization of intrinsic cardiac nervous system in left ventricular hypertrophy
Authors: Gomez Maria , Reynoso Alejandro , Giles Ruth , Flores Gonzalo
Published: June 2021

The intrinsic cardiac nervous system (ICNS) is organized in ganglion connected by nerve fibers. Little information about their changes in heart hypertrophy is available. In this study we characterized the morphological changes of the ICNS in hearts with experimental hypertrophy with the Golgi‐Cox and Glyoxylic acid methods. Hearts from Spontaneous Hypertension (SH) and Wystar Kyoto (WK) rats at 4 and 8 months age were processed. Hearts were extracted, washed, and incubated for 30 days in Golgi Cox solution, sectioned by vibrotome (200 microns), processed and analyzed by the Sholl method under light microscopy adapted to a camera lucida. Diameters of ganglion and nerve fiber from endocardium, myocardium and epicardium from left ventricles were recorded in microns. Histofluorescence for catecholamine at 2 % was made and analyzed qualitatively. Data are expressed as mean ± SEM and analyzed with t‐test, P < 0.05 was considered significant. Clearly increase in number and size of ganglion and nerve fibers was observed in the left ventricle from SH rats. Ganglion are increasing in size from epicardium to endocardium (51.8±5.4, to 114.1± 8.8 micron) in WK and in SH rats, the ganglion size were higher, particularly in endocardium (66.30±7.79, and 138± 14, respectively). Histofluorescence for catecholamine in left ventricle hypertrophy was increased. Ours results demonstrate increases in size and number of nerve structures in left ventricle hypertrophy, and suggest that the adaptative tissular changes generated in hypertrophy also involve the intrinsic cardiac nerve system.
(Pro)renin receptor deletion prevents the development of DOCA‐salt hypertension in neuron‐specific (pro)renin receptor knockout mice
Authors: Li Wencehng , Peng Hua , Ichihara Atsuhiro , Feng Yumei
Published: June 2021

We previously found the presence of (pro)renin receptor (PRR) in the brain. To elucidate the physiological importance of brain PRR, we developed a neuron‐specific PRR knockout mouse model (Nefh‐PRR). Using real time PCR and immunofluorescence, we observed PRR deletion throughout the brain. Blood pressure (BP) and heart rate (HR) were recorded continuously by telemetry in conscious Nefh‐PRR and control mice (n=5/group) before and during 21 days of deoxycorticosterone acetate‐salt (DOCA, 50mg, 0.9% NaCl drinking solution + tap water) treatment. At baseline, Nefh‐PRR mice exhibited normal BP (mmHg), sympathetic and parasympathetic tone. DOCA‐salt induced hypertension in control mice (137.0±10.5), while Nefh‐PRR mice remained normotensive (114.1±2.9; P<0.05) at the end of experiments. DOCA‐salt significantly increased cardiac (delta HR: −219±6 vs. −66±10 bpm) and vasomotor sympathetic tone (delta BP: −63.7 ± 10.2 vs. −28.7 ± 1.2), and decreased parasympathetic tone (delta HR: 25±7 vs. 147±6 bpm) in control mice. Nefh‐PRR mice exhibited lower sympathetic tone (delta HR: −155±6 bpm; P=0.002, delta BP: − 32.4±5; P=0.05) and higher parasympathetic tone (delta HR: 101±27 bpm; P=0.03) compared to control mice following DOCA‐salt treatment. These data indicate neuron‐specific PRR knockout prevents DOCA‐salt induced hypertension via improvement of autonomic function. (NIH 1P30HL101285, AHA 11SDG7360050)
Placental ischemic stimulated CD4+ T helper cells increase blood pressure and placental and renal oxidative stress during pregnancy
Authors: Wallace Kedra , Scott Jeremy , Novotny Sarah , Heath Judith , Moseley Janae , LaMarca Babbette
Published: June 2021

Adoptive transfer of CD4+ Tcells from placental ischemic (RUPP) rats causes hypertension, elevated inflammatory cytokines, endothelin‐1, and agonistic autoantibodies to the angiotensin II receptor, all of which contribute to oxidative stress during preeclampsia. We hypothesized that hypertension in response to RUPP CD4+ T cells is associated with oxidative stress. To answer this question, CD4+ Tcells from RUPP or normal pregnant rats (NP) were injected into NP recipients on gestational day (GD) 13. GD19 MAP, urine and tissues analyzed. MAP increased from 101±2mmHg in NP to 115±3mmHg in NP+RUPP CD4+ T cell rats (P<0.05) but did not change in NP+NP CD4+ T cell rats (92±3mmHg). Placental NADPH oxidase stimulated ROS increased from 420.7±51 relative light units (RLU) in NP to 726.2±43 RLU in NP+RUPP CD4+ T cell rats (P<0.01). Cortical NADPH oxidase stimulated ROS increased from 634.6±54 RLU in NP to 781.6±19 RLU in NP+RUPP CD4+ T cell rats vs. 611.9±35 in NP+NP CD4+ T cell rats (P<0.05). Urinary isoprostanes increased 64.49± 11.7 to 112±19.8pg/mg/ml in NP+RUPPCD4+ T cell(P<0.05), but was unchanged in NP+NPCD4+ T cell rats; 53.52±17.45pg/mg/ml. These data support the hypothesis that hypertension in response to RUPP induced CD4+ T cells is associated with oxidative stress, one mechanism whereby immune activation mediates hypertension during pregnancy.
Role of Binding and Nucleoside Diphosphate Kinase A (NDPK‐A) in the Regulation of CFTR by AMP‐Activated Protein Kinase (AMPK)
Authors: King J Darwin , Lee Jeffrey , Rieman Claudia E. , Mehta Anil , Muimo Richmond , Hallows Kenneth R.
Published: June 2021

A better understanding of CFTR Cl− channel regulation could suggest new therapies for cystic fibrosis. AMPK binds to the CFTR C‐terminal tail, phosphorylates, and inhibits PKA‐activated CFTR gating, processes that are not well understood. PKA‐induced CFTR gating was unchanged by AMPK addition in excised inside‐out patch‐clamp recordings, suggesting the importance of other factor(s) in intact cells. NDPK‐A interacts with both AMPK and CFTR in overlay blots of airway epithelia. Binding studies in Xenopus oocytes and transfected HEK‐293 cells revealed that a peptide fragment of CFTR that binds AMPK (CFTR‐1420‐57) disrupted this interaction. Transduction of this peptide into Calu‐3 cells enhanced forskolin‐induced whole‐cell CFTR conductance. Injection of CFTR‐1420‐57 into Xenopus oocytes blocked AMPK‐dependent inhibition of cAMP‐stimulated CFTR conductance, as measured by two‐electrode voltage clamp. AMPK activation inhibited CFTR conductance with co‐expression of wild‐type NDPK‐A in Xenopus oocytes, but co‐expression of a catalytically inactive H118F mutant blocked this inhibition. These results suggest that NDPK‐A exists in a cellular complex with AMPK and CFTR and is required for the AMPK‐dependent regulation of CFTR. We propose that NDPK‐A may make ATP readily available for AMPK‐mediated phosphorylation and regulation of CFTR under conditions of metabolic stress. (Supported by NIH and CFF)
Activation of AT2 receptors reduces renal AT1 receptor function and enhances ACE2 activity in obese Zucker rats
Authors: Ali Quaisar , Hussain Tahir
Published: June 2021

Earlier, we reported that AT2R activation inhibits renal Na+K+‐ ATPase (NKA), promotes natriuresis and in the long‐term regulates blood pressure (BP) in obese rats (OZR). In the present study, we investigated whether AT2R activation influences other components of the renin angiotensin system and thereby regulates renal function and BP in obesity. We treated OZR with AT2R agonist CGP (1 μg/kg/min, for 2‐wks). The CGP‐treatment caused decrease in BP, which was associated with reduction in Na‐balance, early increase in urinary nitrates and cGMP and no change in GFR compared to obese controls. Under anesthesia, pressor response to Ang II and candesartan‐induced natriuresis was reduced in CGP‐treated OZR compared to control OZR. In‐vitro study in the proximal tubules from OZR revealed that AT2R activation, via cGMP pathway reduced AT1R‐mediated NKA stimulation. While no changes was seen in renal expression of AT1R, AT2R or ACE, ACE2 expression/activity was 4‐fold increased and PRA was decreased in CGP‐treated as compared to control OZR. We conclude that activation of AT2R reduces AT1R function, PRA and increases ACE2/ACE, which collectively may have contributed to natriuresis and BP reduction. These findings highlight the importance of AT2R in reducing the anti‐natriuretic AT1R function and enhancing the protective ACE2 axis of RAS that could serve as a therapeutic target for treating obesity related hypertension. NIH RO1‐DK61578
Comparative studies of the desert rodent Dipodomys merriami and Munich‐Wistar rat urine concentrating mechanisms
Authors: Babaria Arati C , Dantzler William H , Pannabecker Thomas L
Published: June 2021

Comparative studies of the mammalian renal medulla suggest that variations in the architecture of the thin limb of Henle's loop contribute to variations in ability to produce concentrated urine. For this study, tubules and blood vessels of the renal inner medulla were identified by indirect immunofluorescence using antibodies and lectins that recognize segment‐specific proteins. Variations in axial expression of the water channel aquaporin 1 and the Cl channel ClC‐K1 in the descending thin limb, have previously suggested that equilibration of luminal fluid by water reabsorption occurs along a greater proportion of each loop length, and Cl reabsorption occurs along a shorter proportion of each prebend loop length in Dipodomys than in the Munich‐Wistar rat. Interstitial nodal spaces adjacent to CDs exist in both species and preferential solute diffusion into these spaces may play a significant role in driving fluid reabsorption from CDs. In the terminal papilla, the ATL‐to‐CD surface area ratio is markedly greater in the Munich‐Wistar rat, suggesting that NaCl reabsorption may have less of an impact on water reabsorption from terminal CDs in Dipodomys.
The effects of pyrene exposure on gill O2 respiration and osmoregulation in channel catfish
Authors: Rehman Azeem , Goertzen Ned J. , Gayam Sajjan , Stabenau Erich K.
Published: June 2021

The present study examined whether pyrene exposure alters gill function in channel catfish, a species with impermeable skin and gills that function as the primary gas exchange and osmoregulatory organ. Following a 7‐day, in‐water treatment of catfish to control or pyrene water, animal O2 consumption was measured. Blood samples were then collected and analyzed for the hematocrit, ion and protein concentrations, plasma osmolality, and hemoglobin concentration and red cell water content. Pyrene exposure produced significant decreases in water O2 uptake and plasma Na+ concentration, and significant increases in hematocrit and plasma osmolality in pyrene‐exposed fish. However, there were no significant changes to the red cell water content, and the hemoglobin, plasma protein, and K+ concentrations. These data suggest that in‐water pyrene exposure significantly damaged the gill tissue, thereby leading to reduced O2 uptake and altered osmoregulation. Catfish were kindly provided by the Jake Wolf Fish Hatchery and the Fountain Bluff Fish Farm in Illinois. Funding was provided by the Sherry Endowment and Bjorklund Research Funds, and the Bradley University Biology Department and LAS Dean's Office.
Cardiovascular plasticity during hypoxic development in snapping turtle and alligator embryos
Authors: Eme John , Tate Kevin B , Slay Christopher E , Kohl Zachary F , Hicks James W , Crossley Dane A
Published: June 2021

The cardiovascular (CV) system is the first operational organ system, and reptiles represent evolutionary intermediates between water‐breathing vertebrates and birds and mammals. Reptile eggs are subject to environmental stressors during development, including hypoxia, and we have shown that hypoxia impacts reptilian CV function and maturation. Using Snapping turtles and American alligators, we investigated in ovo species differences following chronic incubation in hypoxia (10% O2). Hypoxia alters tonic regulation of baseline CV variables, causing hypotension in both species and relative cardiac enlargement. Assessment of CV reflexes induced via acute hypoxia or pharmacological stimuli indicate reflexes can be plastic and blunted by chronic hypoxic incubation. Alligator embryos possess adrenergic tone originating from circulating catecholamines only, and cholinergic tone is absent until near hatching. Turtle embryos display adrenergic and cholinergic tone at 70% of development, and can utilize autonomic tone to control heart rate over the last 30% of incubation. Our data indicate that maturation of CV function exhibits phenotypic plasticity in response to hypoxic incubation in reptile species, and that turtle embryos display a more ‘advanced’ developmental program than alligator embryos. NSF CAREER IBN IOS‐0845741 to DAC