bluuebunny/crossref_metadata_2025 huggingface dataset:
Preclinical evaluation of the mitochondria‐targeted antioxidant mitoquinone to treat sepsis‐induced acute kidney injury
Authors: Patil Naeem K , MacMillan‐Crow Lee Ann , Mayeux Philip R
Published: June 2021

Sepsis is caused by a severe microbial infection and can lead to acute kidney injury (AKI). This results in a high mortality rate of 70%. No specific therapies exist for septic AKI. To test if mitochondrial injury contributes to septic AKI, we used a clinically relevant cecal ligation and puncture (CLP) murine model. At 4h and 18h after CLP, the activity of manganese superoxide dismutase was decreased 50%, suggesting decreased mitochondrial antioxidant defense. Intravital microscopy showed a 50% fall in perfusion of renal capillaries at 18h after CLP (P < 0.05 versus control mice). Also, renal mitochondrial superoxide levels were more than doubled at 18h. We hypothesize that protecting the renal mitochondria using mitochondrial specific mitoquinone (MitoQ) will attenuate sepsis induced AKI. MitoQ (10mg/kg, i.p) given at the time of sepsis restored renal perfusion, decreased mitochondrial superoxide and preserved renal morphology at 18h post CLP. MitoQ also improved survival rate. In addition, MitoQ administered in a clinically relevant delayed dosing paradigm at 6h post CLP, also protected the renal microcirculation and inhibited mitochondrial oxidant generation. These data show that mitochondrial superoxide, a previously unrecognized mediator of septic AKI, can be targeted by mitochondrial antioxidants to attenuate septic AKI, even with delayed treatment. Supported by NIH DK075991 and AHA 12PRE12040174.
Moving From the Lab to the Law
Authors: Plotkin Robert
Published: June 2021

A successful transition from the lab to the law builds on lessons learned and rigor developed as a scientist, but also requires the acquisition of new skills; a shift in work habits, tempo, and rhythm; and a shift in attitude from the pursuit of truth for its own sake to the development of pragmatic solutions that draw on the law to serve the best interests of one's own client, to resolve a dispute between two parties, or to generate legal rules to promote a policy goal. This talk will explain these requirements for a successful transition to a legal career in more detail and provide audience members with guidelines for determining whether a career in the law is right for them.
Effects of acute doxorubicin treatment on oxidative stress markers and expression of the vitamin D receptor in liver of fasted animals
Authors: Dirks‐Naylor Amie Jeanette , Bero Joseph D. , Mabolo Raean , Tran Ngan T.K. , Yang Sendra , Kouzi Samir A.
Published: June 2021

Doxorubicin (DOX) is a chemotherapeutic agent known to cause cardiac and hepatic toxicity. Mechanisms of toxicity include induction of oxidative stress and apoptosis. While mechanisms of cardiotoxicity have been extensively studied, much less is known about hepatic toxicity. Although the effect of DOX on markers of oxidative stress in the liver has been previously described, none of the studies controlled for the anorexic effects of DOX. Anorexia can deplete glutathione and cause oxidative stress in the liver. Thus, we aimed to delineate the effects of DOX‐induced oxidative stress from the effects of anorexia. Furthermore, the effect of DOX on vitamin D receptor (VDR) expression is not known. Therefore, a second aim was to make this determination. Male F344 rats were injected IP with 20 mg/kg of DOX or saline. Once treated, all animals were fasted with no food or water until sacrifice 24 hours later. Total glutathione content was elevated and protein carbonyls were decreased, with no change in Mn superoxide dismutase content, compared to control animals at 24 hr post treatment. These results differ from those previously reported and may be due to the differences in experimental design. Furthermore, content of VDR was increased in the DOX group compared to controls. In summary, improved oxidative stress status and increased VDR expression at 24 hr may be due to a rebound response.
Characterization of Efflux Transporters Involved in Distribution and Disposition of Apixaban
Authors: Zhang Donglu , Herbst John J , Kolb Janet , Wang Lifei , Shou Wilson , Balimane Praveen , He Kan , Frost Charles , Humphreys w Griffith
Published: June 2021

The studies reported here were conducted to investigate the transport characteristics of apixaban and to understand the impact of transporters on apixaban distribution and disposition. In P‐gp and BCRP‐cDNA transfected cell monolayers, as well as Caco‐2 cell monolayers, the apparent efflux ratio of basolateral to apical (PcB‐A) versus apical to basolateral permeability (PcA‐B) of apixaban was 10–38 compared to the apixaban efflux ratios of 1–4 in control cells. The P‐gp and BCRP‐facilitated transport of apixaban was concentration‐and time‐dependent and did not show saturation over a wide range of concentrations (1–100 μM). The efflux transport of apixaban was also demonstrated by the lower mucosal to serosal permeability than that of the serosal to mucosal direction in the isolated rat jejunum segments. Apixaban did not inhibit digoxin transport in Caco‐2 cells. Ketoconazole decreased the P‐gp‐mediated apixaban efflux in Caco‐2 and P‐gp‐cDNA transfected cell monolayers, but did not affect the apixaban efflux to a meaningful extent in BCRP‐cDNA transfected cell monolayers. Co‐incubation of a P‐gp inhibitor (ketoconazole or cyclosporin A) and a BCRP inhibitor (Ko134) provided more complete inhibition of apixaban efflux in Caco‐2 cells than separate inhibition by individual inhibitors. These results demonstrate that apixaban is a substrate for efflux transporters P‐gp and BCRP, which helps explain the observed disposition characteristics of the drug in rat studies that include its low brain penetration, low fetal exposure, and extensive excretion into milk.
The ABC transporter Mrp4/Abcc4 is required for Leydig cell protection from chemotherapeutic drugs
Authors: Morgan Jessica A , Schuetz John D
Published: June 2021

The ABC transporter Mrp4 is expressed in both human and mouse testes, specifically in Leydig cells, the primary testosterone (T) synthesizing cells in males. In an Mrp4 KO model, we demonstrated that Mrp4 is required for T production as signaling molecules regulating Leydig cell T production are disregulated, producing cAMP dependent downregulation of multiple genes involved in T biosynthesis. Because chemotherapeutic drugs used to treat childhood leukemia can produce detrimental reproductive effects related to impaired T production, we hypothesized that Mrp4 in Leydig cells may play a protective role against drugs that are Mrp4 substrates, including 6‐mercaptopurine (6MP). We show that in vivo administration of 6MP to WT and KO mice produces a substantial reduction in KO Leydig cell number, decreased testicular T concentration, and increased apoptosis as determined by caspase 3. However, as Leydig cells are post‐mitotic, the mechanism of death is unrelated to 6MP nucleotide incorporation into DNA. Under in vitro conditions, we show that cultured KO Leydig cells are susceptible to the cytotoxic effects of 6MP by reduction in cell number and viability. Caspase 3 inhibition blocks death by 6MP in KO Leydig cells, indicating the activation of an apoptotic pathway. Our data suggests that 6MP is toxic to Leydig cells by activating a caspase‐dependent pathway, and Mrp4 protects Leydig cells by export of 6MP nucleotides.
Encapsulation in ceramide lipid nanoparticles enhances bortezomib‐induced effects on metabolic activity
Authors: Park Gabriel Chin , Joshi Medha , Gilchrist Annette
Published: June 2021

Bortezomib (Velcade) is a first‐in‐class proteasome inhibitor approved for multiple myeloma that induces apoptosis in several cancer cell lines. Using U2OS, a human osteosarcoma cell line, and RAW 264.7, a mouse pre‐osteoclast cell line, we established bortezomib treatment resulted in a time‐and dose‐dependent decrease in metabolic activity, indicative of enhanced apoptosis. To improve its delivery we examined two types of liposomal formulations namely cationic and ceramide. Liposomes were prepared by dry film evaporation technique, characterized in terms of particle size, zeta potential, and encapsulation efficiency, and then examined for their effects on metabolic activity using undifferentiated and differentiated cells. In the case of RAW 264.7 the cells were differentiated using M‐CSF and RANKL, while for U2OS cells _‐glycerol phosphate and ascorbic acid were used. For both cell lines, encapsulation of bortezomib into ceramide lipid nanoparticles enhanced apoptosis of undifferentiated and differentiated cells. These results are in contrast to encapsulation of bortezomib into cationic lipid nanoparticles which did not significantly enhance the drugs’ apoptotic effect. Thus, encapsulation of bortezomib into ceramide lipid nanoparticles may enhance its effects on osteoclast cells, which are upregulated in multiple myeloma as well as osteosarcomas. Funding to GP is provided by Chicago College of Pharmacy Student Summer Research Grant and funding for this research provided by a Chicago College of Pharmacy's Startup Research Grant
Development of claudin‐1‐specific ligand
Authors: Nagase Shotaro , Yamashita Mayo , Iida Manami , Kondoh Masuo , Watari Akihiro , Fukasawa Masayoshi , Yagi Kiyohito
Published: June 2021

Claudins (CLs) are a family of tetratransmembrane proteins consisting of 27 members and are potential targets for enhancing mucosal drug absorption, treating cancer, and mucosal vaccination using the C‐terminal fragment of Clostridium perfringens enterotoxin (C‐CPE), a CL‐3/‐4 binder. The development of efficient CL binders is important for their future clinical application. We previously developed a broadly specific CL binder by using C‐CPE as a prototype; however, we did not successfully generate a CL subtype‐specific binder. Here, we focused on developing a CL subtype‐specific antibody. Because of the low antigenicity of CLs, we performed three different immunization procedures. BXSB mice with an autoimmune disorder were immunized with CL‐1‐displayed budded baculovirus, CL‐1‐expressing cells, or plasmid DNA encoding CL‐1. The DNA‐based immunization showed the highest production of serum anti‐CL‐1 antibodies. Therefore, B cells were isolated from the DNA‐immunized mice, and the B cells were fused with myeloma cells to form hybridomas. Hybridomas were screened for the production of the anti‐CL‐1 antibody. The antibodies bound to CL‐1‐expressing cells, but not to CL‐2, ‐4 or ‐5‐expressing cells. These results indicate that immunization of BXSB mice with CL DNA is a potentially useful method for the development of specific CL ligands.
Cytochrome P450 2C9 contributes to pulmonary vasoconstriction in exercising swine
Authors: Zhou Zhichao , de Beer Vincent J , Duncker Dirk J , Merkus Daphne
Published: June 2021

The role of endothelium‐derived hyperpolarizing factor (EDHF) in the pulmonary circulation, particularly the involvement of cytochrome P‐450 (CYP) 2C9 as an EDHF to regulate pulmonary vascular tone is incompletely understood. We investigated the contribution of CYP 2C9 to regulation of pulmonary vascular tone in swine at rest/during treadmill exercise. Inhibition of CYP 2C9 with sulfaphenazole had no effect on the systemic and pulmonary circulations either at rest or during exercise. Surprisingly, after blockade of both nitric oxide synthase (NOS) and cyclooxygenase (COX) with N‐nitro‐L‐arginine and indomethacin, sulfaphenazole produced significant systemic and pulmonary vasodilation, indicating a CYP 2C9‐mediated vasoconstrictor influence in these vascular beds both at rest and during exercise. In isolated pulmonary small arteries mounted on wire myograph, sulfaphenazole had no effect on the vasodilation produced by bradykinin under control conditions, but markedly enhanced the bradykinin‐induced vasodilation after pretreatment with NOS/COX blockade. The latter effect was no longer observed after pretreatment with reactive oxygen species (ROS) scavenger MPG. In conclusion, under basal conditions, CYP 2C9 is not critical for the regulation of pulmonary vascular tone either at rest or during exercise, but exerts a vasoconstrictor influence after NOS/COX blockade that appears ROS mediated.
The Effects of AT1 Receptor Blockade on Skin Microcirculatory Blood Flow and Thromboxane A2 (TXA2) Production in Young Healthy Women
Authors: Cavka Ana , Grizelj Ivana , Cosic Anita , Koller Akos , Jelakovic Bojan , Lombard Julian H , Phillips Shane A , Drenjancevic Ines
Published: June 2021

AT1 receptor activation has an important role in maintaining the vascular release of prostaglandins responsible for mediating hypoxic dilation in skeletal muscle microvessels in normotensive rats. High salt diet leads to impairment of vasodilator responses that could be restored by infusion of subpressor dose of ANG II via its interaction with AT1 receptors. In addition to AT1 receptors blockade, losartan blocks thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor. The aim of this study was to determine the effect of one week AT1 receptor blockade by losartan in 10 healthy normotensive women (20±2 years old) on law salt diet (LSD) on post occlusive hyperemic blood flow response in skin microcirculation by Laser Doppler Flowmetry (LDF) and to determine the plasma renin activity (PRA) and plasma thromboxane A2 (TXA2) concentration. Participants maintained LSD (<40 mmol of Na/day) and took 50 mg of losartan/day for 7 days. After one week of losartan administration the post occlusive hyperemic tissue blood flow was unchanged. However, plasma PRA and TXA2 levels were significantly increased compared to pre‐losartan values. Our results indicate that although one week of AT1 receptor blockade increases the plasma TXA2 concentration and PRA, the skin microcirculatory blood flow remained unchanged, possibly due to losartan blockade of TXA2 receptors and ANG II interaction with AT2 receptors.
Flow modulation and recruitment in a theoretical model for blood flow regulation in heterogeneous microvascular networks
Authors: Fry Brendan C. , Secomb Timothy W.
Published: June 2021

A theoretical model is presented for metabolic blood flow regulation in realistic heterogeneous network structures, derived from previously published experimental results from hamster cremaster muscle in control and dilated states. The model is based on modulation of arteriolar diameters, according to the length‐tension characteristics of vascular smooth muscle. Responses of smooth muscle cell tone to myogenic, shear‐dependent, and metabolic stimuli are included. Blood flow is simulated, including unequal hematocrit partition at diverging vessel bifurcations. Convective and diffusive oxygen transport in the network is simulated, and oxygen‐dependent metabolic signals are assumed to be conducted upstream from distal vessels to arterioles. Simulations were carried out over a range of tissue oxygen demand. With increasing demand, arterioles dilated, blood flow increased, and the numbers of flowing arterioles and capillaries – defined by red cell flux above a small threshold value – increased. Unequal hematocrit partition at bifurcations contributed to this behavior. These results imply that vessel recruitment, as observed in the hamster cremaster preparations, can occur as a consequence of local control of arteriolar tone. Supported by NIH grant HL070657.
In isolated vessels H2S is a less effective scavenger of exogenous superoxide than SOD
Authors: Springo Zsolt , Solymar Margit , Cseplo Peter , Toth Peter , Berta Gergely , Hamar Janos , Koller Akos
Published: June 2021

Recent studies suggest that hydrogen sulfide (H2S) exhibits potent antioxidant capacity and improves vascular and tissue functions in oxidative stress conditions. Thus we aimed to compare the direct antioxidant efficacy of H2S and superoxide dismutase (SOD).

Isometric force of isolated rat carotid arteries and gracilis veins was measured with a wire myograph. The vasomotor effect of the superoxide‐generator pyrogallol (10−5M) was obtained in control conditions, and then in the presence of SOD (120 U/ml) or H2S (10−5M or 10−4M), respectively. Spectrophotometric measurements were performed to detect the effect of SOD and H2S on the auto‐oxidation of pyrogallol to superoxide

Pyrogallol increased the isometric force of carotid arteries (9.7±0.8mN), which was abolished by SOD (5.3±0.8mN), but was not affected by 10−5M H2S (9.1±0.5mN), whereas 10−4M H2S significantly reduced it (8.1±0.7mN). Pyrogallol significantly increased the isometric force of gracilis veins (1.3±0.2mN), which was abolished by SOD (0.9±0.2mN), whereas 10−5M (1.3±0.2mN) or 10−4M H2S (1.2±0.2mN) did not significantly affect it. Pyrogallol‐induced superoxide production was measured by a spectrophotometer (A420=0.19±0.0). SOD reduced absorbance (A420=0.02±0.0), but 10−5M H2S did not (A420=0.18±0.0), whereas 10−4M H2S slightly, but significantly reduced it (A420=0.15±0.0).

This in vitro vascular bioassay study suggests that the superoxide scavenging efficacy of H2S is substantially less than that of SOD, suggesting that the beneficial effects of H2S on vascular function in conditions of oxidative stress could be due primarily to secondary mechanisms activated by H2S.
Platelets of humans with type 2 diabetes (DM2) exhibit fewer prostacyclin receptor (IPR) binding sites as well as decreased iloprost (ILO)‐mediated cAMP accumulation
Authors: Knebel Stephanie , Sprague Randy , Ellsworth Mary , Stephenson Alan
Published: June 2021

Humans with DM2 exhibit enhanced platelet aggregation that has been suggested to result from increased synthesis of the proaggregatory prostanoid, thromboxane by platelets, as well as from increased platelet thromboxane receptor sensitivity. The proaggretory effects of thromboxane are opposed by the antiaggregatory prostanoid, prostacyclin, released from the endothelium. Platelets express the prostacyclin receptor (IPR) and it has been suggested that reduced IPR activity may contribute to increased thromboxane sensitivity in DM2. Here we investigated the hypothesis that platelets of humans with DM2 exhibit decreased IPR expression as well as reduced responsiveness to the prostacyclin analog, iloprost (ILO) when compared to healthy humans. Platelets of humans with DM2 and controls were isolated from whole blood. Using 3H‐ILO, we determined that the number of IPR binding sites (Bmax) is reduced in platelets of humans with DM2. Furthermore, when platelets of humans with DM2 were stimulated with ILO, increases in cAMP were significantly reduced. The decreased IPR binding capacity and response to prostacyclin in platelets of humans with DM2 may contribute to the increased tendency for platelet aggregation in humans with DM2. Financial support provided by the American Diabetes Association (BS‐150).
Sensory nerve‐dependent prolonged opening of pannexin‐1‐based channels controls eNOS phosphorylation in mesenteric arteries
Authors: Gaete Pablo Simon , Lillo Mauricio , Figueroa Xavier
Published: June 2021

In this work, we analyzed if perivascular sensory nerves (PSN) or pannexin 1 (Panx1)‐based channels control eNOS activity in resistance arteries. PSN of isolated arterial mesenteric bed of rat were activated with capsaicin (1 μM, 20 min) and acetylcholine (ACh)‐induced vasodilation and NO production were assessed 1 h later. Panx1‐based channel activity was evaluated by ethidium uptake and eNOS phosphorylation (p‐eNOS) by Western blot. Ethidium uptake was undetectable in control conditions, but stimulation with capsaicin or calcitonin gene‐related peptide (CGRP) induced robust dye uptake for more than 1 h. ACh‐induced NO‐dependent vasodilation and NO production, decreased drastically 1 h after capsaicin treatment, which was associated with a reduction in p‐eNOSSer1177 but not in p‐eNOSTh495. CGRP8–37 (CGRP receptor blocker) and probenecid (Panx1‐based channel blocker) inhibited both CGRP‐ and capsaicin‐activated ethidium uptake. These blockers also prevented the reduction in p‐eNOSSer1177 and NO‐dependent dilation. Consistent with this, Panx1‐based channel blockade with 10Panx prevented the inhibition in NO production observed after capsaicin treatment. These results suggest that capsaicin‐sensitive sensory nerves activation induces a prolonged opening of Panx1‐based channels, which leads to a reduction of phosphorylation‐mediated eNOS activity.

FONDECYT 1100850, Anillos ACT‐71, AT2012
Vascular endothelial cell protein expression of adiponectin is related with vascular endothelial function in healthy older adults
Authors: Yoo Jeung‐Ki , Hwang Moon‐Hyon , Kim Han‐Kyul , Luttrell Meredith J. , Meade Thomas H. , English Mark W. , Christou Demetra D.
Published: June 2021

Adiponectin (APN) is associated with enhanced vascular endothelial function in humans. Recently, there is some evidence that human renal glomerular endothelial cells express APN mRNA and protein but this has not been established in human vascular endothelial cells. We sought to 1) measure vascular endothelial cell protein expression (VECPE) of APN and circulating plasma APN; and 2) examine their relation to vascular endothelial function in healthy older adults. We measured vascular endothelial function (brachial artery flow‐mediated dilation [FMD] using ultrasonography), APN VECPE (biopsy coupled with quantitative immunofluorescence) and plasma APN (Mercodia, ELISA) in 23 adults (64±2 years; mean±SE, 55 – 79 years; 11 men and 12 women) free from clinical disease. APN VECPE was not related with plasma APN (r=−0.14, P=0.6). Greater APN VECPE was related with greater FMD (r=0.43, P=0.047), lower baseline (r=−0.45, P=0.03) and post‐occlusion shear stress (r=−0.46, P=0.03) and greater FMD/shear stress (r=0.56, P=0.007). Both plasma and APN VECPE were related with impaired renal function (r=0.44 to 0.62, P≤0.04). In conclusion, APN VECPE is associated with enhanced vascular endothelial function and is elevated with impaired renal function in older adults free from clinical disease.

Supported by NIH Grant AG032067 & AHA Grant 0865117F.
Drag reducing polymers diminishing the near vessel wall cell poor region
Authors: Brands Judith , Kameneva Marina , Pacella John
Published: June 2021

Drag reducing polymers (DRP) are blood soluble long‐chain macromolecules that have been shown to enhance microvascular perfusion during severe coronary stenosis. The mechanism for the enhanced perfusion is not clear. Recently, it was discovered that nanomolar concentrations of DRP in red blood cell (RBC) suspensions flowing in a straight microchannel significantly reduce the near wall cell poor region (CPR) thickness. Therefore, we hypothesized that DRP redistribute RBCs within the vasculature reducing the CPR, which may in turn enhances perfusion. In rat cremaster tissue, CPR was measured in arterioles (range 25–44 μm) at baseline and 5, 15, 25 and 35 minutes after administration of DRP (blood concentration of 1ppm, N=5) or saline (control, N=6). In the control group, there was a 20% increase in CPR compared to baseline 15–35 minutes after infusion of saline (P<0.05). After DRP, CPR was significantly reduced compared to the CPR measured in the control group (72% of control measurement) and reduced to 87% compared to corresponding baseline at all time points (P<0.05). These data demonstrated that in vitro observations showing a decrease in CPR do occur in vivo, and that redistribution of RBC within the microvascular network may contribute to DRP perfusion enhancing effects via an increase in wall shear stress and a reduction in plasma skimming. Supported by AHA 12POST9400001 and NIH K08 1 K08 HL086730–01
Effects of time of day on 24‐h post‐exercise blood pressure and mood in normotensive humans
Authors: Sone Ryoko , Yamazaki Fumio , Tan Nobusuke
Published: June 2021

The duration of blood pressure (BP) lowering after a bout of exercise, i.e. post‐exercise hypotension (PEH), has not been sufficiently observed. The effects of the time of day on the PEH have not been clarified. Moderate exercise that is easy to induce PEH has been reported to improve mood. So, we examined the effect of exercise in the morning and evening on the 24‐h post‐exercise BP and mood. Nine normotensive males performed running at 60% of the maximal oxygen uptake for 30 min from 8:00 (AM) and 16:00 (PM), respectively, and we subsequently measured the BP for 24h. Control experiment without exercise (C) was also conducted. Compared with the C condition, systolic BP in a supine position (SBPsup) was lower in the AM and PM in the daytime (p<0.05), and tended to be lower in the AM, but did not differ in the PM at nighttime. The SBPsup averaged over 24h was ~3 mmHg lower in the AM and PM than under the C condition. A similar observation was also obtained concerning the SBP on sitting in the daytime, and the magnitude of SBP lowering was correlated with the fatigue score of the profile of mood states (p<0.05). Therefore, the post‐exercise BP response at nighttime depends on the time of day, and BP lowering in the daytime may relate to the fatigue sensation.
COMBINED EXERCISE TRAINING ATTENUATES CARDIAC AND AUTONOMIC DYSFUNCTIONS IN AN EXPERIMENTAL MODEL OF MENOPAUSE AND DIABETES
Authors: Sanches Iris Callado , Conti Filipe Fernandes , Soares Marcos Rafael Rodrigues , Quinteiro Hugo Garcia , Sirvente Raquel , Irigoyen Maria‐Cláudia , De Angelis Kátia
Published: June 2021

The objective of this study was to evaluate the effects of combined (aerobic+resistance) exercise training (ET) in ovariectomized diabetic rats. Female Wistar rats were divided (n=8 each group) into: euglycemic (E), diabetic (STZ, 50 mg/kg, iv) (D), and sedentary (SOD) or trained (TOD) ovariectomized diabetic rats. The combined ET included sessions on alternate days of aerobic exercise on treadmill and resistance exercise in ladder (40–60% of maximum capacity, 1h/d, 5d/wk, 8wk). Cardiac morphology and function were assessed by echocardiography. The animals were cannulated for arterial pressure (AP) recording and baroreflex sensitivity (BRS) assessment. The heart rate and the AP variability were analyzed in the time and frequency domain (FFT). Diabetes in ovariectomized rats increased 9% the glycemia and 20% the myocardial performance index (MPI), and reduced 14% the left ventricular shortening (SOD vs. D). The ET increased exercise capacity, attenuated the STZ‐induced LV morphometric changes, hypotension, bradycardia and BRS dysfunction, enhanced 65% the PI‐VAR, reduced 31% the MPI and 55% the SAP variability (TOD vs. SOD). In conclusion, combined exercise training induced cardiac, hemodynamic and autonomic benefits in diabetic ovariectomized rats, reinforcing the role of this approach in the management of risk factors observed in ovarian hormone deprivation associated with hyperglycemia.
Effects of Oryeongsan improved vascular relaxation in hypertension models
Authors: Kho Joung Hyun , Kim Hye Yoom , Kwon Oh Jeong , Tan Rui , Cho Kyung Woo , Kang Dae Gill , Lee Ho Sub
Published: June 2021

Body fluid volumes, which are regulated by the kidneys, are related to blood volume and arterial blood pressure. Oryeongsan (ORS), composition of five herbal medicines, has been widely used to traditional prescription for the treatment of body fluid disorders. The purpose of this study is to investigate the effect of ORS on vascular reactivity and related mechanism in the hypertension model. The experimental models were designed two types of hypertension as follows; 2 kidney 1 clip (2K1C) model and L‐NAME‐induced model. Both 2K1C‐ and L‐NAME‐ induced models were treated with two different dose of ORS (10 and 100 mg/kg/day) for 2 weeks. Blood pressure was markedly increased in both 2K1C and L‐NAME‐induced hypertension models. Interestingly, treatment with ORS significantly improved the impairments of acetylcholine (ACh)‐, sodium nitroprusside (SNP)‐, and atrial natriuretic peptide (ANP)‐induced vascular relaxation of aortic rings in both hypertension models. Treatment with ORS also significantly increased cyclic GMP (cGMP) levels of aortic rings (p<0.01). In addition, both 2K1C and L‐NAME‐induced hypertension models increased ANP, aldosterone levels, and renin activity in plasma, by contrast, treatment with ORS attenuated those levels and activity. These results suggest that ORS ameliorates vascular dysfunction in 2K1C and L‐NAME‐induced hypertension models.
Type 2 diabetic patients with treatment‐resistant hypertension display urine plasmin excretion that correlate with blood pressure and activate ENaC current
Authors: Friis Ulla Glenert , Buhl Kristian Bergholt , Oxlund Christina Stolzenburg , Svenningsen Per , Bistrup Claus , Abildgaard Ib , Jensen Boye L
Published: June 2021

Background

It was hypothesized that urine samples from patients with proteinuria (see below) would be able to activate EnaC through plasmin.

Materials

A cross sectional study design was applied to type 2 diabetic patients diagnosed with treatment resistant hypertension (RHTN). Urine samples were analyzed for albumin, creatinine, plasmin(ogen) and ability to activate inward currents in single collecting duct cells.

Results

Albuminuric T2DM patients with RHTN displayed significantly elevated levels of urinary plasmin(ogen) (844 ± 297 plg/crea, n=5) compared to normoalbuminuric‐T2DM‐RHTN patients (1.24 ± 0.9 plg/crea, n=5, P=0.011). The CCD cell line M1 showed significantly increased inward current when superfused with urine from albuminuric T2DM‐RHTN (58.8% ± 14%, n=5) compared to normoalbuminuric T2DM‐RHTN (16.2% ± 5.8%, n=5, P=0.0121).

Conclusion

A significant proportion of patients with type‐2 diabetes and treatment‐resistant hypertension excrete plasminogen proportional to albumin in urine which confers the ability of urine samples to evoke ENaC currents in vitro. The findings are in agreement with the notion that plasmin in the pre‐urine potentially aggravates hypertension trough inappropriate ENaC stimulation in type 2 diabetic treatment‐resistant hypertensive patients.
Downregulation of Brain Gα12 Blunts Angiotensin II‐induced Hypertension
Authors: Gao Juan , Kapusta Daniel R
Published: June 2021

We have shown that down‐regulation of brain Gα12 proteins blunts the pressor response to intracerebroventricular (icv) Angiotensin II (Ang II). Also, Gα12 expression is increased in brains of rats chronically infused s.c. with Ang II. We hypothesize that knock‐down of brain Gα12 will prevent chronic Ang II‐induced hypertension. Rats (n= 5–7/group) received Ang II s.c. (350 ng/kg/min) and were infused i.c.v. with a scrambled (SCR; 50 μg/day) or Gα12 oligodeoxynucleotide (ODN; 50 μg/day). Heart rate (HR) and blood pressure (BP, telemetry) were recorded during the 2 week infusion period. In the end, responses to atropine, propranolol and chlorisondamine were examined and baroreflex activity was analyzed. I.c.v. Gα12 ODN infusion lowered mean BP in Ang II infused rats compared with SCR ODN infusion (14‐ day peak; 133 ± 12 vs 176 ± 11 mmHg, p < 0.05). Compared to the SCR ODN group, Ang II infused rats that received i.c.v. Gα12 ODN showed a significantly (p<0.05) lower basal HR (344 ± 8 vs 397 ± 16 bpm), a greater increase in HR to atropine (Δbpm, 92±12 vs 51±9), and baroreflex sensitivity was improved (HR Gmax, Gα12 ODN, 28.4 ± 8.5 vs SCR ODN, 8.8 ± 2.1 bpm/mmHg). I.c.v. SCR or Gα12 ODN infusion did not alter BP in rats chronically infused s.c. with saline. These data indicate that Ang II impairs the parasympathetic branch of the baroreflex and contributes to the development of hypertension through a central Gα12 pathway. AHA 12GRNT12060613
Brain (pro)renin receptor knockdown modulates the body fluid homeostasis during angiotensin II‐dependent hypertension
Authors: Cao Theresa , Li Wencheng , Seth Dale , Feng Yumei
Published: June 2021

We previously reported that brain‐targeted PRR knockdown attenuates Angiotensin (Ang) II‐dependent hypertension and decreases vasopressin (AVP) release. To address the role of PRR in body fluid homeostasis, adeno‐associated virus‐mediated PRR shRNA (AAV‐PRR‐shRNA) was intracerebroventricularly injected to non‐transgenic (NT, n = 5) and human renin and angiotensinogen transgenic (RA, n= 6) mice. Urine excretion and water intake were recorded using metabolic cages. Ang II levels were measured at baseline and two weeks after PRR knockdown. At baseline, RA mice exhibited higher water intake (5 ± 0.6 VS. 3 ± 0.5 mL/day, p< 0.05), urine excretion (3.3 ± 0.5 VS 1.9 ± 0.1mL/day, p< 0.05), and urinary sodium excretion (0.3 ± 0.1 VS. 0.1 ± 0.1mmol/day, p< 0.05) compared to NT mice. PRR knockdown decreased water intake (3.3 ± 0.6 mL/day, p< 0.05) and urinary sodium excretion (0.2 ± 0.1mmol/day, p< 0.05) in RA mice. In addition, PRR‐shRNA significantly decreased Ang II levels in both the hypothalamus (1470 ± 90 VS. 1051 ± 77 fmol/mg, p< 0.05) and plasma (347 ± 41 VS. 156 ± 42 fmol/ml, p< 0.05) compared to baseline in RA mice; however, it did not alter Ang II levels in NT mice. This data indicates that brain‐targeted PRR knockdown modulates the body fluid homeostasis and is associated with reduction of Ang II levels, and brain PRR plays a regulatory role in the body fluid homeostasis during Ang IIdependent hypertension.
Extracellular Matrix Genes Fmod and Prelp are Candidate Genes for Salt‐Sensitive Hypertension in Females
Authors: Núñez Lizbeth , Hoffman Matthew , Xiao Bing , Flister Michael , Quinn Carol Moreno
Published: June 2021

We have narrowed a 3.7 Mb congenic interval on rat chromosome 13 that attenuates salt‐sensitive hypertension in females but not in male rats. The congenic interval was reduced to a 290 Kb region of Brown Norway (BN) that confers protection against hypertension on the Dahl Salt‐Sensitive (SS) background. This region contains 3 genes and 57 non‐coding sequence variants between SS and BN. Mean arterial pressure (MAP) was measured by telemetry on 8% NaCl diet. Renal damage was assessed by 24h protein excretion. Gene expression was determined by RT‐qPCR. MAP (176±5 vs 180±7 mmHg) and protein excretion (401±32 vs 354±59 mg/day) was not different between SS and congenic males on high‐salt diet. Conversely, MAP was significantly attenuated in congenic females compared to SS (152±5 vs 174±5 mmHg, P<0.005). Likewise, protein excretion was reduced in congenic females (111±20 mg/day) compared with SS (225±20 mg/day). Of the 3 genes in the candidate region, only Fmod and Prelp were expressed in the kidney. Compared with SS, the congenic females had 27‐fold lower expression of Fmod and a 5‐fold lower expression of Prelp in the renal medulla, whereas expression in the cortex did not change. Our results suggest a novel protective role of Fmod and Prelp in gender‐specific salt‐sensitive hypertension.
Sex‐dependent effect of prenatal and early postnatal red palm oil supplementation on cardiac ischemic tolerance of adult rats
Authors: Frantisek Kolar , Neckar Jan , Thamahane‐Katengua Emma , van Rooyen Jacques
Published: June 2021

Female Wistar rats were fed a standard diet supplemented with 0.25 ml/day of red palm oil (RPO) for 4 wks before mating, during pregnancy and until weaning at the offspring age of 4 wks; the second group was fed a control diet. Body mass of supplemented male and female offspring was higher compared to their respective controls and the faster growth continued after the cessation of RPO supplementation till adulthood. At the age of 14 wks, myocardial antioxidant enzyme levels were assessed and separate groups of offspring were subjected to 20 min of coronary artery occlusion and 3 h of reperfusion. RPO supplementation resulted in a more pronounced down‐regulation of superoxide dismutase 2 and glutathione peroxidase 1 in females than in males. The incidence and severity of ischemic ventricular arrhythmias was much lower in control females than in males. RPO markedly reduced the total number of arrhythmias and arrhythmia score in males to the same level as observed in control females, mainly due to a suppression of ventricular tachycardia. In contrast, RPO had no additive antiarrhythmic effect in females. Myocardial infarct size did not significantly differ between males and females and was not affected by RPO. The results indicate that RPO supplementation during early ontogeny may protect the adult rat heart against malignant ischemic arrhythmias in a sex‐dependent manner.

Supported by URF, CPUT, SA and Carotino Malaysia.
Overexpression of renin in the collecting duct causes hypertension
Authors: Ramkumar Nirupama , Ying Jian , Stuart Deborah , Kohan Donald E
Published: June 2021

The role of collecting duct (CD)‐derived renin in regulating arterial pressure (BP) is unknown. To examine proof of concept, gene targeting was used to obtain mice with renin overexpression in the CD. Mice were generated in which the sequence: loxP ‐ PGK promoter‐NeoR ‐ transcriptional stop sequence – loxP ‐ Ren1 cDNA – bovine growth hormone polyA was knocked in to the ROSA26 locus. These mice were crossed with mice transgenic for aquaporin 2 AQP2) promoter‐Cre recombinase, resulting in excision of the PGK‐Neo‐Stop and CD‐specific overexpression of renin (CD renin mice). Mice heterozygous for the knock‐in allele and AQP2‐Cre were studied. BP was recorded by telemetry and plasma and urine were collected in metabolic cages on normal, high and low Na diets. DNA recombination and PCR for ROSA‐renin mRNA confirmed kidney‐specific expression. Medullary renin mRNA was increased 5‐fold and urinary renin excretion was increased 2.5‐fold in CD renin mice. CD renin mice had salt‐sensitive hypertension that was most apparent on a high Na diet (mean BP 105 ± 2 mmHg vs. 95 ± 2 mmHg in wild type). Plasma renin concentration (PRC) was reduced by ~50% and ~80% in CD renin mice on a normal or high Na diet, respectively, as compared to WT animals. There were no differences in BP or PRC between CD renin and WT mice fed a low Na diet. These findings indicate that CD‐derived renin has the potential to modulate BP and that this effect is due to local, and not systemic, renin activity.
Renin knock out modulates sodium reabsorption in the Dahl salt‐sensitive rats
Authors: Pavlov Tengis S , Levchenko Vladislav , Ilatovskaya Daria V , Moreno‐Quinn Carol , Staruschenko Alexander
Published: June 2021

Dahl salt‐sensitive (SS) rats fed a high salt diet exhibit increased blood pressure and renal damage. We have shown recently that ENaC expression and activity were significantly enhanced in SS rats fed a high salt diet. The renin‐angiotensin‐aldosterone system (RAAS) regulates blood pressure and water electrolyte homeostasis, and renin is a critical component of this system. Ren−/− rats were used to further investigate the role of the renin and RAAS in regulation of sodium transport in salt‐sensitive hypertension. Knock out rats were created using zinc‐finger nucleases designed to target the renin gene in the SS rat. As it has been shown previously, Ren−/− rats have lower body weight and blood pressure whereas no difference is observed in plasma and urine Na+ concentration compared to WT littermates. Here we demonstrate that aldosterone level is significantly lower in Ren−/− rats compared to WT littermates. Aldosterone level is also suppressed in SS rats fed high salt diet compared to animals fed low salt. Single‐channel patch clamp analysis of the freshly isolated collecting ducts revealed decreased ENaC activity in Ren−/− rats. Expression of ENaC subunits was also assessed by immunohistochemistry and Western blotting. We conclude that renin knock out causes low blood pressure, decrease in aldosterone level and suppression of ENaC activity in the aldosterone‐sensitive distal nephron. Supported by AHA, ADA and NIH.
Macula Densa NOS1 Protects Against Acute Kidney Injury (AKI) Mediated by Primary Cilia
Authors: Fu Yiling , Lu Yan , Juncos Luis A. , Ge Ying , Pollock Jennifer S. , Wei Jin , Roman Richard J. , Cheng Liang , Liu Ruisheng
Published: June 2021

We hypothesize ischemia‐reperfusion induced AKI damages primary cilia on the MD and reduces NOS1 activity. After 18 min of bilateral ischemia‐reperfusion injury (IRI) in C57BL/6 mice (WT), the number and length of primary cilia were decreased. When increasing tubular flow in isolated JGA, NO (unit/min) in the MD was elevated from 120.2±21.5 to 164.0±13.8 in control, and from 72.3±15.6 to 86.4±14.7 after AKI. In cultured MD cells, elevating flow increased NO from 174.8 ± 21.5 to 276.1 ± 24.3, while no effect when cilia were removed by siRNA(48.6 ± 17.5 vs. 62.7 ± 15.2). Two strains of NKCC2‐tissue‐specific KO mice we developed (Cilia KO in which the primary cilia on the MD and thick ascending limb were deleted and the NOS1 KO in which all splice variants of NOS1 in the MD were deleted) received IRI. After 24 hrs, plasma creatinine (mg/dl) was 0.075±0.03 in sham, 0.56±0.02 in WT, 1.25±0.05 in Cilia KO and 0.89±0.03 in NOS1 KO. Urine KIM‐1(pg/24h) was 10.7±2.9 in sham, 31.2±5.5 in WT, 274.7±9.3 in Cilia KO and 1340.3±362.2 in NOS1 KO. Morphologically, we observed mild injury in WT and more severe cortical injury in both the Cilia KO and NOS1 KO while no injury in sham. Tubuloglomerular feedback (TGF) was enhanced (4.5±0.3 vs 8.4±0.7 mmHg) and NO in the MD was absent in intact NOS1 KO compared with WT. We conclude renal IRI damages cilia and reduces NO; KO of cilia/NOS1 exacerbates AKI probably via enhanced TGF. (R01‐HL086767 AHA11POST7750023)
Active ENaC channels are selectively recycled
Authors: Edinger Robert S , Butterworth Michael B , Johnson John P
Published: June 2021

ENaC resides in a vesicle pool which trafficks to the apical membrane in response to forskolin stimulation. Following forskolin withdrawal ENaC is internalized but recycles in response to further forskolin stimulation. The active state of ENaC is determined to some degree by cleavage of α and γ subunits with cleaved subunits having greater activity. We examined the relative trafficking of cleaved and uncleaved channels in FRT cells transfected with either wild type (WT) or cleavage resistant (CR) channels. CR channels lack both cleavage sites in both α and γ subunits. We first demonstrated that WT channels recycle in FRT cells in response to forskolin exactly as they do in endogenously expressing cell lines. FRT cells expressing WT channels have both full length and cleaved subunits, while CR cells show only full length channels. Cells expressing CR subunits have markedly diminished basal transport rate compared to WT. Both CR and WT cells respond to initial forskolin stimulation with an increase in current, proportional to basal levels. Following forskolin withdrawal, both decrease to baseline, but only WT cells respond to a second forskolin challenge representing recycling of apical channels. This suggests that cleavage state of ENaC may alter its recycling to the apical membrane.
Src‐family tyrosine kinase (SFK) phosphorylates With‐No‐ Lysine Kinase4 (WNK4) and modulates the inhibitory effect of WNK4 on ROMK channels.
Authors: Lin Dao‐Hong , Yue Peng , Yarborough OrLando , Lifton Richard , Rinehart Jesse , Wang Wen‐Hui
Published: June 2021

The aim of the study is to determine whether SFK phosphorylates WNK4 and roles of tyrosine (Tyr) phosphorylation site in modulating inhibitory effect of WNK4 on ROMK. Western blots demonstrated that c‐Src increased WNK4's Tyr phosphorylation and that c‐Src‐induced Tyr phosphorylation sites included Tyr1092, Tyr1094 and Tyr1143. Mass spectrometry further confirmed that c‐Src increased the phosphorylation of WNK4 at Tyr1143. SFK and PTP1D were coimmunoprecipitaed with WNK4. Mutation of Tyr1092 and Tyr1143 decreased the association of c‐Src and PTP‐1D to WNK4, respectively. Also, the inhibitory effect of WNK4 on ROMK was largely abolished in WNK4Y1092F–transfected cells and was absent in cells transfected with WNK4Y1092/1094F. Expression of c‐Src inhibited SGK1‐induced phosphorylation of WNK4 at Ser1196 but had no effect in the cells transfected with WNK4Y1092/1094F. WNK4Y1143F inhibited ROMK as potently as those of wt WNK4 and the inhibitory effect of WNK4Y1143F was not abolished by SGK1 which reversed the inhibitory effect of wt WNK4 on ROMK. However, SGK1 reversed WNK4Y1143F‐induced inhibition of ROMK in the cells treated with SFK inhibitors, suggesting that elimination of PTP‐1D‐WNK4 association may enhance the effect of SFK on WNK4. We conclude that WNK4 is the substrate of SFK and that the association of c‐Src and PTP‐1D with WNK4 at Tyr1092 and Tyr1143 plays a role in modulating the effect of WNK4 on ROMK.
Effect of nonpeptide V2 receptor antagonist SR121463 on phosphoproteome of rat IMCD
Authors: Wilson J. L. , Hoffert J.D. , Pisitkun T. , Knepper M.A.
Published: June 2021

We previously identified large numbers of phosphorylation sites that change in response to the V2 vasopressin receptor (V2R)‐ selective analog dDAVP in isolated IMCD suspensions (Hoffert et al. PMID: 22108457). Here, we used the same mass‐spectrometry‐based approach to carry out large‐scale phosphopeptide identification and quantification in isolated rat IMCD suspensions in the presence and absence of the V2R antagonist SR121463 (10 nM pre‐incubation followed by dDAVP, 1 nM). In brief, protein extracts were subjected to in‐solution trypsinization, iTRAQ labeling, and phospho‐peptide enrichment by immobilized metal affinity chromatography. 2454 phosphopeptides in 1163 proteins were successfully quantified by LC‐MS/MS (Velos‐Orbitrap). Interestingly, although many of the same phosphopeptides were quantified as in the previous study (Hoffert et al above), the results were not simply a mirror image of the dDAVP‐response. In particular, 164 phosphopeptides with a proline in position +1 that decreased in response to dDAVP, were unchanged or decreased in response to SR121463. This indicates that SR121463 does not block the ability of V2R occupation to inhibit proline‐directed kinases including MAP kinases and cyclin‐dependent kinases.
Regulation of posttranslational modifications of Npt2a (renal type IIa sodium phosphate cotransporter)
Authors: Lederer Eleanor DeLand , Murray Rebecca , Lesousky Nina , Khundmiri Syed Jalal , Weinman Edward
Published: June 2021

NpT2a, a proximal tubule apical membrane (AM) protein, plays a critical role in regulation of phosphate homeostasis. We and others have shown that 1) NpT2a is a glycosylated protein, 2) glycosylation is required for translocation to AM, and 3) NHERF1 deficiency causes aberrant AM expression. We hypothesized that NHERF1 associates with NpT2a prior to AM insertion and facilitates glycosylation. To test this hypothesis, we examined NpT2a‐NHERF1 association in Golgi (G) by density gradient centrifugation of opossum kidney cells. Cells transfected with GFP‐NpT2a at 16C showed retention of NpT2a in the G fractions while cells in 37C showed plasma membrane (PM) localization. NHERF1 was present in both G and PM fractions at both temperatures. NpT2a IP from G and PM fractions showed association with NHERF1. GFP‐NpT2a lacking the terminal – TRL motif lacked glycosylation and failed to associate with NHERF1 or traffic to the AM. Confocal imaging confirmed failure of the TRL deletion mutant to localize to the AM. We conclude that NpT2a associates with NHERF1 in the G through the C‐terminal PDZ binding domain and that this association is essential for NpT2a glycosylation and AM trafficking. Supported by VA
PHA2D proteins, KLHL3‐CUL3 and WNK1, Form An Endocytic Switch Signal that Controls the Renal K+ Excretory Channel, ROMK
Authors: Welling Paul , Kim Bo‐Young , Wade James B , Coleman Richard , Fang Liang
Published: June 2021

Mutations in Kelch like 3 (KLHL3) and Cullin 3 (CUL3) have been recently reported to cause a familial disorder of hyperkalemia and hypertension (Pseudohypoaldosteronism type II, PHAII) but the precise mechanism remains unclear. Here, we report that KLHL3/CUL3 and another PHAII gene, WNK1, tune endocytosis of the Renal Potassium channel, ROMK, by regulating the clathrin adaptor, ARH. We found that KLHL3 and CUL3 are critical subunits of a ubiquitin ligase complex that controls ARH abundance in the distal nephron. CUL3 interacts with ARH in a KLHL3‐dependent manner, driving ubiquitination and proteasomal degradation of ARH to limit ROMK endocytosis in states of dietary potassium excess. WNK1, which is activated in states of potassium restriction, stimulates phosphorylation of ARH at S14 and this prevents rapid degradation of ARH by KLHL3/CUL3. Thus, KLHL3/CUL3 and WNK1 exert opposing activities on ARH to control ROMK abundance on the apical surface in concert with physiological demands. Uncontrolled activation of ARH and ROMK endocytosis provides an explanation for urinary potassium retention and hyperkalemia in PHAII.