bluuebunny/crossref_metadata_2025 huggingface dataset:
Generation and characterization of hPepT1 transgenic mice
Authors: Dalmasso Guillaume , Nguyen Hang Thi Thu , Sitaraman Shanthi V , Merlin Didier
Published: June 2021

Human PepT1 (hPepT1), a di‐tripeptide transporter highly up‐regulated in inflammatory bowel disease (IBD), plays a key role in mediating immune response by transporting bacterial peptides. Currently there is no animal model to study the role of hPepT1 in intestinal disorders. To obtain strong and ubiquitous transgene expression in mice, we constructed a β‐actin promoter‐hPepT1 expression vector. β‐actin was chosen as it is constitutively expressed during embryonic development and adulthood. hPepT1 transgenics mice show a strong ubiquitous hPepT1 expression in Brain, Kidney, Lung, Spleen, Small Intestine, Colon, Liver, and Stomach. The external examination, macroscopic examination of body cavities, organs and tissues did not show significant changes. At the microscopic examination, several tissues such as Skin, Heart, Aorta, Larynx, trachea, Lungs, Thymus, Exocrine pancreas, Kidney, cerebral cortex, spinal cord, or ganglia of peripheral nervous system were unremarkable. Interestingly, minor histological changes in the gastro‐intestinal tract were observed to localize in the duodenum, ileum and large intestine. We found diffuse submucosal edema and multifocally presence of lymphocytes and macrophages in the lamina propria of these tissues. Overall, the hPepT1 transgenic mice should provide a good in vivo model to study the physiopathological relevance of hPepT1 over‐expression in IBD.
Adenosine 2b receptor blockade inhibits pro‐inflammatory events in the intestine
Authors: Kolachala Vasantha L , Matham Vijay Kumar , Wang Lixin , Srinivasan Shanthi , Gewirtz Andrew T , Linden Joel , Merlin Didier , Sitaraman Shanthi V
Published: June 2021

Background: Adenosine 2b receptor (A2bR) is the predominant adenosine receptor expressed in the colon. Our earlier studies demonstrated the protective effects of A2bR antagonist ATL‐801 on murine colitis. This study addresses possible mechanism of ATL‐801 protection against murine colitis. Methods: Dextran sodium sulfate (DSS)‐induced colitis and piroxicam‐treated IL‐10−/− mice were used as animal models of colitis. A2bR specific antagonist, ATL‐801, (10mg/Kg) was given in the diet. Colonic organ cultures and T84 cells were used to study IL‐8 secretion. Results: Mice treated with A2bR specific antagonist ATL‐801 in DSS or IL‐10−/−/piroxicam model showed markedly attenuated diarrhea as well as significantly low levels of MPO activity and KC in colon and organ cultures of the mice respectively reflecting inhibition of neutrophil transmigration. ATL‐801 abolished adenosine‐induced chloride secretion (ΔIsc: adenosine 7.9, ATL‐801+adenosine 1.9μA). In addition, ATL‐801 inhibited flagellin‐induced IL‐8 secretion in T84 epithelial cells (p<0.007, n=3). Conclusions: Taken together, these data demonstrate that intestinal epithelial A2bR is an important mediator of inflammatory responses such as chloride secretion and neutrophil recruitment in the intestine and blockade of A2bR by specific antagonist may be an effective therapeutic modality in intestinal inflammatory diseases.
Role Hypoxia‐inducible Factor‐1 α in the Development of Liver Fibrosis
Authors: Copple Bryan L , Moon Jeon‐OK
Published: June 2021

Liver fibrosis is a disease characterized by excessive deposition of extracellular matrix in liver during chronic injury. During early stages of this disease, cells secrete profibrotic proteins that stimulate matrix production. Although it is clear that these proteins are important for the development of fibrosis, what remains unknown is the mechanism by which liver injury stimulates their production. In the present study, the hypothesis was tested that hypoxia‐inducible factor‐1α (HIF‐1α) is activated in the liver during chronic injury and regulates expression of profibrotic proteins. HIF‐1α protein was increased in the livers of mice subjected to bile duct ligation (BDL), a model of fibrosis. To test the hypothesis that HIF‐1α is required for the development of fibrosis, wild‐type and HIF‐1α ‐deficient mice were subjected to BDL. Levels of type I collagen and α‐smooth muscle actin mRNA and protein were increased in wild‐type mice after BDL. These levels were significantly reduced in HIF‐1α ‐deficient mice. Furthermore, platelet‐derived growth factors, fibroblast growth factor‐2, and plasminogen activator inhibitor‐1 mRNA levels were increased to a greater extent in wild‐type mice when compared to HIF‐1α ‐deficient mice. Results from these studies suggest that HIF‐1α is a critical regulator of profibrotic mediator production during the development of liver fibrosis.
Regulation of Early Growth Response Factor‐1 (Egr‐1) by Bile Acids
Authors: Allen Katryn Miller , Copple Bryan
Published: June 2021

Cholestasis is a condition that results when excretion of bile acids from the liver is interrupted. Liver injury occurs in both humans and animals as a result of this disease. Recent studies have shown that inflammation is required for injury during cholestasis. Data from our laboratory has shown that early growth response factor‐1 (Egr‐1) is required for inflammation in vivo during cholestasis, and that Egr‐1 is upregulated in hepatocytes exposed to bile acids in vitro. What remains unknown is the mechanism by which bile acids upregulate Egr‐1. Bile acids modulate gene expression through activation of Farnesoid X Receptor (FXR) or by signaling through the mitogen‐activated protein (Map) kinase cascade. To elucidate the mechanism by which bile acids upregulate Egr‐1, primary mouse hepatocytes were isolated from wild‐type and FXR knockout mice and exposed to either deoxycholic acid (DCA) or chenodeoxycholic acid (CDCA). Egr‐1 mRNA was increased after bile acid treatment, however there was no difference in the level of induction between wild‐type and FXR knockout hepatocytes. Consistent with these in vitro findings, Egr‐1 was upregulated in the liver to a similar extent in wild‐type and FXR knockout mice after bile duct ligation. Next primary mouse hepatocytes were isolated and pretreated with U0126, an inhibitor of MEK‐1, a member of the Map kinase cascade. Pretreatment with UO126 prevented upregulation of Egr‐1 mRNA and protein after DCA and CDCA treatment. Results from this study demonstrate that bile acids upregulate Egr‐1 in hepatocytes via the Map kinase cascade.
Iron‐depletion limits intracellular bacterial growth in macrophages
Authors: Paradkar Prasad , De Domenico Ivana , Kaplan Jerry , Ward Diane McWay
Published: June 2021

Many intracellular pathogens are known to infect macrophages and these pathogens require iron for growth. Here we demonstrate in vitro that the intracellular growth of Chlamydia psittaci, Chlamydia trachomatis and Legionella pneumophila is regulated by the levels of intracellular iron. Macrophages that express cell surface ferroportin, the only known iron exporter, limit the intracellular growth of these bacteria. Hepcidin is an anti‐microbial peptide secreted by the liver in response to inflammation. Hepcidin binds to ferroportin mediating its internalization and degradation. Changes in ferroportin levels are of particular importance in macrophages as they are continually turning over red blood cells and thus acquiring iron. Addition of the exogenous hepcidin to infected macrophages enhanced the intracellular growth of these pathogens. Flatiron mouse macrophages, a mouse model of Ferroportin disease, showed enhanced intracellular bacterial growth independent of the presence of exogenous hepcidin. Macrophages, from wild type or flatiron mice, incubated with the oral iron chelators deferriprone or desferasirox showed reduced intracellular bacterial growth suggesting that these chelators might be therapeutic in chronic intracellular bacterial infections.
Anti‐inflammatory Properties of Murine Mesenchymal Stem Cells: Role of Interleukin 1 Receptor Antagonist
Authors: Pandey Amitabh C , Dutreil Maria F , Phinney Donald G
Published: June 2021

Mesenchymal stem cells (MSCs) are pluri‐potent cells derived from the bone marrow that possess the capacity to regulate the cellular microenvironment by production of various cytokines, chemokines, and matrix proteins. The objective of this study was to identify novel products secreted by MSCs that may modulate the inflammatory response. Interrogation of the murine MSC transcriptome, which we previously catalogued via serial analysis of gene (SAGE), revealed numerous SAGE tags corresponding to interleukin 1 receptor antagonist (IL1RN). Screening of a murine MSC cDNA library by PCR confirmed that cells expressed mRNAs encoding IL1RN. Furthermore, fractionation studies indicated that MSCs are the principal source of IL1RN in murine bone marrow. FACS analysis further revealed that expression of IL1RN was restricted to ∼24% of murine MSCs, which was confirmed by immunostaining. Enzyme‐linked immunosorbent assays (ELISA) demonstrated that murine MSCs secrete approximately 3ng/ml/cell of IL1RN protein. Studies conducted in vitro showed that media conditioned by MSCs was able to block in a dose dependent fashion the ability of IL‐1α to stimulate proliferation of a T cell line. This inhibitory effect of MSC conditioned media could be abrogated by a neutralizing anti‐IL1RN antibody, thereby demonstrating that the effect was specific to IL1RN secreted by cells. Collectively, these studies identify a unique subpopulation of murine MSCs that express high levels of IL1RN, which functions to antagonize the activity of IL‐1α This activity may account, in part, for the anti‐inflammatory effects of MSCs in vivo.
The Novel Impact Of Treadmill Exercise And Sex Difference On Cell Proliferation and Cell Survival In The Dentate Gyrus Of G93A Mice
Authors: Ma Xiaoxing , Hamadeh Mazen J , Zhang Bingjun , Christie Brian , Tarnopolsky Mark A
Published: June 2021

Neurogenesis persists in the adult mammalian brain and can be regulated by physiological stressors influencing cell proliferation or cell survival. We investigated whether sex and treadmill running would affect cell proliferation and newborn cell survival in the dentate gyrus (DG) of G93A mice, a transgenic model over‐expressing mutant human Cu/Zn‐superoxide dismutase (SOD1) and leading to excessive oxidative stress. Proliferating and surviving cells were labeled with bromodeoxyuridine (BrdU) over 7‐days before sacrificing mice @ 1 and 3 weeks. Proliferating cells were significantly higher in males than in females (p=0.009) but cell survival tended to be higher in female vs. male G93A mice (p=0.057). Running G93A mice have less proliferating cells than sedentary G93A mice (p=0.056); however, running wild type mice have significantly more proliferating cells than sedentary wild type mice (p=0.036) and than running G93A mice (p=0.040). In addition, female running G93A mice displayed more surviving cells than male running G93A mice (p=0.033). Wild type running mice showed a significant increase in cell survival vs. sedentary wild type (p=0.028). These results suggested that running promotes cell proliferation in wild type mice, but inhibits cell proliferation in G93A mice. Running promotes cell survival in male and female wild type and only female G93A mice. The sex differences in cell proliferation and survival for the G93A mice are supportive of our previous observation that female G93A mice have better survival and resistance to excessive running stress as compared with males.
SK4 mediated K+ secretion depends on P2Y receptor localization in human mammary epithelial cells
Authors: Schiller Katherine R. , O’Grady Scott M.
Published: June 2021

Immortalized human mammary epithelial cells express multiple P2Y receptors that are localized in both apical and basolateral membranes. Basolateral UTP (10 μM) stimulation of monolayers mounted in Ussing chambers produced an increase in ENaC‐dependent Na+ absorption resulting in part from SK4 channel activation in the basolateral membrane. In contrast apical stimulation with UTP (10 μM) produced a transient decrease in Isc consistent with transepithelial K+ secretion. Inhibition of Ca2+ mobilization evoked by UTP using BAPTA‐AM abolished the decrease in Isc. In addition, pretreatment with a known Ca2+‐activated K+ channel toxin, charybdotoxin (200 nM), blocked the Isc response by >70%. A similar degree of inhibition was produced by pretreatment with clotrimazole (10 μM) and TRAM‐34 (0.5 μM). Apical addition of the selective BK channel inhibitor, iberiotoxin also affected the response to apical UTP by stimulating oscillations in the Isc. These results suggest that apical UTP stimulation elicits K+ secretion that is mediated by BK and SK4 potassium channels. Moreover, apical UTP failed to stimulate ENaC‐dependent Na+ absorption observed following basolateral UTP treatment, suggesting that the Ca2+‐mobilizing effects of apical UTP are limited to the apical membrane.
Role of Golgi apparatus and microtubules for aldosterone and vasopressin–dependent regulation of ENaC activity
Authors: Gresko Nikolay , Rossier Bernard C. , Loffing Johannes
Published: June 2021

Sodium reabsorption in the renal collecting system critically depends on the activity of the epithelial sodium channel (ENaC) at the apical plasma membrane. Previous work of us and others indicated that aldosterone and vasopressin control the activity of ENaC largely by changes of its cell surface expression. The exocytotic trafficking of many membrane proteins involves the Golgi apparatus and occurs along microtubules. In the present study, we used a mouse collecting duct cell line (mCCD cells; JASN 16: 878‐91, 2005) to analyse the role of the Golgi apparatus and of microtubules for ENaC regulation by aldosterone and vasopressin. The mCCD cells were grown on permeable support. Amiloride‐sensitive short‐circuit currents (Isc) were measured in an Ussing‐type chamber as an indicator of ENaC activity. Three hours of aldosterone (300 nM) and vasopressin (DDAVP 10 nM) application increased amiloride‐sensitive Isc from ~13μA/cm2 to ~40μA/cm2 and ~35μA/cm2, respectively. Pretreatment of the cells with the Golgi‐disrupting agent brefeldin A (10μg/ml for 2 hours) or the microtubule‐disrupting colchicine (30 μM for 5 hours) profoundly reduced baseline Isc to ~2.5 μA/cm2, but did not block the response to aldosterone and vasopressin. Both hormones still increased amiloride‐sensitive Isc by more than 300%. The data suggest that intact Golgi apparatus and microtubules are important for maintaining baseline ENaC activity but are not required for the activation of the channel by aldosterone and vasopressin.
Effects of HIV‐1 on Endothelial Gene Expression
Authors: Porter Kristi Michelle , Detorio Mervi , Schinazi Raymond F , Sutliff Roy L
Published: June 2021

Recent data suggest that HIV‐infected persons are at a heightened risk of developing non‐infectious lung disorders. HIV‐related pulmonary hypertension (HIV‐PH), a disorder characterized by increased pulmonary vessel tone and vascular remodeling, is one such example. HIV‐PH is estimated to occur 2500 times more frequently than primary PH in the general population. HIV‐PH is also found to progress more rapidly than primary PH with no association between CD4+ count and disease severity. These observations suggest a correlation between HIV infection and HIV‐PH unrelated to immune dysfunction. However, the cause of HIV‐PH is unknown. We hypothesize that HIV infection plays a critical role in HIV‐PH development by upregulating genes that modulate vasoconstriction and remodeling. To test this hypothesis, we employed co‐cultures of human pulmonary artery endothelial cells (HPAEC) and/or PBMC, either alone or exposed to HIV‐1. Gene array results showed a marked upregulation in the gelatinase, matrix metalloproteinase‐2 (MMP2) in the HPAEC, PBMC and HIV‐1 group as compared to other groups. RT‐PCR confirmed these results and also showed increased expression of angiotensinogen and angiotensin converting enzyme, precursors of the potent vasoconstrictor, angiotensin II. Altogether, our data suggest that HIV‐1‐induced gene alterations may contribute to the increased risk of HIV‐1 patients to develop PH.
Isolation of resistance vessels and detection of Vasopressin receptor (V1a) from different vascular beds in Spontaneous Hypertensive Rat (SHR).
Authors: Samsøe Elisabeth Vaagen , Gharehnia Behzad , Jonassen Anne Kristine , Vågnes Øyvind Brune , Iversen Bjarne Magnus
Published: June 2021

The arginine vasopressin receptor (V1a) is up‐regulated in renal resistance vessels from 4 wk old SHR compared to age matched normotensive Wistar Kyoto rats (WKY). At the age of 20 weeks, the receptor level is not different between the strains. V1a expression in renal resistance vessels seems to be up‐regulated during the development of hypertension.

In the present study we investigated the expression of the V1a in resistance vessels from different organs in SHR and WKY. Resistance vessels were isolated from heart, aorta, small intestine, tail and lower limb using magnetic iron oxide. In addition, the effect of vasopressin injection on coronary blood flow (CBF) was studied in isolated hearts. V1a expression was analysed by western blot and real time PCR.

Results: V1a protein expression was increased in resistance vessels from the heart of young SHR. In resistance vessels from tail, intestine and lower limbs, no difference in expression was found. In 20 wk old rats no difference in CBF response to vasopressin was found between the strains, in 4 wk old rats the situation is not yet clear. In conclusion the V1a expression was upregulated in heart resistance vessels isolated from young SHR compared to age matched control WKY rats. This may indicate involvement in the development of genetic hypertension. No upregulation was found in resistance vessels from other tissues.
Heterogeneous venoconstriction in human lower legs
Authors: Phillips Christien , Horsburgh J , Balanos G , Brown M D
Published: June 2021

Sympathoexcitation reduces venous capacity in the lower leg, based on volume‐pressure relationships of the whole limb during venous congestion. Whether conduit leg veins contribute to this by active constriction has been a matter of debate. Using Doppler ultrasound, we compared responses to a cold pressor test (CPT) of two lower leg veins, the long saphenous (LS) and popliteal (Pop), against that of the whole limb. Calf venous volume (strain gauge plethysmography) was measured in 10 males (26±4 y, 23±2 BMI, mean ± SD) during venous congestion (thigh cuff 50 mmHg, 5 min) while either the LS or Pop vein were imaged longitudinally 5–7 cm below the knee and diameter measured by edge‐detection software. The CPT, applied between min 3 and 4 of congestion, increased mean arterial pressure and heart rate similarly during imaging of LS and Pop (22±3 and 18±3 mmHg, 14±2 and 11±3 bpm respectively, mean ± SEM). It reduced calf volume by 16±6 %, and the cross‐sectional area (CSA) of the LS (filled diameter 3.0±0.3 mm) decreased by 25±6 % (p<0.01). When imaging Pop vein (diameter 9.4±0.8 mm), CPT also reduced calf volume by 16±3 % but Pop CSA did not alter (+4.0±2.4 %, NS). These disparate vein responses despite the similar sympathoexcitatory challenge imply that changes in whole limb volume will reflect constriction in a superficial (LS) but not deep (Pop) conduit vein as well as smaller post‐capillary venules.
Responsiveness and density of alpha‐adrenergic receptors in skeletal muscle of young and old beagles
Authors: DeLorey Darren , Buckwalter John B , Mittelstadt Scott , Anton Maria M , Kluess Heidi A , Tune John D , Clifford Philip S
Published: June 2021

Whether the tonic increase in muscle sympathetic nerve activity that has been associated with aging alters α‐adrenergic receptor protein expression and responsiveness has not been established. The purpose was to investigate the effect of aging on the density and responsiveness of α1‐ and α2–adrenergic–receptors in skeletal muscle. Young (n = 6; 22 ± 3 mo.) and old (n= 7; 119 ± 22 mo.) beagles were instrumented with flow probes on the external iliacs and a catheter in a branch of the femoral artery. Flow‐adjusted doses of selective α1‐ (phenylephrine (PE); 0.05 ug/ml/min) and α2–adrenergic‐receptor (clonidine; 0.1 ug/ml/min) agonists were infused at rest and during treadmill running at 2.5 miles/h and 4 miles/h with 2.5% grade. Hindlimb blood flow (HBF) and mean arterial pressure (MAP) were recorded and vascular conductance (VC) was calculated. Gracillis muscle feed arteries were dissected and receptor protein content was determined by Western Blot. PE infusion resulted in a similar decline in VC in young and old at rest (Young: −59 ± 11 %; Old: −62 ± 13 %) at 2.5 miles/h (Young: −62 ± 10 %; Old: −67 ± 13 %) and 4 miles/h with 2.5% grade (Young: −49 ± 8 %; Old: −54 ± 9 %). Clonidine infusion also produced similar decreases in VC in young and old at rest (Young: −70 ± 14 %; Old: −59 ± 19 %) at 2.5 miles/h (Young: −47 ± 13 %; Old: −52 ± 15 %) and 4 miles/h with 2.5% grade (Young: −43 ± 13 %; Old: −43 ± 12 %). α1D and α2A receptor protein expression was similar, whereas α1B receptor protein expression was 17% greater (p<0.05) in old compared to young. The vascular response to α1‐ and α2–adrenergic‐receptor agonists does not decline with aging. The similar vascular response to PE suggests that the age‐related decline in α1B receptor expression was offset by an increase in receptor responsiveness.
Prostanoid‐induced activation of airway nociceptors via TRPA1
Authors: Taylor‐Clark Tom , McAlexander Michael A , Nassenstein Christina , Ghatta Srinivas , Undem Bradley J
Published: June 2021

Objective: to assess the effect of electrophilic prostanoids on airway sensory nerve activation. Results: Single cell RT‐PCR of airway‐identified mouse sensory neurons demonstrated expression of TRPA1 mRNA only in TRPV1 mRNA‐positive vagal neurons. Known TRPA1 agonists (allyl isothiocyanate (AITC) and cinnamaldehyde) caused robust activation of all capsaicin‐sensitive airway C‐fibers in a mouse ex vivo isolated vagal‐lung preparation. Electrophilic compounds have been shown to activate TRPA1 via covalent modification. Metabolites of prostaglandin (PG) D2 and PGE2 (J‐series and A‐series PGs) including 15‐deoxy‐δ12,14 PGJ2 (15dPGJ2) are capable of covalent modification. HEK cells transfected with human TRPA1, but not wild type, responded robustly in a calcium assay to the A and J‐series PGs. In patch clamp studies, 15dPGJ2 mimicked the effect of AITC on TRPA1‐HEK cells with characteristic outward rectification. Isolated trigeminal neurons also showed robust responses to 15dPGJ2, AITC and capsaicin. 15dPGJ2 responses were inhibited by HC‐030031 (TRPA1 inhibitor) and ruthenium red (nonselective TRP inhibitor) but not iodo‐resiniferatoxin (TRPV1 inhibitor). Finally, 15dPGJ2 caused action potential discharge in airway C‐fibers terminals in an ex vivo isolated vagal‐lung preparation. Conclusion: airway nociceptors can be activated by metabolites of PGD2 and PGE2 via TRPA1. Funded in part by NIH.
Differentiating EGFR activation vs NHE1 inhibition in TRO induced growth arrest of breast cancer cell line MCF‐7
Authors: Friday Ellen , Oliver Robert , Welbourne Tomas , Turturro Francesco
Published: June 2021

Troglitazone acts at both the EGFR and NHE1 to induce ERK activation and a cellular acidosis. Because the former site may promote cell growth whereas NHE1 inhibition is associated with growth arrest, we determined the relative sensitivity of these two sites to TRO over a concentration range from 1–20uM. MCF‐7 cells were incubated over 12min in a weakly buffered KH media and ERK activation (ratio p‐ERK/total ERK) and NHE1 activity ( NH4+/ acid pulse) assayed. At 20uM TRO, ERK activation increased 9.2 fold (p<0.001) vs. time control and NHE1 activity decreased (p<0.01) to 39% of control (0.035±0.006 vs. 0.090±0.003). Intracellular pH at the end of 12 min decreased (p<0.001) from 7.32±0.04 to 6.73±0.05 associated with a large retention of acid ( =20mmol/pHi) within the cells (114±16 vs. 1±14nmole H+/12min, p<0.01); lactate released over the 12min was not increased (66±4 vs. 61±7nmole and accounted for 20% and 48% of the total acid produced respectively) suggesting acid produced from some source other than glycolysis. At 1uM TRO, ERK activation increased 2.4fold (p<0.05) without a decreased NHE1 activity but with a fall (p<0.05) in pHi (7.08±0.06 vs. 7.34±0.05). Acid production increased (223±22 vs. 151±14nmole, p<0.05) over control without an increase in lactate production; similar results were obtained at 5uM TRO. Inhibiting MEKK with PD98059+UO126 prevented both ERK activation and the increased acid production with 20uM TRO and reduced the rate of acidification but not the final pHi or NHE1 inhibition. These results are consonant with a close relationship between EGFR and NHE1 and that the former may support growth via activation of ERK and a subsequent acidogenic metabolic pathway which responds to 20fold lower concentrations of TRO than NHE1 inhibition.
Quantitative analysis of renal inner medullary structure
Authors: Pannabecker Thomas Lloyd , Dantzler William H
Published: June 2021

3‐D architecture of vessels and nephrons of rat renal inner medulla (IM) was analyzed quantitatively. IM outer zone (first 3–3.5 mm) has collecting duct (CD) clusters with borders defined by Euclidean distance map as organizing motif. Descending thin limbs (DTLs) of Henle's loops and descending vasa recta (DVR) are located outside CD clusters on these independently defined borders. DTLs in first 1 mm lack AQP1 and in remaining 2–2.5 mm lack AQP1 for last 60%. Ascending thin limbs (ATLs) and ascending vasa recta (AVR) are inside and outside CD clusters. ∼50% of AVR tightly abut CDs. Nodal spaces inside clusters are bordered laterally by CDs, AVR, and prebends or ATLs, and vertically by interstitial cells. Surface area of prebends plus corresponding length of ATL (probable site of NaCl efflux) decreases vertically with periodic peaks and valleys. Peaks (av. ∼2500 μm2 greater than valleys) may correspond to location of nodal spaces. In IM inner zone (last 1.5–2 mm), CD clusters disappear, all DTLs lack AQP1, all vasa recta are fenestrated, and loops are too few to show periodic prebend oscillations. Number of AVR abutting CDs increases with increasing CD diameter so that abutting surface area is unchanged. Nodal spaces are fewer, but larger. Final 0.5 mm has loops with narrow bends and loops with transverse bends. Transverse bends prevent surface area/medullary volume of prebends plus corresponding ATLs from decreasing. DK16294
An Optimal Mathematical Model for the Urine Concentrating Process of Neonatal Rats
Authors: Marcano Mariano , Bonilla‐Félix Melvin
Published: June 2021

Immature rats have limited abilities to concentrate the urine. A mathematical model for the concentrating mechanism in the neonatal rat medulla was formulated. A nonlinear optimization method was used to identify model parameter sets that maximize a model efficiency measure, which was taken as the ratio of free‐water absorption rate to total active solute transport rate. The optimization method systematically changes the parameter values within their prescribed ranges, seeking maximal efficiency. The variable parameters were: NaCl and urea concentration in the cortico‐medullary junction of descending limbs of Henle (DLH) and collecting ducts (CD); urea permeability in DLH, ascending limbs (ALH) and inner medullary CD (IMCD); NaCl maximum active transport rates in ALH and IMCD; CD water permeability; and the exponential decrease rates of loops of Henle and CD population. Parameter baseline values were obtained from reported means in physiological experiments or estimated using physiological assumptions. By allowing most parameters to fluctuate by ± 25% from baseline values the maximal model efficiency was 15% above baseline, resulting in only 5% increase in urine osmolality. This is consistent with experimental data from animals and isolated CD from immature rats.
Investigating the nephron architecture between the outer and inner medulla of the rat kidney
Authors: Anand Neelesh K , Koushik Sarang S , Milazzo Anthony J , Naqvi Mohammad I
Published: June 2021

The rat kidney consists of one renal pyramid which can be divided into three fundamental regions; the cortex, outer medulla, and inner medulla. Nephrons are organized throughout these three regions in a highly organized fashion. The focus of this study is to identify the architectural relationships between the descending (DVR) and ascending vasa recta (AVR), the ascending thin limbs (ATLs), and collecting ducts (CDs) between the inner and outer medulla. Architectural relationships were identified through digital reconstructions of nephrons and vessels observed in images of physical and optical sections prepared at 1 to 5μm intervals. The short long loops of Henle have glomeruli positioned in the cortex above the border with the outer medulla. Their loops of Henle descend into the inner medulla, looping bends predominantly within the interior of CD clusters. The four to six AVR arranged symmetrically around each inner medullary CD form similar arrangements with outer medullary CDs. Interstitial spaces are formed adjacent to CDs in outer medulla and appear to be associated with thick ascending limbs of Henle. These relationships may contribute to the differences between the inner and outer medullary concentrating mechanisms. (NIH DK16294)
Hyperosmolality activates the integrated stress response in mouse medullary cells
Authors: Cai Qi , Keck Maggie , Brooks Heddwen L
Published: June 2021

The integrated stress response (ISR) is a eukaryotic translation initiation factor‐2 (eIF2a) phosphorylation‐dependent, stress inducible pathway activated by a variety of environmental stresses such as oxidative stress and endoplasmic reticulum stress. However IRS has not been characterized in osmotic stress. In normal conditions, renal medullary cells face high and various interstitial osmolality due to changing concentrations of NaCl and urea. To determine whether hyperosmolality causes IRS, mIMCD3 cells were subjected to hyperosmolality by adding extra NaCl or urea into culture medium. We found that phosphorylated eIF2a increased in the presence of 125, 150 or 200mM NaCl after 0.5, 1, 2 and 3 hours in a dose‐dependent manner. eIF2a phosphorylation also increased in response to 250, 300, 400mM urea at the same time points. High NaCl or urea activated an eIF2a kinase, general regulated non‐depressible‐2 (GCN2), in mIMCD3 cells. 7 day vasopressin infusion into mice raised the osmolality of the renal medulla and increased the mRNA expression of IRS pathway genes ATF3 and ATF4 (2.20 ± 0.37 fold and 1.62 ± 0.03, respectively, P<0.05). We conclude that high concentrations of NaCl or urea activate IRS in renal medullary cells.
MATE1 topology: accessibility of native cysteine residues in MATE1 to an impermeant thiol‐reactive reagent
Authors: Zhang Xiaohong , Wright Stephen
Published: June 2021

Mammalian multidrug and toxic compound extrusion(MATE) proteins have been suggested to mediate polyspecific H+‐coupled transport of organic cations(OCs) in the apical membrane of proximal tubules. Analysis of the putative secondary structure of the rabbit, human, and rat orthologs of MATE1 with computerized algorithms generally predict 13 transmembrane helices (TMH) and an external C‐terminus. Assessment of the accessibility of an antibody against a C‐terminal V5‐epitope suggested the C‐terminus of rbMATE1 is extracellular. To further define MATE structure, the accessibility of native cysteine residues in rbMATE1 to hydrophilic thiol‐reactive reagents was examined. Maleimide‐PEO2‐biotin precipitation (surface biotinylation followed by Western blotting) was performed with a series of cysteine mutations (replaced with alanine) to determine their accessibility to an impermeant hydrophilic thiol‐reactive reagent. Significant decrease of cell surface biotinylation was observed for elimination of cysteines at amino acid positions 62, 270, 352, 356, and 444, suggesting that these residues are located either in the extracellular loops or in transmembrane helices exposed to a hydrophilic cleft. Mutation of cysteine to alanine at residue 251, which is predicted to be in the intracellular loop between TMH 6 and TMH 7, led to a significant loss of tetraethylammonium transport activity and protein expression on the plasma membrane, suggesting an important role of cysteine 251 for ;protein processing of MATE1. Supported by NIH grant DK58251.
Rescue of mechanical function, muscle morphology and signaling by desmin plasmid transfection
Authors: Lieber Richard Louis , Palmisano Michelle , Shah Sameer , Hornberger Troy , Ryan Allen F.
Published: June 2021

The muscle‐specific intermediate filament desmin has been described as the “mechanical integrator” of the muscle cell. We probed this function by transfecting adult desmin knockout (DKO) muscle with plasmid DNA encoding desmin and measuring the resulting structural and functional changes. Mice were transfected by electroporation and, after 7 days, when protein expression peaked, we measured the regularity of the myofibrillar lattice and several responses to mechanical stretch including the deformability of nuclei, stress production, injury and c‐Jun‐kinase (JNK) phosphorylation. Desmin transfection reversed essentially all of the knockout phenotype in a quantitative manner—Z‐disks were re‐aligned, myonuclear deformability was restored, and stress production and the resulting injury response was also returned to near‐wildtype levels in a dose‐dependent, although nonlinear manner. Furthermore, the stretch‐induced activation of JNK phosphorylation, which was not detectable in the DKO muscles, was restored. These results demonstrate that the desmin intermediate filament is primarily responsible for the spatial alignment of Z‐disks and plays a central role in transducing mechanical stress: (1) from the force‐generating cytoskeletal network to the surrounding tissue and (2) to the intracellular signaling apparatus.

Supported by NIH and Department of Veterans Affairs.
Effect of Sodium Ferulate on the Cell Migration Induced by TNF‐α
Authors: Zhang Yemin , Ouyang Jingping , Zhang Deling , Wang Fengjie , Liu Yongming
Published: June 2021

People's Republic of Migration of vascular cells is a crucial response to vascular injury resulting in atherosclerosis. Tumor necrosis factor‐α(TNF‐α) functions as a potent chemoattractant for cells and can change cell cytoskeleton obviously.Sodium Ferulate(SF), a principle from traditional Chinese herb medicine of Angelica, is approved by State Drugs Administration of China as a new drug for the treatment of cardiovascular diseases.This study was to employ ECV304 cells migration model induced by Tumor necrosis factor‐α(TNF‐α) and to make studies of the effect of SF on the migration of ECV304 cells .Through cell scratch wound migration assay and quantification of cell migration, we observed that TNF‐α significantly promoted cell migration of ECV304 and that SF could inhibit the migration induced by TNF‐α treated for 24h.Meanwhile the morphological changes were observed under Giemsa staining and HITACHIS‐570 scanning electron microscope.The morphologies of the migration cells treated only by TNF‐α were markedly changed:cytoplasm radial protruding,numerous pseudopods presenting filament‐like and thin strip‐like contour of some cells presenting.crab.‐ like or.hedgehog.like shape.And the morphologies of the cells treated SF and TNF‐α were similar to the control cells, exhibited regular arrangements, relatively large volume, mostly round or polygon shapes, abundant surface microvillus on the surface.These findings suggest that SF may protect cell pseudopodium and surface microvillus from TNF‐α, and inhibite the migration of ECV304 stimulated by TNF‐α, accordingly it can prevent and treat atherosclerosis.(The research is supported by national natural science foundation of China granted NO.30470680)
The WT1 Interacting Protein: A molecular messenger between the slit diaphragm and podocyte nucleus
Authors: Kim Jane H , Mukherjee Amitava , Schelling Jeffrey R , Konieczkowski Martha , Sedor John R
Published: June 2021

Podocyte differentiation is critical for glomerular filtration barrier function, which is regulated by WT1, a zinc finger transcription factor. We identified a novel protein called the WT1 interacting protein (WTIP) and propose that WTIP normally monitors slit diaphragm assembly and shuttles into the nucleus after podocyte injury. To investigate nucleocytoplasmic translocation, podocytes that stably express an inducible, epitope‐tagged WTIP were treated with LPS, which causes transient nephrotic syndrome in mice. WTIP rapidly disassembles from cell‐contacts, transits along cytoskeletal‐based structures, localizes in podocyte nuclei by 6 hrs but shifts back to plasma membrane after 24 hrs. LPS stimulates podocyte JNK activity in a time‐dependent manner, and a JNK, but not p38 inhibitor, prevents translocation of WTIP to nuclei. Intact actin cytoskeleton and microtubular networks, as well as activity of the motor protein, dynein, are required for LPS‐stimulated WTIP translocation. We propose WTIP translocation to nucleus requires an intricate orchestration of JNK activation, which releases WTIP from podocyte cell contacts and promotes assembly of a dynein‐based complex that transports WTIP to the nucleus for import. Nuclear WTIP may reprogram WT1‐dependent gene expression to facilitate a podocyte phenotype switch that promotes cell survival until an injury is terminated.
Physiopathological characterization of the venom of the freshwater stingray Potamotrygon sp C
Authors: dos Santos Juliane Monteiro , Santos Janaina C. , Lopes Monica F. , Marques Elineide E. , Seibert Carla S.
Published: June 2021

Freshwater stingrays are benthic animals that almost always behave passively and preferentially inhabit shallow water. Their tail barb is rigid and serrated, and is covered by glandular epithelium. Injury from the barb is followed by local symptoms such as bleeding, intense throbbing pain, and edema in the affected member. Nausea, salivation, convulsion, and tachycardia are reported systemic changes. The Potamotrygon sp. C is stingray species most often encountered in rivers of the state of Tocantins, Brazil.

Objective

To evaluate the physiopathological changes induced by poisoning caused by Potamotrygon sp. C.

Methods

Mucus was removed from the epithelial tissue at the barb and from the disc of the animal (far from the barb), and an extract was prepared from the glandular epithelium that covers the barb and analized. Different concentrations of this material (25, 50, 100 μg/ml) were used to evaluate the edematogenic, nociceptive, hemorrhagic, and necrotic effects in male and female Swiss mice.

Results

The nociceptive and edematogenic activity was dose‐dependent, with the most intense response observed for the mucus collected from the disc. No hemorrhagy was observed, and necrosis was limited to small dots on the tissue evaluated.

Conclusion

It was evident that the greatest changes caused by poisoning with Potamotrygon sp. C are related to contact of the barb‐injured tissue with the mucus that covers the animal.
Exploration of gene changes in circadian rhythm in zebrafish eyes using microarray technology
Authors: Sharma Shobhit , Monoz Jesus , Garcia Dana M
Published: June 2021

The objective of this research is to determine changes in gene expression levels between day and night in zebrafish eye using microarray technology. Sampling was performed at midday in light adapted fish and midnight in dark adapted fish with the midday sample used as a control. Preliminary results using duplicate samples reveal 256 genes which are differentially regulated under these two conditions (p value <0.05). Among these 256 genes, 62 showed a 2‐fold or greater change expression. We selected 5 genes for further analysis. Among these genes we found that expression zgc:66156 (encoding a motor protein) in the dark was 50% more than its expression level in the light, and expression of zgc:92682 was up‐regulated 2‐fold in the dark. In contrast, expression of the phosphodiesterase zgc:73359 was decreased about 50%, phosphodiesterase zgc:73310 was decreased more than 50% in dark and erbb2 was decreased 50% in the dark. Results will be further confirmed by quantitative PCR and fluorescent in situ hybridization.
The ontogeny of skeletal muscle adaptations that enable long deep dives in Weddell seals
Authors: Kanatous Shane B , Hawke Thomas J , Trumble Stephen J , Pearson Linnea E , Davis Randall W
Published: June 2021

Our objective was to determine the ontogenetic changes in the skeletal muscles of Weddell seals that transform a non‐diving pup into an elite diving adult. Muscle biopsies were collected from pups, juveniles and adults and analyzed for changes in fiber type, mitochondrial density, myoglobin concentrations, and aerobic, lipolytic and anaerobic enzyme activities. The fiber type results showed a decrease in type I fibers with a significant increase in type IIA fibers as the animals mature. In addition, the volume density of mitochondria and the activity of lipolytic enzymes significantly decreased as the seals matured. To our knowledge, this is the first quantitative account describing a decrease in aerobic fibers shifting towards an increase in fast‐twitch oxidative fibers with a significant decrease in mitochondrial density as the animals mature. These differences in muscle physiology are due to their three stages of life history. During the first few weeks of life, pups are a non‐diving terrestrial mammal that must rely on natal fur for thermoregulation in the harsh conditions of Antarctica. The increased aerobic capacity of pups, associated with increased mitochondrial volumes, acts to provide additional thermogenesis. As these future elite divers mature, their skeletal muscles transform to a more sedentary state in order to maintain the low levels of aerobic metabolism associated with long duration diving.
Development of a sleeve gastrectomy weight‐loss surgical model in obese Zucker rats
Authors: Lopez Peter P , Nicholson Susannah E , Burkhardt Gabriel E , Johnson Robert A , Johnson Fruzsina K
Published: June 2021

Obesity promotes the development of diabetes and cardiovascular disease. The most effective weight‐loss treatment is bariatric surgery, but results greatly vary by procedure. Recently, sleeve gastrectomy (SG) has emerged as a safe weight‐loss procedure for super obese patients. However, the mechanism of weight‐loss by SG and its effects on obesity‐induced complications are yet to be determined. Our goal was to develop an experimental model of SG in genetically obese rats. Male obese Zucker rats (400–500g, leptin‐insensitive) were anesthetized with isoflurane. After a midline laparotomy, the stomach was clamped, the greater curvature was excised and a triple suture line was used to close the gastric remnant. Sham rats underwent laparotomy only. Metabolic parameters were followed for 14 days after surgery. Caloric intake (SG: 136.0±4.4 to 29.2±8.2 vs sham: 134.5±4.9 to 102.5±2.6kcal/day) and body weight (Δmax SG: −88±14 vs sham: +63±5g) decreased in SG rats over 14 days. Blood total and LDL cholesterol levels were lower in rats that lost weight (total SG: 120±10 vs sham: 214±23mg/dL) (LDL SG: 67±9 vs sham: 169±17mg/dL). Furthermore, blood glucose (SG: 145±20 vs sham: 246±23mg/dL) and insulin (SG: 4.1±0.4 vs sham: 5.8±1.0ng/mL) levels were lower in rats that lost weight. These results show that SG promotes weight‐loss in obese Zucker rats. Furthermore, SG‐induced weight‐loss is accompanied by improved plasma cholesterol and glucose/insulin profile. These results suggest that SG is an effective weight‐loss procedure in leptin‐insensitivity to improve the lipid profile and decrease insulin resistance.

Supported by NIH grant HL64577 (RAJ) and in‐house funds (PPL).
Role of PAR2 in Type 2 Diabetes‐induced Endothelial Dysfunction
Authors: Park Yoonjung , Klaahsen Darcey , Zhang Cuihua
Published: June 2021

Previously we have found a critical role of tumor necrosis factor‐α (TNF) in endothelial dysfunction resulting from type 2 diabetes. Because TNF can influence many pathways of inducing oxidative stress and cell injury, our goal was to identify one of the contributing mechanisms. Protease‐activated receptors (PARs), particularly endothelial PAR2 are thought to play a role in inflammatory cardiovasculardisease. We investigated the role of PAR2 in regulating TNF‐induced endothelial dysfunction in Type 2 diabetes. Coronary arterioles of WT (normal) and db/db (diabetic) mice (12–16 weeks) were isolated and pressurized (60 cmH2O). Although vasodilation to endothelium‐independent vasodilator sodium nitroprusside was not different among WT, db/db and dbTNF‐/dbTNF‐ (db/db null for TNF) mice, dilation to endothelium‐dependent agonist ACh was impaired in db/db mice but was greater in dbTNF‐/dbTNF‐compared to db/db (P<0.05). Administration of PAR2 antagonist (FSLLRY‐NH2) partially enhanced ACh‐induced vasodilation in db/db mice. Flow‐induced vasodilation showed similar response to ACh‐induced vasodilation. Both mRNA and protein expression of PAR2 were higher in db/db heart compared to WT heart whereas mRNA and protein expression were reduced in dbTNF‐/dbTNF‐ vs db/db. These results indicate that PAR2 plays a pivotal role in type 2 diabetes‐induced endothelial dysfunction through TNF mechanism.
Metabolic Consequences of Diets with Varying Levels of Omega‐3 and Omega‐6 Fatty Acids in Zucker Rats
Authors: Ajuwon Kolapo Matthew , Franklin Dru M , Banz William J , Winters Todd A
Published: June 2021

Typical western diets are higher in Omega‐6 (n‐6) than Omega‐3 (n‐3) fatty acids. This may be linked to the development of metabolic complications, e.g., diabetes and cardiovascular diseases. We evaluated the metabolic impact of varying the n‐3 and n‐6 fatty acid concentrations in the diets of male obese Zucker rats fed 5 different diets: Low fat (7 % fat) control diet (C); 4 high fat diets ‐ a saturated fat (25 % beef tallow, diet T), a 20% soy oil (50 % omega‐6 linoleic acid, diet S), a 20% fish oil (50 % omega‐3 fatty acids from eicosapentaenoic acid, EPA and docosahexaenoic acid, DHA, diet F) and a mixed diet (M) with 10% fish oil and 10 % soybean oil. High fat diets consist of 60 % fat calories relative to control diet (16 % fat calories). A total of eight animals were assigned to each treatment and animals were fed ad libitum for 8 weeks. We conducted oral glucose tolerance test (OGTT) and measured serum metabolites in the animals. Animals on the S diet had a higher (P < 0.05) area under the curve (AUC) for the OGTT relative to other groups, indicating delayed clearance of glucose. The animals on the S diet also had a higher total blood cholesterol, free fatty acid and insulin concentrations compared to other groups (P< 0.05). Adiposity, measured as percent visceral and epididymal fat pads, was significantly higher in the S group (P< 0.05). These animals also had increased basal phosphorylation level of AKT, perhaps as a result of the higher circulating insulin concentration. Animals on the fish oil diet had the lowest adiposity and circulating concentration of free fatty acids and total cholesterol. The 1:1 mixture of soybean oil and fish oil in diet M resulted in metabolic performances that were similar to the control diet. Thus, despite the high lipid intake of the M animals, the 1:1 balance of the n‐3 and n‐6 fatty acids offsets the negative effects of the high fat intake.