bluuebunny/crossref_metadata_2025 huggingface dataset:
Maternal exercise during pregnancy reduces risk of mammary tumorigenesis in rat offspring (886.1)
Authors: Zheng Wei , Camarillo Ignacio , Clah Leon , Zhou Xuanzhu , Larrick Brienna , Blaize Nicole , Breslin Emily , Donkin Shawn , Gavin Timothy , Newcomer Sean , Teegarden Dorothy
Published: June 2021

Breast cancer is the most common cancer among women. Emerging research indicates that modifying lifestyle factors during pregnancy may convey long‐term health benefits to offspring. This study was designed to determine whether maternal exercise during pregnancy leads to reduced mammary tumorigenesis in female offspring. Pregnant rats were randomly assigned to exercised and sedentary groups, with the exercised group having free access to a running wheel and the sedentary group housed with a locked wheel during pregnancy. Female pups from exercised or sedentary dams were weaned at 21 days of age and fed a high fat diet without access to a running wheel. At 6 weeks, all pups were injected with the carcinogen N‐methyl‐N‐nitrosourea (MNU). Mammary tumor development in all pups was monitored for 15 weeks. Pups from exercised dams had a substantially lower tumor incidence (42.9%) compared to pups from sedentary dams (100%). Of the pups with tumors, the pups from exercised dams had a higher tumor frequency (1.7±0.4 tumors/rat) than pups from sedentary dams (1.2±0.2 tumors/rat). Neither tumor latency nor histological grade differed between the two groups. These data are the first to demonstrate that exercise during pregnancy potentiates reduced tumorigenesis in offspring. This study provides an important foundation towards developing more effective modes of behavior modification for cancer prevention.

Grant Funding Source: Supported by NIH R25CA128770 and ICTSI NIH/NCRR TR000006
Influence of exercise in pregnancy on fetal heart response during labor and delivery (886.5)
Authors: Barbari Nichelle , May Linda , Newton Edward , Livingston Jeffrey
Published: June 2021

Current research supports the health benefits of exercise during pregnancy for mother and baby. However, scant research has focused on how maternal exercise during pregnancy influences fetal heart response during labor and delivery. The purpose of this study was to compare the effects of exercise throughout pregnancy on fetal heart response to the stress of labor and delivery. We hypothesized that during labor and delivery there would be lower baseline fetal heart rate (HR), increased fetal heart rate variability (HRV), and no differences in fetal accelerations and decelerations in fetuses exposed to maternal exercise relative to controls. The Modifiable Physical Activity questionnaire was used to classify controls and exercisers. Based on routine fetal HR recordings, we measured fetal baseline HR, HRV, accelerations, and decelerations and birth measures. Group differences were tested using t‐tests. There were no differences found between the mother’s age, gestational age, gravida and parity. No statistical difference was found in average fetal HR and HRV; however, there is a trend towards increased variability among the exercise group. These data suggest babies exposed to maternal exercise respond the same, or better, during the physiologic stress of labor and delivery. These findings are applicable in regards to the potential to improve pregnancy outcomes in the US.
Do the exercise habits of pregnant women conform to evidence‐based recommendations? (886.7)
Authors: McKenzie Amy , Ellis Lindsay , Fuller Kelly , Guelinckx Isabelle , Perrier Erica , Armstrong Lawrence
Published: June 2021

Professional organizations recognize the health benefits of exercise during pregnancy and recommend pregnant women exercise 3‐7 days·wk‐1 for at least 30 min·day‐1 in the absence of contraindications. Therefore, the purpose of this retrospective analysis was to determine actual exercise habits of women during and following gestation. METHODS: 20 healthy pregnant women (mean±SD; 31±3 y; 165.5±6.5 cm; 68.25±18.63 kg pre‐pregnancy; 12.85±4.48 kg pregnancy weight gain) recorded exercise habits (mode, days·wk‐1, min·day‐1) before each of 6 visits (weeks 10, 13, 26, 37 gestation; 2, 8 postpartum). These data were compared to ACOG and ACSM guidelines. RESULTS: During pregnancy, 8/20 subjects intentionally exercised: 2/8 maintained exercise volume and met the 30 min·day‐1 recommendation, 6/8 voluntarily reduced exercise volume as pregnancy progressed. By 8 weeks postpartum, 10/18 women intentionally exercised, yet only 2/10 met the daily recommendation. CONCLUSIONS: These results demonstrate that a majority of subjects did not exercise during pregnancy (60%) or exercised at a volume below (30%) evidence‐based recommendations, consistent with previous research. As this pattern continues to be observed in pregnancy, future research should identify barriers to physical activity (e.g., exercise history, weight carriage, unsubstantiated concerns for baby’s health) and methods to enhance exercise adherence.

Grant Funding Source: Supported by Danone Research
Hypoxia‐induced angiogenesis in the conditional HIF‐1α deficient and HIF‐2α deficient mice (887.3)
Authors: Xu Kui , Sun Xioayan , Tsipis Constantinos , LaManna Joseph
Published: June 2021

Hypoxia‐inducible factors (HIFs) are transcriptional regulators that mediate hypoxic adaptation. We investigated the neuronal or glial cell‐specific role of HIF‐1α or HIF‐2α in the angiogenic adaptive response to chronic hypoxia by using the specific conditional knockout (KO) mice for HIF‐1α or HIF‐2α. The wild‐type (WT), conditional HIF‐1 KO (neuronal and glial) and conditional HIF‐2 KO mice (neuronal and glial) mice were exposed to the equivalent of 8% oxygen for 3 weeks in a hypobaric chamber at a pressure of 300 torr (0.4 ATM). Littermates of each type were kept normoxic in the same location. At the end of 3 weeks mice were perfusion‐fixed. Cerebral capillary density was identified from brain sections by GLUT‐1 immunohistochemistry staining and was quantified from the positive counts per unit area (number/mm2) in cortex. The normoxic baseline of capillary density was similar in the WT, neuronal HIF‐1 KO and the glial HIF‐2 KO mice (428 ± 66, 452 ± 54 and 440 ± 45, respectively, mean ±SD); the glial HIF‐1 KO and the neuronal HIF‐2 KO groups had higher normoxic baselines (483 ± 25 and 498 ± 55, respectively) than the WT mice. As expected, the capillary density was significantly increased by about 23% in the WT mice after 3 weeks of hypoxia, compared to that of their normoxic littermates. The capillary density was trended higher (about 10 to 15%) following hypoxia in the neuronal HIF‐1 KO, glial HIF‐1 KO and glial HIF‐2 KO groups; however, the capillary density remained unchanged in the neuronal HIF‐2 KO group. In addition, the hematocrit was similar in all groups before and after hypoxia. Or data suggest that HIF‐1 and HIF‐2 may regulate hypoxia‐induced angiogenesis differentially and cell‐specifically, and the neuronal HIF‐2 is required for hypoxic angiogenesis.

Grant Funding Source: Supported by NIH grant NS 38362
Ketamine decreases immune gene expression in ovine fetal frontal cortex exposed to acute hypoxic hypoxia (887.5)
Authors: Chang Eileen , Rabaglino Maria , Richards Elaine , Wood Charles
Published: June 2021

Fetal hypoxic hypoxia (HH), a consequence of maternal hypoxia, stimulates glutamatergic neurotransmission. We hypothesize that excitotoxicity induced by HH can be reduced by pretreatment with ketamine (KET), an NMDA receptor antagonist. The objective was to evaluate the genomic response of fetal frontal cortex under HH with KET. KET (3 mg/kg) was administered intravenously to chronically catheterized ovine fetuses (n=3‐5/group; 123±3 d gestation) 10 min prior to 30 min HH. Fetal frontal cortex was collected 24 hrs post‐HH. mRNA was extracted, labeled, and hybridized to ovine Agilent 15.5k microarray. Significant differences in gene expression between control±KET and HH±KET groups were determined by F‐statistics using the limma package for R (P<0.05). Differentially up‐/down‐regulated genes were analyzed by gene ontology and network analysis, identifying statistically significant overrepresentation of biological processes (P<0.05). Significantly up‐/down‐regulated genes: HH: 244/470 and HH+KET: 556/246. There were 120 common genes up‐regulated by HH and down‐regulated by HH+KET. The enriched biological processes for the overlap genes were involved in regulation of immune system processes (TLR2, MYD88, NFKBIA, IL8, IL6R, IL6ST, CD5, CD14, ICAM1, CCL2, CCL4L1). In conclusion, acute HH increased genomic expression of immune pathways in fetal frontal cortex, and ketamine reduced the response suggesting potential therapy for attenuating neuronal hypoxic damage in fetuses and neonates.
T‐type calcium channels in carotid body oxygen sensing (889.1)
Authors: Makarenko Vladislav , Peng Ying‐Jie , Piao Lin , Nanduri Jayasri , Prabhakar Nanduri
Published: June 2021

Previous studies showed that low voltage gated T‐type Ca2+ channels play an important role in hypoxia‐evoked catecholamine secretion from adrenal chromaffin cells. The objective of the present study was to investigate whether T‐type Ca2+ channels contribute to hypoxic sensing by the carotid body, a key sensory organ for detecting arterial blood O2 levels. Real time quantitative PCR analysis of rat carotid body revealed relatively high levels of α1H as compared to α1G and α1I subunits of T‐type Ca2+ channels. Immunocytochemical analysis showed much of the α1H protein in glomus cells. Mibefradil (1µM), a T‐type Ca2+channel blocker significantly attenuated the carotid body sensory and glomus cell secretory and [Ca2+]i responses to hypoxia. Similar attenuation of glomus cell secretory as well as [Ca2+]i responses were also seen in α1H knockout mice. These results demonstrate that T‐type Ca2+ channels contribute to carotid body and glomus cells response to hypoxia.

Grant Funding Source: Supported by NIH‐HL‐90554
EPR oximetry as a method for repetitive measurements of in vivo kidney tissue oxygenation (890.2)
Authors: Franzén Stephanie , Pihl Liselotte , Khan Nadeem , Gustafsson Håkan , Palm Fredrik
Published: June 2021

Kidney tissue oxygenation is heterogeneous with oxygen levels normally being greater in the superficial cortex and lowest in the inner medulla. Decreased oxygenation in the kidney has been implied in the pathology of several diseases. However, there is currently no method available to repetitively monitor kidney oxygenation regionally using minimally invasive procedures. Therefore, we evaluated implantable lithium phthalocyanine (LiPc) probes, which closely correlate between EPR peak‐to‐peak line width and oxygen availability. LiPc probes were implanted in the cortex (left kidney) and medulla (right kidney) in the same mouse and EPR spectra were acquired using an L band scanner (1 GHz). Mice were measured repetitively over 45 days and during inhalation of air (21% oxygen) or an acute measurement of air and nitrogen mixture (10% oxygen). A 1 G/cm gradient was applied in order to separate the signals from the two probes. The signals were derived from 40 consecutive sweeps and a peak‐to‐peak line width comparison of the EPR spectra was used to convert the signal to an applicable oxygen tension in MATLAB. Cortical and medullary oxygenation was stable over the 45 day period (56±7 mmHg and 43±6 mmHg respectively). However, acute 10% oxygen inhalation significantly reduced oxygenation in both tissues (cortex 56±6 to 34±2 mmHg n=15 p<0.05 and medulla 42±5 to 29±3 mmHg n=7 p<0.05). In conclusion, EPR oximetry using LiPc probes implanted in discrete kidney structures can be used to repetitively measure regional tissue oxygenation. This minimally invasive method is particularly well suited for conditions of reduced tissue oxygenation since this increases the signal intensity which enables the quantification of the EPR signal to absolute oxygenation values.
Telemetry‐based recording of renal cortical oxygenation in response to angiotensin II and phenylephrine in the conscious rat (890.3)
Authors: Koeners Maarten , Emans Tonja , Ow Connie , Evans Roger , Joles Jaap , Malpas Simon
Published: June 2021

Previously we have developed unique technology which allows wireless measurement of the partial pressure of oxygen (PO2) in the renal medulla in unrestrained rats. The aim of our current study was to determine whether this modality can reliably measure tissue PO2 in the cortex and its responses to angiotensin‐II (AngII) and phenylephrine (PE). Cortical PO2 values were stable over time (3 weeks) and comparable between animals (Wistar, n = 8). Responses to systemic hypoxia and hyperoxia were reproducible over this time period, and were similar to those previously observed in the renal medulla. Injection of AngII in conscious rats via an implanted catheter in the femoral vein decreased cortical PO2 dose‐dependently. At a dose of 500 ng/kg a maximum decrease of 45 ± 10% was observed. In contrast equi‐pressor doses of PE had no or little effect on cortical PO2. A maximum decrease in PO2 of 8 ± 5% was observed at 1500 ng/kg. There was little evidence of fibrosis or scarring after three weeks of electrode implantation. This technology provides the opportunity to examine the temporal relationships between renal cortical tissue hypoxia and the progression of renal disease.

Grant Funding Source: EU, FP7, Marie Curie Actions, International Outgoing Fellowship
Decreased kidney oxygenation due to mitochondrial uncoupling: a mechanism to injury after renal transplantation (890.7)
Authors: Friederich‐Persson Malou , Papazova Diana , Joles Jaap , Verhaar Marianne
Published: June 2021

*Equal contribution The mechanisms to reduced graft function after renal transplantation are poorly understood. Mitochondrial uncoupling via uncoupling proteins (UCP) increases oxygen consumption and can contribute to the decreased kidney oxygen tension that is regarded as part of a common pathway to nephropathy. The aim was to investigate mitochondrial uncoupling, graft oxygenation and function after renal transplantation.

Male Lewis rats were left untreated as controls (CON, N=6) or underwent syngenic renal transplantation (Tx, N=6). Tx was performed with end‐to‐end anastomoses of vessels and urether, with 30 min and 30‐40 min cold and warm ischemia time respectively and removal of right kidney. Two weeks after Tx, we measured renal function as glomerular filtration rate (GFR: inulin clearance), renal blood flow (RBF: PAH clearance/1‐hct) and kidney oxygen tension (pO2). Mitochondria uncoupling was evaluated in a separate cohort (6/group).

Tx resulted in mitochondrial uncoupling evident as a larger decrease in oxygen consumption after UCP blockade (‐15.8±2.1 vs. ‐1.9±0.8 pmol O2/s p<0.05). Furthermore, Tx resulted in increased GFR (2.4±0.2 vs. 1.8±0.4 ml/min/kidney, P<0.05), RBF (14.4±1.4 vs. 11.4 ± 1.7 ml/min/kidney p<0.01), proteinuria (9±1 vs. 4±0 mg/day p<0.05) and decreased pO2 (. 30.3±12.6 vs. 58.8±15.3 µM p<0.01) but did not affect mean arterial pressure (98±11 vs. 99±5 mmHg ns).

The present study demonstrates that mitochondrial uncoupling occurs early after Tx and is associated with decreased kidney oxygen tension despite hyperperfusion. This hypoxia may contribute to progressive damage in transplanted kidneys.
Calcium sensing receptor modulates vacuolar H+‐ATPase activity in a cell model of proximal tubule (OKP cells) (891.1)
Authors: Fernandez Ricardo , Dos Santos Priscilla
Published: June 2021

The aim of the present study was to analyze the effects of CaSR stimulation on the activity of the vacuolar H+‐ATPase in a cellular model of proximal tubule, OKP cells. H+‐ATPase activity was accessed by a biochemical method. Intracellular pH (pHi) was monitored with BCECF and changes in [Ca2+]i was determined using Fluo‐4 AM. A significant increase of H+‐ATPase activity was observed when the CaSR was stimulated with agonists such as 300μM Gd3+ and 200μM neomycin. This activity was also stimulated in a dose‐dependent fashion when Ca2+ concentration was increased between 10‐4mM and 2mM. Neomycin induced a significant alkalinization of pHi (∆pHi 0.57±0.04) that was significantly reduced in the presence of 10‐8M concanamycin. Gd3+ and neomycin produced a sustained rise of cytosolic ionized calcium (4.77±0.47 and 12.79±0.86%, respectively, p<0.0001 vs basal), an effect that disappeared when extracellular calcium was removed in the presence of 0.1μM thapsigargin. Inhibition of phospholipase C activity with U73122 (5x10‐8M) reduced the increase in [Ca2+]i induced by neomycin. These data allow us to conclude that CaSR stimulation of OKP cells induces an increase in vacuolar H+‐ATPase activity. This effect involves an increase in intracellular calcium, resulting from the combination of Ca2+ release from intracellular stores and the influx of Ca2+ from the extracellular medium, an effect that requires PLC activity.

Grant Funding Source: Supported by CAPES (Brazil)
Acute effects of angiotensin II on calcium influx in the podocytes of the freshly isolated glomeruli of the wild type and TRPC6‐deficient mice (892.1)
Authors: Ilatovskaya Daria , Palygin Oleg , Ma Rong , Birnbaumer Lutz , Staruschenko Alexander
Published: June 2021

Podocytes play a key role in regulating glomerular permeability and maintaining glomerular structure. It was previously reported that angiotensin (Ang II) enhances albuminuria and increases calcium flux in podocytes by activating TRPC6 channels. The goal of this study was to examine acute effects of Ang II on intracellular calcium levels and endogenous ion channels activity in their native setting, podocytes of freshly isolated mouse glomeruli. Single channel patch‐clamp experiments demonstrated that an ion channel with distinct TRPC6 properties (22 pS) was present in the wild type animals, however it was not identified in TRPC6 knockout mice. Ang II acutely activated these native channels in the podocytes of wild type, but not TRPC6 deficient mice; the effect was mediated by changes in the channel open probability. Data obtained in CHO cells transiently expressed with TRPC6 and AT1R confirmed these findings. We further measured alterations in intracellular calcium stimulated by Ang II in the presence and absence of the TRPC inhibitors gadolinium chloride and SKF 96365, and TRPC6 activator flufenamic acid; these experiments were performed in Fluo4/FuraRed loaded podocytes of the intact glomeruli of the wild type and TRPC6‐deficient mice. Our data have provided the first direct evidence of Ang II‐dependent activation of TRPC6 channels and calcium influx in podocytes, which might play a significant role in the pathogenesis of glomerular diseases.

Grant Funding Source: Supported by NIH HL108880, DK079968, Z01‐ES‐101684, ADA 1‐10‐BS‐168, ASN Ben J Lipps Fellowship
Low dietary potassium intake stimulates NCC to cause sodium retention (892.16)
Authors: Terker Andrew , Yang Chao‐Ling , Ellison David
Published: June 2021

Low dietary K+ stimulates the renal sodium chloride cotransporter (NCC), whereas high salt intake inhibits it. As most people in the world consume a diet that is both low in K+ and high in salt, we investigated how NCC responds to conflicting Na+ and K+ signals. High salt (HS)/ low K+ (LK) intake increased NCC and pNCC compared with HS/normal K+ (NK) and redistributed SPAK, OSR1, and WNK4 to cytoplasmic punctae. These effects were preserved in both SPAK‐/‐ and AT1aR‐/‐ mice. A HS/LK diet also decreased urinary Na+, compared with a HS/NK diet. This anti‐natriuretic effect, however was absent in NCC‐/‐ mice. As mineralocorticoids have been reported to stimulate NCC, we confirmed that fludrocortisone (F) increases NCC and phosphorylated NCC (pNCC) in mice. A high K+ diet, which normalized plasma K+, prevented the effects of F on pNCC. F also caused substantially more hypokalemia in NCC‐/‐ mice, than in wild type mice, suggesting that NCC is essential to preserve plasma K+. Amiloride, which caused hyperkalemia and volume contraction, decreased NCC and pNCC, suggesting K+ was determining NCC status. Mice treated with both F and A had plasma K+ and both NCC and pNCC levels equal to A alone. In HEK293 cells, low K+ medium increased pNCC and pSPAK/pOSR1, an effect that was dependent on WNK kinases as shown through siRNA studies. Transfection with mutant KCNJ10 channels that depolarize cells, including multiple EAST sydrome mutants, reduced pNCC compared with wild type KCNJ10. Treatment with barium also reduced pNCC. The results suggest that low plasma K+ stimulates NCC directly, independent of any intermediating signaling hormones; this effect likely requires WNK‐SPAK/OSR1. Our data suggest that the DCT responds to plasma K+ and predominantly modulates renal K+ excretion.

Grant Funding Source: Supported by NIDDK, the Department of Veterans Affairs, and the American Heart Association
TRPV4 function in the distal nephron is regulated by dietary potassium and sodium intake (892.2)
Authors: Boukelmoune Nabila , Mamenko Mykola , Zaika Oleg , O'Neil Roger , Pochynyuk Oleh
Published: June 2021

It becomes recognized that Ca2+‐permeable TRPV4 channel is predominantly expressed in the distal nephron and its activity is essential for [Ca2+]i elevations in response to increased tubular flow. Here, we probe whether TRPV4 function in the kidney is regulated by dietary intake of major electrolytes. We found that renal TRPV4 expression was increased by high potassium diet (5% K+) and decreased by dietary potassium restriction (0.003 % K+). Saturation of systemic mineralocorticoid status with DOCA had little effect on TRPV4 protein abundance pointing to an aldosterone‐independent nature of this regulation. Increased TRPV4 expression by high potassium intake was associated with significantly augmented [Ca2+]i responses to elevated flow as was detected using Fura‐2 Ca2+ imaging in freshly isolated split‐opened distal nephrons. Furthermore, dietary sodium load (2% Na+) similarly increased renal TRPV4 expression and enhanced flow‐induced [Ca2+]i responses in distal nephron cells. Interestingly, protein levels of other Ca2+‐permeable channel, TRPC3, also expressed in the distal nephron, were not altered by variations in dietary potassium and sodium intake. We conclude that increased ultrafiltrate delivery to the distal nephron as a result of high potassium and sodium intake elicits respective augmentation of TRPV4 expression and therefore enhanced cellular responses to distal tubular flow.

Grant Funding Source: DK095029
Shear stress and cellular stretch induces intracellular calcium increases in the thick ascending limb: role of transient receptor potential vanilloid 4 (892.3)
Authors: Cabral Pablo , Garvin Jeffrey
Published: June 2021

The thick ascending limb (TAL) reabsorbs 30 % of the filtered NaCl. Thus it plays a pivotal role in the regulation of salt homeostasis. Luminal flow enhances both shear stress and cellular stretch. The mechanosensitive Transient Receptor Potential Vanilloid 4 (TRPV4) channel mediates increases in intracellular calcium (Cai) in response to shear stress and cellular stretch in many cells. However, it is not known whether shear stress and/or cellular stretch mediate flow‐induced Cai increases in TALs. We hypothesized that shear stress and cellular stretch mediate flow‐induced Cai increases in TALs. We measured Cai using the ratiometric Ca‐sensitive dye Fura‐2 in isolated and perfused rat TALs. Increasing luminal flow increased Cai from 54 ± 7 to 410 ± 116 nM, n=6; p<0.03. The TRPV4 antagonist RN 1734 (50 uM) blocked this effect (from 29 ± 7 to 80 ± 11 nM, n=5). To assess the contributions of shear stress and stretch we measured Cai at a location close to the perfusion pipet where shear stress and stretch would be high and at a distal site at the end of the tubule where shear stress would be the same as the proximal site but stretch would be greatly reduced. Luminal flow increased Ca by 0.75 ± 0.11 ratio units (RU) in the proximal region. In the distal region, flow increased Ca by 0.45 ± 0.10 RU (n=5), 40% less than in the proximal region (p<0.002). The general TRPV4 antagonist ruthenium red blocked the increase in both regions. We conclude that: 1) flow increases intracellular Ca via TRPV4 in TALs; and 2) shear stress mediates most of the effect but cellular stretch also likely contributes.

Grant Funding Source: Supported by NHLBI of NIH to J.G (HL 070985; HL 090550; HL 028982). AHA to P.C (13POST16280001)
Potassium channels in cancer cells: a tool to reprogram immortality (893.14)
Authors: Gentile Saverio
Published: June 2021

K+ channels are traditionally known for controlling a variety of cell functions including electrical excitability, secretion and contraction. However, recent investigations have revealed that several K+ channels can be expressed in cancer cells of different histogenesis, suggesting that K+ channels can play a fundamental role in proliferation and in cancer biology. Yet, very little is known about function of K+ channels in cancer.

Our studies revealed for the first time that stimulation of K+ channels (e.g. Kv11 or Kv7) activity with selective agonists determined an irreversible inhibition of cell proliferation in different cancer cells by arresting the cell cycle in G0/G1‐phase. We have characterized a set of biochemical pathways linking K+ channels to inhibition of tumor markers (cyclin E2, AKT) or stimulation of tumor suppressor (p21; p16) activities. We concluded that stimulation of K+ channel activity can lead to activation of a cellular senescence program in cancer cells.

Cellular senescence is widely recognized as a potent antineoplastic mechanism however, very little is known about the molecular cascade controlling this phenomenon. In addition, the role of ion channels in determining cellular senescence has never been investigated. Therefore, our finding provides novel evidence for K+ channel functions that stretch beyond their traditional roles. In addition, our data offer the prospect to design an antiproliferative strategy for cancer treatment by using K+ channels agonists
Differential mechanisms of ammonia transport in the liver and skeletal muscle (893.30)
Authors: Worrell Roger , Dasarathy Srinivasan
Published: June 2021

Hyperammonemia is a consistent abnormality in cirrhosis due to impaired ureagenesis and contributes to both encephalopathy and sarcopenia, or loss of skeletal muscle mass. Recent data suggests that the skeletal muscle functions as a metabolic partner for the liver in ammonia disposal. We determined the mechanism of ammonia transport in the skeletal muscle using biopsy samples from cirrhotic patients and C2C12 murine myotubes exposed to 10mM ammonium acetate. Intracellular pH measurement was done using pH‐sensitive dye, 2',7'‐biscarboxyl‐ethyl‐5(6)‐carboxyfluorescein (BCECF). In response to ammonium acetate as well as ammonium chloride, there was a rapid ammonia influx into the myotubes. Quantifying the Rh group of ammonia transporters showed that human skeletal muscle had very low level mRNA expression for RhB. C2C12 myotubes expressed RhBG and RhCG mRNA. Interestingly, the expression of RhBG and RhCG mRNA in C2C12 myotubes was significantly lower during hyperammonemia than in control cells. Human liver tissue robustly expressed only RhBG mRNA with no detectable RhCG mRNA. Alternative ammonia transporters in the skeletal muscle including NKCC1 and K+/H+‐ATPase likely play a role. These data suggest that ammonia transport in the liver is mediated differently from that in the skeletal muscle. These data are of high clinical significance since under normal conditions, the liver is the major organ for ammonia transport while in cirrhosis, skeletal muscle uptake of ammonia is significantly increased. Understanding the regulation of ammonia transport has the potential to develop mechanistic and targeted therapies for hyperammonemia.

Grant Funding Source: Supported by NIH DK 083414 to SD and NIH DK079979 to RTW
Activation of renal NHE3 by angiotensin II requires NHERF1 (893.33)
Authors: He Pejiian , Yun Chris
Published: June 2021

Na+/H+ exchanger (NHE3) plays a pivotal role in the reabsorption of Na+ and HCO3‐ in the kidney. The NHE3 activity is tightly regulated by hormones and growth factors to maintain fluid and blood pressure homeostasis. We have previously shown that NHE3 activity and trafficking is stimulated by low dose angiotensin II (Ang II) dependent on IRBIT and CaMKII. However, it remains unclear how IRBIT mediates the exocytosis of NHE3 protein. In this study, we found that IRBIT binds to NHERF1 in OKP cells, which was enhanced by Ang II. Overexpression of IRBIT or Ang II treatment increased NHERF1 and NHE3 binding, whereas knockdown of IRBIT attenuated the interaction. Knockdown of NHERF1 expression by lentiviral shRNA completely blocked the stimulatory effect of Ang II on NHE3 exocytosis and transport activity. Moreover, inhibition of CaMKII by KN‐93 abolished the Ang II‐induced NHERF1‐NHE3 interaction and membrane expression of NHE3, IRBIT and NHERF1. In conclusion, activation of NHE3 exocytosis by Ang II is dependent on the formation of a macromolecular complex containing IRBIT, NHERF1 and NHE3.

Grant Funding Source: American Heart Association‐Scientist Development Grant
Role of Na+‐HCO3‐ cotransporter NBCe1 (Slc4a4) in colonic pHi regulation and anion transport (893.38)
Authors: Liu Xuemei , Yu Qin , Riederer Brigitte , Tian De‐An , Tuo Biguang , Aalkjaer Christian , Shull Gary , Seidler Ursula
Published: June 2021

Background: The electrogenic Na+‐HCO3‐ cotransporter NBCe1 (Slc4a4) is strongly expressed in intestinal epithelium. Aim and methods: We used miniaturized Ussing chambers and fluorescent pH‐indicator BCECF‐loaded colonic crypts to study the importance of NBCe1 in pHi control, HCO3‐ as well as anion (Cl‐ and HCO3‐) secretory rates in Slc4a4 KO and WT mice. Results: Steady state pHi and pHi‐recovery was significantly lower in proximal and distal colonic surface/cryptal mouth cells but not in and colonic crypt base cells, which also displayed high expression levels of the electroneutral NBCn1(Slc4a7) (and was significantly lower in slc4a7‐deficient colonic crypt cells). Forskolin‐stimulated HCO3‐ secretory rates were similar between WT and KO mucosa in all intestinal segments. The Cl‐‐dependent luminal alkalinization rate, which is a function of the anion exchanger slc26a3 (DRA) expressed in the colonic surface cells, were strongly decreased after carbonic anhydrase inhibition in the NBCe1‐deficient but not the WT colonic mucosa. Conclusions: In the intestine, the electrogenic NBCe1 is a mechanism to counteract surface enterocyte acid loads that occur with absorptive processes, but is not important for agonist‐induced electrogenic HCO3‐ secretion, possibly because the concomitant K+ channel activation reduces the driving for electrogenic Na+‐HCO3‐ cotransport.

Grant Funding Source: Supported by DFG Se 13‐4 and 9‐6 to Ursula Seidler
Genetic variation in SLC30A2 (ZnT2): associations with mammary cell function and milk zinc concentration in breastfeeding women (893.41)
Authors: Kelleher Shannon , Hennigar Stephen , Soybel David , Alam Samina
Published: June 2021

ZnT2 imports zinc (Zn) into vesicles for secretion from mammary epithelial cells (MEC) into milk. Four mutations in ZnT2 have been identified that abrogate Zn secretion into milk, resulting in severe neonatal Zn deficiency; however, the role of genetic variation in ZnT2 on MEC function and milk Zn level has not been explored. DNA from buccal swabs and milk from healthy, exclusively breastfeeding women (n=54) were obtained at 4 mo postpartum. Milk Zn level was stratified into tertiles (high, >1.7 μg/mL; normal, 1‐1.6 μg/mL; low, <0.9 μg/mL) and the ZnT2 coding region was sequenced. ~50% of women had genetic variation in ZnT2. Four SNPs (rs142587047, rs149340896, rs150251854, rs145406127) and 17 novel non‐synonymous variants were identified and only found in women in the high and low tertiles. 8 select variants were generated to functionally characterize defects in MECs in vitro. Variants responsible for low Zn secretion were confined to the ER/Golgi apparatus and mitochondria and induced apoptosis and G1 arrest. Variants responsible for high Zn secretion were mislocalized to the cell membrane and induced G1 arrest and entry into S and G2 phase. Altered function corresponded to a redistribution of subcellular Zn as assessed by FluoZin3. This suggests that genetic variation in ZnT2 is common and affects ZnT2 function and milk Zn levels. Defects in ZnT2 may compromise infant health and modify a woman’s risk for breast disease.
Interaction of organic anion transporter polypeptides 1B1 and 1B3 with cytostatics (896.7)
Authors: Hagos Yohannes , Marada Venkata , Flörl Saskia , Kühne Annett , Burckhardt Gerhard
Published: June 2021

One of the main functions of liver is the systemic elimination of many exogenous and endogenous substances. In order to perform this function, the initial step is their uptake from blood into the hepatocytes by specific transporter proteins. Many of such proteins constitute the solute carrier (SLC) superfamily. Our work deals with two proteins belonging to the SLC superfamily, the organic anion transporting polypeptides 1B1 and 1B3 which are highly expressed on the basolateral membranes of hepatocytes. We studied these two proteins in stably transfected human embryonic kidney cells using estrone sulfate and cholecystokinin octapeptide 8 (CCK8) as substrates for OATP1B1 and OATP1B3 respectively. Cis‐inhibition experiments were performed to evaluate the interactions of these proteins with cytostatics. Our experiments showed that OATP1B1 interacts strongly with vinblastine and paclitaxel. Ki values of OATP1B1 for vinblastine and paclitaxel were calculated to be 16.87 µM and 0.85 µM respectively. On the other hand, OATP1B3 dependent CCK8 uptake was inhibited in the presence of a variety of cytostatics viz., chlorambucil, bendamustine, doxorubicin, mitoxantrone, vincristine, vinblastine, paclitaxel and etoposide. From our experiments it is clear that these transporters play a crucial role in the uptake of cytostatics into hepatocytes and hence in systemic elimination of these drugs from the body.

Grant Funding Source: Deutsche Forschungsgemeinschaft GRK 1034
Uncontrollable stress causes downregulation of CCL2 in colon of the endometriosis rat model (898.4)
Authors: Bonilla Stephanie , Rivera Jocelyn , Cruz Myrella , Isidro Raymond , Appleyard Caroline , Chompre Gladys
Published: June 2021

Background: Endometriosis is characterized by the growth of endometrial tissue in areas such as the peritoneal cavity and colon causing pain and discomfort. We have previously demonstrated that rats with explants of endometrial tissue in the peritoneal cavity subjected to stress showed a significant increase in the size of endometrial lesions. Chemokine ligand 2 (CCL2) has been found to be increased in plasma in women with endometriosis perhaps contributing to the inflammatory process but the effects of stress on this cytokine are still unclear. Aim: Examine the relationship between CCL2 expression and stress in a rat model of endometriosis. Methods: Endometriosis was surgically induced in female Sprague‐Dawley rats by transplanting uterine tissue onto the intestinal mesentery. Sham rats had sutures only. Animals were exposed to either an uncontrollable (ENDO‐UN) or controllable (ENDO‐C) stress protocol for 10 days, or no stress (ENDO‐NS). At sacrifice (day 60), all rats were examined for presence of endometrial cysts, and colons were collected for mRNA extraction and histology analysis. The relative expression of CCL2 was measured by PCR, and macrophage numbers were determined by immunofluorescence for CD68. Results: As might be expected CCL2 transcript levels were higher in colonic tissue from animals with ENDO‐NS compared to sham however they were significantly decreased in ENDO‐UN (p<0.05). mRNA expression of CCL2 positively correlated with CD68 positive cells (p<0.05). Surprisingly, higher levels of corticosterone and microscopic damage accompanied the lower levels of CCL2 in stressed animals. Conclusion: In this model uncontrollable stress decreases colonic CCL2 expression, possibly via corticosterone‐induced immunosuppression. It is unclear whether decreased CCL2 and/or increased corticosterone levels induced by stress contribute to an increased severity of colonic damage.

Grant Funding Source: R15A006373, RR003050/MD007579
Developmental regulation of nuclear receptor mRNAs in livers of FXR‐null mice (901.1)
Authors: Cui Julia Yue , Peng Lai , Yoo Byunggil , Tien Yun‐chen , Lu Hong , Zhong Xiao‐bo , Klaassen Curtis
Published: June 2021

The farnesoid X receptor (FXR/Nr1h4) is a critical transcriptional regulator for genes involved in bile acid processing, as well as lipid and glucose metabolism. However, very little is shown regarding the role of FXR in modulating the transcription of other nuclear receptors (NRs) that regulate drug metabolism and energy homeostasis. The purpose of this study was to unveil the importance of FXR in the developmental regulation of all NRs during liver development using FXR‐null mice. Six developmental ages from day‐2 to day 60 were selected, and the cDNA libraries were sequenced (100 cycles paired‐end) using an Illumina’s HiSeq2000 sequencer. Reads were mapped to the mouse mm10 genome (Tophat), and differential mRNA expression estimated by Cuffdiff (FDR<0.05). About half of the NRs were differentially expressed throughout liver development. Most notably, the hepatocyte nuclear factor 4α (HNF4α), which is the master regulator for liver development, is consistently the highest expressed NR at all ages, and interestingly, its mRNA level is markedly decreased in livers of FXR‐null mice, suggesting that FXR may influence the expression of HNF4α. The expression of oxysterol sensor liver X receptor α (LXRα/Nr1h3) and the xenobiotic sensor pregame X receptor (PXR/Nr1i2) is also consistently decreased in FXR‐null livers during development. Conversely, the expression of the retinoid‐related orphan receptor α (RORα), which was recently shown to regulate the expression of multiple drug metabolizing enzymes, is consistently increased in FXR‐null mice during liver development. In fact, RORα is the top most up‐regulated NR within the entire NR family in FXR‐null livers at all ages examined. In conclusion, the present study has unveiled the critical roles of FXR deficiency interferes with the mRNA ontogeny of critical NRs that are involved in lipid and drug metabolism during liver development.

Grant Funding Source: NIH
CFTR mutation/downregulation induces inflammation in mouse small intestine (902.1)
Authors: Liu Kaisheng , Zhang Xiaohu , Chan Hsiao Chang
Published: June 2021

Cystic fibrosis (CF) is the most common autosomal recessive disease in Caucasians which is caused by the mutations of CFTR (cystic fibrosis transmembrane regulator) gene. CF presents a multitude of clinical manifestations with chronic inflammation in the lung as one of the hallmarks. In our study, we also found increased inflammation in the small intestine of CFTR DF508 mutation mice. The length of the small intestine, the thickness of the smooth muscle layer and the depth of the crypt were all increased significantly in DF508 mutants. The NF‐κB signaling pathway was found activated with the upregulation of p105/50 and increased nuclear translocation of p65. COX‐2 expression was increased significantly, which is a key player of inflammation. Further more, downregulation of CFTR in intestinal cell line Caco‐2 cells by transfection of CFTR siRNA also induced increased inflammation markers including TNFα, IL6, IL8 and IL18. Meanwhile, the mRNA of COX‐2 increased 124.84±4.26%. Both in vivo and in vitro results indicate that defect in CFTR may directly link to the activation of inflammatory pathways, such as NF‐κB and COX‐2, which may promote abnormal growth of the small intestine.
The effects of alpha lipoic acid on apoptosis, NF‐κB and iNOS mRNA expression in experimental colitis (902.3)
Authors: Özkurt Mete , Zencirci İlhan , Necdet Fatih , Erkasap Nilüfer , Tosun Murat , Demirüstü Canan , İhtiyar Enver , Erkasap Serdar
Published: June 2021

Objective: Ulcerative colitis (UC) is characterized with a mucosal immune defect causing immunologic system hyperactivation against environmental agents and antigens, leading to lymphocyte proliferation, cytokine release, accumulation of other cells, excessive neutrophil production and ultimately development of cell injury. In our study, we aimed to show the effects of alpha lipoic acid (α‐LA) on UC to improve apoptosis, and p53, NF‐κB and iNOS mRNA expressions in a rat model of colitis.

Methods: There were 3 study groups, with 10 rats in each group. Group 1: control group, Group 2: UC, after induction of colitis, rats were followed for 4 weeks without treatment and Group 3: UC + LA, rats were treated with α‐LA 100 mg/kg intraperitoneally (i.p) for 4 weeks following induction of colitis by injection of acetic acid. After completion of the experiments, colon tissue samples of all rats were obtained surgically. Apoptosis was determined by TUNEL method and iNOS mRNA gene expression by using real‐time polymerase chain reaction (RT‐PCR). And also, p53 and NF‐κB expressions were analyzed by immunohistochemistry.

Results: In our results, apoptotic cells were significantly less in UC+LA group than in UC group, even if the expressions of p53 and NF‐κB were significantly different in both groups. There were no differences in gene expressions of iNOS between the groups Conclusion: The results of our study showed that α‐LA treatment has beneficial effects in terms of apoptosis and inflammation in experimental colitis.
Experimental spinal cord injury upregulates vagal nodose TRPV1 expression (902.4)
Authors: Swartz Emily , Deiter Gina , Holmes Gregory
Published: June 2021

The gastrointestinal (GI) tract is critically dependent upon adequate blood flow. Spinal cord injury (SCI) produces dramatic GI and vascular dysregulation, including vascular hypotension and, possibly, visceral hypoperfusion. We have previously shown that T3‐SCI in rats results in diminished visceral blood flow, increased expression of inflammatory markers and consequent gastrointestinal dysmotility. Vagal afferent injury has been shown to induce changes in cholecystokinin (CCK) and transient receptor potential vanilloid type 1 receptor (TRPV1) expression within the nodose ganglion. We hypothesized that SCI leads to increased activation of TRPV1 channels along the vagus nerve.

Nodose ganglia TRPV1 and CCK mRNA was quantified using qRT‐PCR, at 1‐day, 3‐days, and 7‐days post‐SCI and control. We used a superior mesenteric artery (SMA) occlusion model to show a worst case scenario of ischemia‐reperfusion to the GI tract. Our data show a significant increase in nodose TRPV1 and CCK of SCI and SMA rats compared to controls. The increased TRPV1 indicates GI inflammation and the increased CCK suggests that CCK increases as per vagal afferent injury. Our data present similarities to experimental ischemia‐reperfusion of the GI tract, in which SMA occlusion causes a dramatic inflammatory response within the GI tract. We propose that our model of SCI represents a continuum of GI ischemia and that the increase in inflammatory mediators and TRPV1 activation may play a role in the post‐SCI pathophysiology of gastric vagal afferents.

Grant Funding Source: Supported by: NINDS 49177
Properdin mediates complement activation and is protective against infectious colitis (902.6)
Authors: Jain Umang , Thomas Nikhil , Stover Cordula , Stadnyk Andrew
Published: June 2021

Background: Properdin amplifies complement activation and also acts as a pattern recognition receptor, facilitating the removal of microbes and apoptotic/necrotic cells in complement‐dependent and ‐independent mechanisms. C. rodentium is a natural bacterial pathogen of mice leading to colitis; we asked if properdin plays a role during C. rodentium colitis.

Methods: C. rodentium was gavaged into C57BL/6 (wildtype, WT) or properdin gene knockout (P‐/‐) mice and colitis was quantified by histopathological scoring of the colon and measurements of inflammatory mediators and apoptosis. In some experiments WT or P‐/‐ serum was injected into infected mice and the effects on colitis assessed.

Results: C. rodentium infected P‐/‐ mice exhibited levels of gut C3a higher than in infected WT mice but significantly less C5a than in WT mice. P‐/‐ mice suffered exacerbated colonic inflammation compared to infected WT characterized by excessive ulceration, infiltration by CD4+ T cells, neutrophils and macrophages, and increased levels of interferon‐γ. Using immunofluorescence, properdin associated with apoptotic cells and infected P‐/‐ mice had increased numbers of apoptotic cells. Lastly, properdin sufficient serum resulted in increased C5a levels and fewer apoptotic cells associated with less inflammation.

Conclusion: We discovered that properdin modulates complement activation during infectious colitis, evident by a lack of C5 convertase activity associated with a paradoxical increase in inflammation. We conclude that properdin regulates colitis in a C5a‐independent mechanism possibly related to a failure to remove apoptotic cell in the mucosa.

Grant Funding Source: NSERC, CCFC
The effects of enteral nutrition on gut microbiota in IL‐10‐/‐ mice (903.3)
Authors: Somero Jillian , Gulati Ajay , Uno Jennifer
Published: June 2021

Enteral nutrition delivered by an exclusive formula‐based diet can both reduce intestinal inflammation and induce mucosal healing in individuals with Crohn’s disease (CD); however, the mechanism by which this occurs remains unclear. Recent work also indicates that individuals with CD have altered enteric bacterial composition relative to healthy individuals. Here we investigate if an exclusive formula diet alters the bacterial composition in the stool of wild‐type (WT) and IL‐10‐/‐ mice. WT and IL‐10‐/‐ mice were placed on a formula‐based (ED) or regular diet (RD) for four weeks. Histologic analysis of these mice indicated no effects of the ED on the WT group; however, unexpectedly, the IL10‐/‐ mice on the ED showed increased inflammation with 25% of the mice developing rectal prolapse. Gut cultures corresponded with histology with the ED IL10‐/‐ mice consistently exhibiting increased production of IL‐12p40 and TNF (n=3, p= 0.05). To examine the bacterial composition of the microbiota, bacterial DNA was isolated and primers were used to PCR amplify the 16S gene of specific bacterial groups. Bacterial levels were calculated as a percentage relative to total 16S bacteria. Il10‐/‐ mice on the ED showed modest, although not significant increases in total bacteria as well as decreases in genus‐specific Bifidobacterium. Furthermore, significant decreases were observed in F. prausnitzii while increases were observed in Lactobacillus (n=4, p=.05). Collectively, these data indicate a dysbiosis in bacterial communities in the genetically susceptible IL10‐/‐ mice on the enteral relative to the regular diet, which may provide insight into the underlying mechanism involved in the intestinal changes associated with enteral nutrition.

Grant Funding Source: Elon University