bluuebunny/crossref_metadata_2025_split huggingface dataset:
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
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
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